The Effect of Hyperbilirubinaemia on Hepatic Cholesterol Metabolism in the Gunn Rat

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Bulmer, Andrew
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Du Toit, Eugene
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Cardiovascular Disease (CVD) is one of the world’s leading causes of morbidity and mortality, contributing to approximately 30% of the total deaths in Australia. CVD affects the heart and its associated vasculature and affects people of all ages and socio-economic backgrounds. Hypercholesterolaemia is key risk factor associated with the onset of CVD, particularly through its direct contribution to the development of atherosclerosis. Elevation in circulating cholesterol contributes significantly to the progression of atherosclerotic plaques leading to cardiovascular events such as embolism, myocardial infarction and stroke. Recent meta analyses show that people with Gilbert’s syndrome, a mild benign form of hyperbilirubinemia, experience less severe CVD and have an improved plasma lipid profile compared to normobilirubinaemic individuals. This finding has sparked research into the possible cardioprotective effects of bilirubin, which was previously only considered diagnostic test for the presence of liver dysfunction. Since then, the powerful antioxidant potential of bilirubin has been extensively investigated, and currently is considered a major mechanism contributing to the cardioprotective effect of the molecule. However, recent evidence suggests that mild hyperbilirubinemia may improve lipid status. This study aimed to determine whether hyperbilirubinemia is associated with lipid lowering effects in Gunn rats, a mutant model of chronic unconjugated hyperbilirubinaemia. By analysing endogenous cholesterol synthesis and biliary lipid secretion, amongst other parameters, this study aimed to develop a deeper understanding of impaired bilirubin excretion (i.e. UGT1A1 dysfunction) on cholesterol/lipid metabolism. Methods: 36 age- and litter-matched 10-week-old adult hyperbilirubinaemic Gunn rats (male n=9, female n=10; homozygous) and controls (male n=8, female n=9; heterozygous) were compared in order to determine potential differences in cholesterol metabolism associated with UGT1A1 dysfunction. All rats were exposed to a 19-day protocol, where regular blood samples were collected (day 0, 7, 19) and animals placed in metabolic cages for 5, 24 hour periods (day -1, -4, 10, 14, 18). In the final 5 days, the water supply was supplemented with a stable isotope (C13) label of acetate (2% w/v), to which animals had ad libitum access. On the final day, the bile duct was cannulated (following appropriate anaesthesia) and bile collected for 30 minutes. Analysis of tissue including blood, liver and faeces was undertaken to determine possible differences in blood and bile biochemistry. Repeated blood samples collected in the final 5 days of the protocol were analysed using GC-MS to calculate fractional cholesterol biosynthesis. Analysis of serum samples were also completed in 30, 3-week-old juvenile Gunn (male n=10, female n=7; homozygous) and control (male=n=8, female n=5; heterozygous) animals to assess any differences associated with sexual maturity. Results: Gunn rats had significantly reduced body weight (Gunn = 190.92, control = 245.9, p<0.01; g) and food intake (control = 20.0 ± 1.84, Gunn =16.4 ± 2.06, p<0.001; g). Analysis of plasma biochemistry revealed a notable reduction of lipids HDL (control = 1.36 ± 0.25, Gunn =0.20 ± 0.09, p<0.001; mmol/L), total cholesterol (control = 1.56 ± 0.34, Gunn = 0.60 ± 0.12, p<0.001; mmol/L) and an increase in plasma bile acids (control = 16.3 ± 7.34, Gunn = 41.7 ± 27.14, p <0.05; μmol/L) .These observations were specific to female animals only. Analysis of biliary constituents and relative bile secretion revealed significantly greater biliary cholesterol secretion in female Gunn rats, compared to female controls (females; control = 73.7 ± 40.0, Gunn 153.3 ± 27.7, p=0.006; mmol/L). However, no changes in hepatic cholesterol content and faecal cholesterol excretion were observed. Fractional cholesterol biosynthesis was significantly increased in female Gunn rats, however, no difference was observed in males. Analysis of juvenile serum biochemistry in Gunn rats, revealed non-significant reductions in plasma cholesterol and bile acids, but significant reduction in phospholipids (controls 150 ± 31.8, Gunn 118 ± 23.1 , p<0.05; mg/L ), compared to controls. Conclusion: This study is the first to assess of fractional cholesterol synthesis and biliary lipid output, providing insight into cholesterol metabolism of adult Gunn rats. It was originally hypothesised that endogenous synthesis is increased in Gunn rats, compared to controls. Female Gunn rats displayed an increased rate of fractional endogenous synthesis, compared to control females. The males on the other hand, did not show any significant differences. Increased endogenous synthesis in female Gunn rats suggests reduction of circulating cholesterol might be due to alternative mechanisms, with increased circulating bile acid concentrations suggesting the upregulation of CYP7A1, redirecting cholesterol to bile acid synthesis, or the downregulation of ABCA1, reducing cholesterol efflux from the periphery. Increased biliary cholesterol secretion was observed in female Gunn rats, compared to female controls, but no difference observed in males. Sex specific effects of UGT1A1 deficiency, seen in the current study, suggest that sexual hormones are influential in the interaction between chronic hyperbilirubinaemia and cholesterol metabolism. This was not addressed in depth, in the current study. Further confirmation of this hypothesis, using stored tissue samples from this study, could implicate new roles for hyperbilirubinaemia/UGT1A1 dysfunction in perturbing lipid metabolism and protecting from CVD.

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Thesis (Masters)
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Master of Medical Research (MMedRes)
School of Medical Science
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Hepatic cholesterol metabolism
Gunn rat
Cardiovascular disease
Gilbert’s syndrome
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