|dc.description.abstract||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
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.||en_US