Mitochondrial Function, Fatty Acid Metabolism, and Body Composition in the Hyperbilirubinemic Gunn Rat

Abstract

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.

Sexual dimorphism: increased sterol excretion leads to hypocholesterolaemia in female hyperbilirubinaemic Gunn rats

“…However, in contrast to human studies, the hypocholesterolaemic effects appear to be sex-specific in rats with a smaller reduction observed in male Gunn rats (Wallner et al 2013). Similarly, we have observed profound sexual dimorphism in body composition, mitochondrial function and fat metabolism in hyperbilirubinaemic Gunn rats, the reason for which remains unknown (Vidimce et al 2021). However, these studies suggest that UCB and/or UGT1A1 impairment may reduce circulating cholesterol concentrations, distinct from UCB's antioxidant properties (Bulmer et al 2013).…”

“…This suggests that bilirubin regulates lipid metabolism through PPARα in male Gunn rats, but an alternative mechanism must be responsible for the changes observed in females. However, unlike studies with hyperbilirubinaemic mice that show PPARα-mediated decreases in adiposity (Hinds et al 2017), minimal changes to fat metabolism occur in male Gunn rats (Vidimce et al 2021).…”

“…Cholesterol was extracted from homogenized liver and faecal samples by three sequential solvent extractions with isopropyl‐alcohol at a 50:1 mg:ml ratio of sample (liver or faeces) to isopropyl‐alcohol as previously published (Vidimce et al . 2021). During each extraction, homogenates were vortexed vigorously and sonicated at RT for 10 min prior to centrifugation (43,000 g , 20°C, 10 min).…”

Long-Term Effects of Biliverdin Reductase a Deficiency in Ugt1−/− Mice: Impact on Redox Status and Metabolism

“…Compared to wild-type mice, the body weight in the 9-months-old mice was significantly reduced in female but not in male Ugt1 −/− mice ( Figure S9A ). A similar gender-specific effect was seen in the hyper-bilirubinemic Ugt1 −/− rat caused by increased hepatic mitochondrial biogenesis in females [ 44 ]. Furthermore, the double knockout females also had a reduced body weight while their serum bilirubin levels were normal.…”

Serum metabolomics analysis reveals increased lipid catabolism in mildly hyperbilirubinemic Gilbert's syndrome individuals

“…The capacity of bilirubin to shape a health beneficial lipid and glucose phenotype has been demonstrated in several other animal models [7,11,[47][48][49] and in vitro studies [40,50]. Numerous large prospective epidemiological studies reported that bilirubin is associated with beneficial characteristics (improved BMI, blood lipids and insulin resistance) and a reduced risk of CVD and DMT2 [9,10,14,15,[51][52][53][54].…”