The leading cause of death in patients with non-alcoholic fatty liver disease (NAFLD) is cardiovascular disease (CVD). However, the mechanisms are unknown. Mice deficient in hepatocyte PPARα ( PparaHepKO) exhibit hepatic steatosis on a regular diet, making them prone to manifesting NAFLD. We hypothesized that the PparaHepKO mice might be predisposed to poorer cardiovascular phenotypes due to increased liver fat content. We use PparaHepKO and littermate control mice fed a regular chow diet to avoid complications with a high-fat diet, such as insulin resistance and increased adiposity. After 30 weeks on a standard diet, male PparaHepKO mice exhibited elevated hepatic fat content compared to littermates as measured by Echo MRI (11.95 ± 1.4 vs. 3.74 ± 1.4%, p<0.05), hepatic triglycerides (1.4 ± 0.10 vs. 0.3 ± 0.01mM, p<0.05), and Oil Red O staining, despite no changes in body weight, fasting blood glucose, and insulin. The PparaHepKO mice also displayed elevated mean arterial blood pressure (121 ± 4 vs. 108 ± 2 mmHg, p<0.05), impaired diastolic function, cardiac remodeling, and enhanced vascular stiffness. To determine mechanisms controlling the increase in stiffness in the aorta, we used state-of-the-art PamGene technology to measure kinase activity. Our data suggest that the loss of hepatic PPARα induces alterations in the aortas that reduce the kinase activity of tropomyosin receptor kinases (TRKs) and p70S6K kinase, which might contribute to the pathogenesis of NAFLD-induced CVD. These data indicate that hepatic PPARα protects the cardiovascular system through some as-of-yet undefined mechanism.

Bilirubin levels in obese humans and rodents have been shown to be lower than in their lean counterparts. Some studies have proposed that the glucuronyl UGT1A1 enzyme that clears bilirubin from the blood increases in the liver with obesity. UGT1A1 clearance of bilirubin allows more conjugated bilirubin to enter the intestine, where it is catabolized into urobilin, which can be then absorbed via the hepatic portal vein. We hypothesized that when bilirubin levels are decreased, the urobilin increases in the plasma of obese humans, as compared to lean humans. To test this, we measured plasma levels of bilirubin and urobilin, body mass index (BMI), adiposity, blood glucose and insulin, and HOMA IR in a small cohort of obese and lean men and women. We found that bilirubin levels negatively correlated with BMI and adiposity in obese men and women, as compared to their lean counterparts. Contrarily, urobilin levels were positively associated with adiposity and BMI. Only obese women were found to be insulin resistant based on significantly higher HOMA IR, as compared to lean women. The urobilin levels were positively associated with HOMA IR in both groups, but women had a stronger linear correlation. These studies indicate that plasma urobilin levels are associated with obesity and its comorbidities, such as insulin resistance.

Several population studies have observed lower serum bilirubin levels in patients with non-alcoholic fatty liver disease (NAFLD). Yet, treatments to target this metabolic phenotype have not been explored. Therefore, we designed an N-Acetylgalactosamine (GalNAc) labeled RNAi to target the enzyme that clears bilirubin from the blood, the UGT1A1 glucuronyl enzyme (GNUR). In this study, male C57BL/6J mice were fed a high-fat diet (HFD, 60%) for 30 weeks to induce NAFLD and were treated subcutaneously with GNUR or sham (CTRL) once weekly for six weeks while continuing the HFD. The results show that GNUR treatments significantly raised plasma bilirubin levels and reduced plasma levels of the bilirubin catabolized product, urobilin. We show that GNUR decreased liver fat content and ceramide production via lipidomics and lowered fasting blood glucose and insulin levels. We performed extensive kinase activity analyses using our PamGene PamStation kinome technology and found a reorganization of the kinase pathways and a significant decrease in inflammatory mediators with GNUR versus CTRL treatments. These results demonstrate that GNUR increases plasma bilirubin and reduces plasma urobilin, reducing NAFLD and inflammation and improving overall liver health. These data indicate that UGT1A1 antagonism might serve as a treatment for NAFLD and may improve obesity-associated comorbidities.

Studies have indicated that increasing plasma bilirubin levels might be useful for preventing and treating hepatic lipid accumulation that occurs with metabolic diseases such as obesity and diabetes. We have previously demonstrated that mice with hyperbilirubinemia had significantly less lipid accumulation in a diet-induced non-alcoholic fatty liver disease (NAFLD) model. However, bilirubin’s effects on individual lipid species are currently unknown. Therefore, we used liquid chromatography-mass spectroscopy (LC-MS) to determine the hepatic lipid composition of obese mice with NAFLD treated with bilirubin nanoparticles or vehicle control. We placed the mice on a high-fat diet (HFD) for 24 weeks and then treated them with bilirubin nanoparticles or vehicle control for 4 weeks while maintaining the HFD. Bilirubin nanoparticles suppressed hepatic fat content overall. After analyzing the lipidomics data, we determined that bilirubin inhibited the accumulation of ceramides in the liver. The bilirubin nanoparticles significantly lowered the hepatic expression of two essential enzymes that regulate ceramide production, Sgpl1 and Degs1. Our results demonstrate that the bilirubin nanoparticles improve hepatic fat content by reducing ceramide production, remodeling the liver fat content, and improving overall metabolic health.

For unknown reasons, plasma bilirubin levels are lower in obese humans and rodents. One possible explanation is that the hepatic expression of enzymes that regulate its half-life, such as the UGT1A1 UDP-glucuronosyltransferase, is higher in the obese compared to lean persons. Bilirubin is marked for excretion within the liver by the conjugation of two glucuronyl groups by UGT1A1, where it is then transported to the intestines by the biliary system. Once in the intestines, the gut microbiota containing bilirubin reductase remove the glucornyl groups and modify the structure to produce urobilin which can be absorbed via the hepatic portal vein and enter the systemic circulation. Previous correlation studies indicate that urobilin might be a factor that induces visceral obesity and cardiovascular disease. However, the physiological function of urobilin is unknown. The purpose of our study was to determine if there is a correlation between bilirubin and urobilin compared to adiposity and insulin resistance in lean and obese women and men. We hypothesized that bilirubin would be lower and urobilin higher in the plasma of obese humans compared to lean. We found that the plasma levels of urobilin and bilirubin are inversely correlated in women and men. Urobilin levels were positively associated with adiposity (BMI R2=0.29 and body fat percentage R2=0.25) and insulin resistance (blood glucose R2=0.05, insulin R2=0.22, and HOMA IR R2=0.18) in obese women. Urobilin was only correlated with adiposity in males (BMI R2=0.025 and body fat percentage R2=0.03). However, the males were not insulin resistant and had no significant difference in HOMA IR compared to lean men. In both women (BMI R2=0.065 and body fat percentage R2=0.05) and men (BMI R2=0.24 and body fat percentage R2=0.13), plasma bilirubin levels were negatively associated with adiposity. We observed that plasma bilirubin levels are negatively associated with adiposity, which has been previously reported. The new findings in our study indicate that urobilin is positively associated with adiposity and insulin resistance. Based on these data, we propose that bilirubin and urobilin are maintained at inverse levels in humans via the UGT1A1 axis and that activity is higher in the obese, reducing plasma bilirubin and providing more substrates to produce urobilin in the gut. These events cause an elevation in plasma urobilin levels that commence obesity-associated comorbidities. Future work to determine the role of urobilin in obesity-associated comorbidities such as insulin-resistant diabetes is warranted and may provide a biomarker for the early This work was supported by the National Institutes of Health R01DK121797 (T.D.H.J.). The sample collection was supported by the Center for Clinical and Translational Science (CCTS) grant UL1TR001998 at the University of Kentucky. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.