A Novel Fluorescence-Based Assay for the Measurement of Biliverdin Reductase Activity

Abstract

Biliverdin reductase (BVR) is the enzyme responsible for the last step in the production of bilirubin from the breakdown of heme. Bilirubin is one of the most potent antioxidant molecules in the body. Monitoring BVR activity is essential in studying the antioxidant capacity of cells and tissues. Traditional methods of determining BVR activity have relied on the measurement of bilirubin converted from biliverdin using absorbance spectroscopy. The approach has limited sensitivity and requires large quantities of cells or tissues. We have developed a novel fluorescence-based method utilizing the eel protein, UnaG, for the detection of bilirubin produced by BVR. The UnaG protein only fluoresces by the induction of bilirubin. We have also used this approach to measure intracellular bilirubin content of cultured cells. We validated this assay using cell lysates from mouse liver and immortalized murine hepatic cell line (Hepa1c1c7) and kidney cell line (MCT) in which BVR isoform A (BVRA) was either knocked out via CRISPR or stably overexpressed by lentivirus. Also, we tested the method using previously reported putative BVRA inhibitors, Closantel and Ebselen. These studies show a new method for measuring bilirubin intracellularly and in lysates.

Biliverdin Reductase A (BVRA) Knockout in Adipocytes Induces Hypertrophy and Reduces Mitochondria in White Fat of Obese Mice

“…Bilirubin has strong antioxidant properties, and we have shown that it also signals through the nuclear hormone receptor PPARα to up-regulate genes associated with increased fatty acid oxidation and glucose sensitization [72,73]. Bilividen is rapidly converted to bilirubin by BVRA [74], and biliverdin treatment in diabetic [75] or obese [76] mice improves metabolic dysfunction. However, biliverdin treatment in human HepG2 hepatocytes with PPARα knockdown attenuates transcriptome responses [73].…”

Bilirubin Nanoparticles Reduce Diet-Induced Hepatic Steatosis, Improve Fat Utilization, and Increase Plasma β-Hydroxybutyrate

“…We had previously shown that mice with the Gilbert's mutation UGT1A1*28 and hyperbilirubinemia were resistant to high-fat diet-induced hepatic steatosis by inhibiting de novo lipogenesis and activation of β-oxidation (Hinds et al, 2017). We have also shown that exercise elevated plasma bilirubin by suppressing hepatic UGT1A1 and increasing biliverdin reductase-A (BVRA) (Hinds et al, 2020), the enzyme that generates bilirubin (Adeosun et al, 2018;O'Brien et al, 2015), which improved hepatic glycogen storage and PPARα target genes (Hinds et al, 2020). Here, we found that PEG-BR reduced SCD1 and FAS while activating PPARα and ACOX1, improving hepatic steatosis in obese mice.…”

Rats Genetically Selected for High Aerobic Exercise Capacity Have Elevated Plasma Bilirubin by Upregulation of Hepatic Biliverdin Reductase-A (BVRA) and Suppression of UGT1A1

“…Indeed, there has been a correlation showing that plasma bilirubin levels are lower in obese humans [3][4][5]. The enzyme that produces bilirubin, BVRA [6,7], is also reduced in obese humans compared to lean matched controls [8]. Studies indicate that bilirubin levels might increase with exercise [9,10] and fasting [11][12][13][14], implying a possible role for metabolic regulation.…”