ABCB10 exports mitochondrial biliverdin, driving metabolic maladaptation in obesity.

Although the role of hydrophilic antioxidants in the development of hepatic insulin resistance and nonalcoholic fatty liver disease has been well studied, the role of lipophilic antioxidants remains poorly characterized. A known lipophilic hydrogen peroxide scavenger is bilirubin, which can be oxidized to biliverdin and then reduced back to bilirubin by cytosolic biliverdin reductase. Oxidation of bilirubin to biliverdin inside mitochondria must be followed by the export of biliverdin to the cytosol, where biliverdin is reduced back to bilirubin. Thus, the putative mitochondrial exporter of biliverdin is expected to be a major determinant of bilirubin regeneration and intracellular hydrogen peroxide scavenging. Here, we identified ABCB10 as a mitochondrial biliverdin exporter. ABCB10 reconstituted into liposomes transported biliverdin, and ABCB10 deletion caused accumulation of biliverdin inside mitochondria. Obesity with insulin resistance up-regulated hepatic ABCB10 expression in mice and elevated cytosolic and mitochondrial bilirubin content in an ABCB10-dependent manner. Revealing a maladaptive role of ABCB10-driven bilirubin synthesis, hepatic ABCB10 deletion protected diet-induced obese mice from steatosis and hyperglycemia, improving insulin-mediated suppression of glucose production and decreasing lipogenic SREBP-1c expression. Protection was concurrent with enhanced mitochondrial function and increased inactivation of PTP1B, a phosphatase disrupting insulin signaling and elevating SREBP-1c expression. Restoration of cellular bilirubin content in ABCB10 KO hepatocytes reversed the improvements in mitochondrial function and PTP1B inactivation, demonstrating that bilirubin was the maladaptive effector linked to ABCB10 function. Thus, we identified a fundamental transport process that amplifies intracellular bilirubin redox actions, which can exacerbate insulin resistance and steatosis in obesity.

Production of a human mitochondrial ABC transporter in E. coli.

Membrane proteins play important roles in health and disease. Despite their importance, the study of membrane proteins has been significantly limited by the difficulties inherent to their successful expression, purification, and stabilization once they have been extracted from the cell membrane. In addition, expression of human membrane proteins commonly requires the use of expensive and/or time-consuming eukaryotic systems, hence their successful expression in bacteria will be obviously beneficial for experimental research. Furthermore, since lipids can have critical effects on the activity of membrane proteins and given the composition similarities between the inner mitochondrial membrane and the bacterial plasma membrane, production of mitochondrial membrane proteins in E. coli represents a logical choice. Here, we present a novel protocol to produce a human mitochondrial ATP-Binding Cassette (ABC) transporter in E. coli. The function of the three known human mitochondrial ABC transporters is not fully understood, but X-ray crystallography models of ABCB10 produced in insect cells are available. We have successfully expressed and purified ABCB10 from E. coli. The yield is close to that of another bacterial ABC transporter routinely produced in our laboratory under similar conditions. In addition, we can efficiently reconstitute detergent purified ABCB10 into lipid nanodiscs. Measurements of ATPase activity of ABCB10 produced in E. coli show an ATP hydrolysis rate similar to other human ABC transporters. This novel protocol facilitates the production of this human mitochondrial transporter for biochemical, structural, and functional analysis, and can likely be adjusted for production of other mitochondrial transporters.

Stimulation of the human mitochondrial transporter ABCB10 by zinc-mesoporphrin.

Heme biosynthesis occurs through a series of reactions that take place within the cytoplasm and mitochondria, so intermediates need to move across these cellular compartments. However, the specific membrane transport mechanisms involved in the process are not yet identified. The ATP-binding cassette protein ABCB10 is essential for normal heme production, as knocking down this transporter in mice is embryonically lethal and accompanied by severe anemia plus oxidative damage. The role of ABCB10 is unknown, but given its location in the inner mitochondrial membrane, it has been proposed as a candidate to export either an early heme precursor or heme. Alternatively, ABCB10 might transport a molecule important for protection against oxidative damage. To help discern between these possibilities, we decided to study the effect of heme analogs, precursors, and antioxidant peptides on purified human ABCB10. Since substrate binding increases the ATP hydrolysis rate of ABC transporters, we have determined the ability of these molecules to activate purified ABCB10 reconstituted in lipid nanodiscs using ATPase measurements. Under our experimental conditions, we found that the only heme analog increasing ABCB10 ATPase activity was Zinc-mesoporphyrin. This activation of almost seventy percent was specific for ABCB10, as the ATPase activity of a negative control bacterial ABC transporter was not affected. The activation was also observed in cysteine-less ABCB10, suggesting that Zinc-mesoporphyrin's effect did not require binding to typical heme regulatory motifs. Furthermore, our data indicate that ABCB10 was not directly activated by neither the early heme precursor delta-aminolevulinic acid nor glutathione, downsizing their relevance as putative substrates for this transporter. Although additional studies are needed to determine the physiological substrate of ABCB10, our findings reveal Zinc-mesoporphyrin as the first tool compound to directly modulate ABCB10 activity and raise the possibility that some actions of Zinc-mesoporphyrin in cellular and animal studies could be mediated by ABCB10.