Goldberg 2019 Biochem J
|Goldberg EJ, Buddo KA, McLaughlin KL, Fernandez RF, Pereyra AS, Psaltis CE, Lin CT, Hagen JT, Boykov IN, Nguyen TK, Gowdy KM, Ellis JM, Neufer PD, McClung JM, Fisher-Wellman KH (2019) Tissue-specific characterization of mitochondrial branched-chain keto acid oxidation using a multiplexed assay platform. Biochem J 476:1521-37.|
Abstract: Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism-evident by gene transcription, metabolite profiling, and in vivo flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown. In vitro experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model in vitro BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.
© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.
Labels: MiParea: Respiration
Organism: Mouse Tissue;cell: Heart Preparation: Isolated mitochondria Enzyme: TCA cycle and matrix dehydrogenases Regulation: ADP, ATP, PCr;Cr Coupling state: LEAK, ET Pathway: F, N, S, NS, Other combinations, ROX HRR: Oxygraph-2k