Bioenergetics Profiling Core
Core Director: Craig Beeson, Ph.D.
Cellular redox species are produced directly or indirectly via bioenergetic metabolic reactions. Although the leak of electrons is often cited as the primary source of superoxide and hydroxy radicals, the leak is only a small contributor to the many different species involved in cellular redox reactions. Indeed, the fundamental basis of bioenergetics involves the oxidation (loss of electrons) of reduced nutrients and subsequent production of metabolites with a range of redox potentials (i.e., nicotinamides, reduced/oxidized metalloproteins, and thiol-containing species with varied redox potentials. Profiling the flux of these various metabolites through their attendant metabolic reactions is fundamental to any studies aimed at understanding cellular redox reactions.
The Bioenergetics Core provides several of the leading technologies that enable researchers to quantify the fluxes of these metabolic reactions in cells, tissues, organoids and small animal models such as zebrafish embryos and nematodes. The central technologies include high resolution respirometry using the XF technology from Seahorse Biosciences/Agilent. Dr. Beeson was involved in the original design of the XF technology profiles extracellular fluxes of oxygen, lactate and CO2 as the samples are interrogated with pharmacological and/or genetic manipulations. The instrumentation utilizes 96-well microplates to provide sufficient sample numbers to provide robust, statistically validated flux profiles of glycolysis, mitochondrial respiration, fatty acid oxidation, glutamine utilization and other related metabolic processes. Rapid, high-throughput imaging optimized to the XF plate architecture provides normalization of cell/tissue numbers, health, and other.
Isotopomer analyses of Krebs cycle intermediates and their byproducts utilizes LC-GC/MS. Typical 13C-labels at particular positions of, for example glucose, are introduced to cells or tissue small samples are periodically quenched, lysed and derivatized to volatile esters. The key advantage is that only the low molecular weight acids readily enter the gas phase for analyses that enable determination of enrichment of the 13C at positions that reveal the fluxes through specific pathways and/or the conversions to key species such as 2-hydroxy-glutamic acid – and oncometabolite produced by a mutated form of isocitrate dehydrogenase seen in many tumors that have escaped from therapeutic pressures. The oncometabolites affect epigenetic mechanisms that regulate tumor cell growth and proliferation.
A key feature of the core is that Dr. Beeson and his team also provide extensive training, data analyses support and aid in experimental design – it is fundamentally a collaborative unit.