Mitochondrial glutathione depletion reveals a novel role for the pyruvate dehydrogenase complex as a key H2O2-emitting source under conditions of nutrient overload.
Once regarded as a "by-product" of aerobic metabolism, the production of superoxide/H2O2 is now understood to be a highly specialized and extensively regulated process responsible for exerting control over a vast number of thiol-containing proteins, collectively referred to as the redox-sensitive proteome. Although disruptions within this process, secondary to elevated peroxide exposure, have been linked to disease, the sources and mechanisms regulating increased peroxide burden remain poorly defined and as such are difficult to target using pharmacotherapy. Here we identify the pyruvate dehydrogenase complex (PDC) as a key source of H2O2 within skeletal muscle mitochondria under conditions of depressed glutathione redox buffering integrity. Treatment of permeabilized myofibers with varying concentrations of the glutathione-depleting agent 1-chloro-2,4-dinitrobenzene led to a dose-dependent increase in pyruvate-supported JH2O2 emission (the flux of H2O2 diffusing out of the mitochondrial matrix into the surrounding assay medium), with emission rates eventually rising to exceed those of all substrate combinations tested. This striking sensitivity to glutathione depletion was observed in permeabilized fibers prepared from multiple species and was specific to PDC. Physiological oxidation of the cellular glutathione pool after high-fat feeding in rodents was found to elevate PDC JH2O2 emission, as well as increasing the sensitivity of the complex to GSH depletion. These findings reveal PDC as a potential major site of H2O2 production that is extremely sensitive to mitochondrial glutathione redox status.
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, USA; Department of Physiology, East Carolina University, Greenville, NC 27858, USA. Electronic address: k.fisher-wellman@garvan.org.au.
Kelsey H
KH
Fisher-Wellman
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, USA; Department of Physiology, East Carolina University, Greenville, NC 27858, USA.
Laura A A
LAA
Gilliam
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, USA; Department of Physiology, East Carolina University, Greenville, NC 27858, USA.
Chien-Te
CT
Lin
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, USA; Department of Physiology, East Carolina University, Greenville, NC 27858, USA.
Brook L
BL
Cathey
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, USA; Department of Kinesiology, East Carolina University, Greenville, NC 27858, USA.
Daniel S
DS
Lark
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, USA; Department of Physiology, East Carolina University, Greenville, NC 27858, USA; Department of Kinesiology, East Carolina University, Greenville, NC 27858, USA.
P
P
Darrell Neufer
D000328:Adult; D000818:Animals; D005260:Female; D005978:Glutathione / Q000737:chemistry*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008931:Mitochondria, Muscle / Q000201:enzymology / Q000378:metabolism*; D009243:NAD / Q000096:biosynthesis / Q000737:chemistry; D010084:Oxidation-Reduction; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012119:Respiration; D013481:Superoxides / Q000737:chemistry*
D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; D013481:Superoxides; D006861:Hydrogen Peroxide; D005978:Glutathione
Free radicals; Glutathione; Mitochondria; Pyruvate dehydrogenase complex; Reactive oxygen species; Skeletal muscle