guanosine-diphosphate and alpha-glycerophosphoric-acid

Whether active UCP1 can reduce ROS production in brown-fat mitochondria is presently not settled. The issue is of principal significance, as it can be seen as a proof- or disproof-of-principle concerning the ability of any protein to diminish ROS production through membrane depolarization. We therefore undertook a comprehensive investigation of the significance of UCP1 for ROS production, by comparing the ROS production in brown-fat mitochondria isolated from wildtype mice (that display membrane depolarization) or from UCP1(-/-) mice (with a high membrane potential). We tested the significance of UCP1 for glycerol-3-phosphate-supported ROS production by three methods (fluorescent dihydroethidium and the ESR probe PHH for superoxide, and fluorescent Amplex Red for hydrogen peroxide), and followed ROS production also with succinate, acyl-CoA or pyruvate as substrate. We studied the effects of the reverse electron flow inhibitor rotenone, the UCP1 activity inhibitor GDP, and the uncoupler FCCP. We also examined the effect of a physiologically induced increase in UCP1 amount. We noted GDP effects that were not UCP1-related. We conclude that only ROS production supported by exogenously added succinate was affected by the presence of active UCP1; ROS production supported by any other tested substrate (including endogenously generated succinate) was unaffected. This conclusion indicates that UCP1 is not involved in control of ROS production in brown-fat mitochondria. Extrapolation of these data to other tissues would imply that membrane depolarization may not necessarily decrease physiologically relevant ROS production. This article is a part of a Special Issue entitled: 18th European Bioenergetics Conference (Biochim. Biophys. Acta, Volume 1837, Issue 7, July 2014).

Topics: Adipose Tissue, Brown; Animals; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cold Temperature; Electron Spin Resonance Spectroscopy; Glycerophosphates; Guanosine Diphosphate; Hydrogen Peroxide; Immunoblotting; Ion Channels; Male; Membrane Potential, Mitochondrial; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Mitochondrial Proteins; Oxygen Consumption; Proton Ionophores; Pyruvic Acid; Reactive Oxygen Species; Succinic Acid; Superoxides; Uncoupling Protein 1

It has been proposed that the alpha-glycerophosphate (alpha-GOP) shuttle plays a crucial role in regulation of glycolysis in beta-cells by linking reoxidation of cytosolic NADH to formation of ATP in the electron transport chain (J. Biol. Chem. 265: 8287, 1981). Direct evidence for this suggestion is still lacking, however. In this work the operation of the alpha-GOP shuttle was investigated in the insulin-secreting cell line HIT-T15. The constituent enzymes of the pathway were found to be present in HIT cells. Flavin-linked alpha-GOP dehydrogenase was associated with the mitochondrial fraction, whereas NAD+-dependent alpha-GOP dehydrogenase was localized in the cytosol. In the presence of amobarbital (used to preserve the function of the alpha-GOP shuttle under conditions where oxidation of NADH by the respiratory chain was blocked), glucose increased insulin secretion, O2 consumption, and the cell [ATP]/[ADP] when compared with amobarbital alone. These results indicate that the alpha-GOP shuttle contributes to ATP generation in HIT cells and that its activation may be necessary for the initiation of insulin secretion by glucose.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Amobarbital; Cell Line; Clone Cells; Cytosol; Electron Transport; Glucose; Glycerolphosphate Dehydrogenase; Glycerophosphates; Glycolysis; Guanosine Diphosphate; Guanosine Triphosphate; Insulin; Insulin Secretion; Islets of Langerhans; Lactates; Lactic Acid; NAD; Oxidation-Reduction; Oxygen Consumption; Pyruvates; Pyruvic Acid