The importance of calcium ions for determining mitochondrial glycerol-3-phosphate dehydrogenase activity when measuring uncoupling protein 1 (UCP1) function in mitochondria isolated from brown adipose tissue

Abstract

Glycerol-3-phosphate is an excellent substrate for FAD-linked mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) in brown adipose tissue mitochondria and is regularly used as the primary substrate to measure oxygen consumption and reactive oxygen consumption by these mitochondria. mGPDH converts cytosolic glycerol-3-phosphate to dihydroxyacetone phosphate, feeding electrons directly from the cytosolic side of the mitochondrial inner membrane to the CoQ-pool within the inner membrane. mGPDH activity is allosterically activated by calcium, and when calcium chelators are present in the mitochondrial preparation medium and/or experimental incubation medium, calcium must be added to insure maximal mGPDH activity. It was demonstrated that in isolated brown adipose tissue mitochondria (1) mGPDH enzyme activity is maximal at free calcium ion concentrations in the 350 nM–1 μM range, (2) that ROS production also peaks in the 10–100 nM range in the presence of a UCP1 inhibitory ligand (GDP) but wanes with further increasing calcium concentration, and (3) that oxygen consumption rates peak in the 10–100 nM range with rates being maintained at higher calcium concentrations. This article provides easy-to-follow protocols to facilitate the measurement of mGPDH-dependent UCP1 activity in the presence of calcium for isolated brown adipose tissue mitochondria.