FIGURE 13.4 FFA activates mitochondrial uncoupling. Pancreatic clonal MIN6 ß cells were infected with recombinant adenovirus expressing UCP1, UCP2, and control green fluorescence protein for 48 hr, then permeabilized and subjected to measurement of mitochondrial membrane potential in a buffer containing 2.5 |i,M safranin. Mitochondrial membrane potential as reflected by the fluorescence was monitored by a FluoroCount plate reader at excitation/emission wavelengths of 530/590 nm. Respiratory substrate glycerol-3-phosphate (Gl-3-P, 7.5 ^M) was first added to induce mitochondrial hyperpolarization. To induce OA-mediated uncoupling effects, free fatty acid oleate (OA) was added to build up OA concentration from 25 to 75 ^M. To confirm the depolarization of mitochondrial membrane, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was added to a final concentration of 5.6 ^M. Vertical arrows denote time points of chemical additions. (Courtesy of Vasilij Koshkin, Wheeler Laboratory.)

Depol. and Uncoupling


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