Abstract 1295: Mitochondrial Uncoupling in db/db Mouse Hearts is Mediated by the Adenine Nucleotide Translocator (ANT)
Diabetes increases myocardial FA oxidation and MVO2, but underlying mechanisms are partially understood. We used perfused hearts, permeabilized fibers (fibers) and isolated mitochondria from diabetic db/db mice to assess mitochondrial (MITO) uncoupling and to elucidate underlying molecular mechanisms. MITO uncoupling and reduced cardiac efficiency (CE) was absent in hearts perfused with glucose alone. However, in db/db hearts perfused with 1.0 mM Palmitate and 11mM glucose, RPP was reduced by 31% p< 0.02, MVO2 was increased by 45% p< 0.03, and CE reduced by 51% p<0.004. In fibers, state 3 respirations with palmitoyl carnitine were similar (16.4±1.3 and 16.7±1.5 nmol/min/mg), but ATP production rates were reduced by 36% in db/db fibers so that ATP/O ratios were 1.5±0.2 in db/db vs. 2.5±0.1 in controls (p<0.005), indicating FA-induced mitochondrial uncoupling. H2O2 production was also increased by 2.6 fold (p<0.001) in isolated db/db MITO exposed to the complex 1 substrate pyruvate. To determine if uncoupling proteins (UCP2 or UCP3), or ANT activity contributed to MITO uncoupling in db/db hearts, 500μM GDP and 5μM atractyloside (ATR) were sequentially added to fibers at state 4 respiration conditions (1μg/mL oligomycin). In the presence of succinate/rotenone, GDP inhibited state 4 respirations in WT MITO by 68% but only by 44% in db/db so that respiration rates remained elevated in db/db fibers (8.1±0.8 vs.5.9±0.7, p<0.03). ATR further reduced O2 consumption in db/db fibers but had no additional effect in WT, resulting in equivalent respiration rates (5.1±0.4 vs.5.4±0.8, p=0.8). To confirm these results, proton leak was measured in isolated MITO, incubated in the presence or absence of exogenous palmitate± GDP or ATR. Basal proton leak kinetics were similar in db/db and WT and palmitate increased proton leak by 2-fold in both groups. GDP reduced proton leak in palmitate-exposed WT MITO, but was without effect in db/db MITO. In contrast, ATR normalized proton leak kinetics in palmitate-exposed db/db MITO. Thus mitochondrial uncoupling characterizes the hearts of db/db mice and is mediated via FA and ROS-mediated activation of ANT.