Figure 2. Pyruvate Dehydrogenase Activity is Diminished and Phosphorylation Enhanced in Respiring Cardiac Mitochondria from Mice Fed High Dietary Fat.

Cardiac mitochondria (0.25 mg/mL) were incubated with 100 µM pyruvate and 1.0 mM malate. At 2.0 min, state 3 respiration was initiated by the addition of 0.25 mM ADP. A. PDH activity at 1.5 min, during state 2 respiration, and at 4.5 min, during state 3 respiration, in cardiac mitochondria from mice fed control or high fat diet for 1 d (n = 5). B. PDH activity at 4.5 min, during state 3 respiration, for cardiac mitochondria from mice fed a control or high fat diet for indicated times. Data are presented as the % decrease relative to controls (n = 5, 6, and 6 for 1 d, 1 wk, and 20 wk). C. Mitochondria were incubated with 100 µM or 10 mM pyruvate and 1.0 mM malate in the presence or absence of 25 µM palmitoylcarnitine. At 2.0 min, ADP was added (0.25 mM) to initiate state 3 respiration and PDH activity was assayed at 4.5 min (n = 5). D. Western blot analysis (representative of n = 5) of phosphorylation status for each site on the PDH E1α subunit during state 2 and 3 respiration in cardiac mitochondria isolated from mice on indicated diets for 1 d. E. Relative phosphorylation status during state 3 respiration in cardiac mitochondria isolated from mice fed a control (arbitrary unit 1) versus high fat diet for 1 d quantified by densitometric analysis of the Western blots (n = 5). All data are presented as the mean ± SEM with p values: * < 0.05; ** < 0.01; and *** < 0.001.