Figure 1.
High Dietary Fat Reduces the Rate of Pyruvate Supported Respiration in Isolated Cardiac Mitochondria.
C57BL/6 mice were fed a control or high fat diet for 1 d to 20 wk. Cardiac mitochondria were then isolated and incubated at 0.25 mg/mL with 25 µM palmitoylcarnitine and 1.0 mM malate or 100 µM pyruvate and 1.0 mM malate as respiratory substrates. State 3 respiration was initiated by the addition of 0.25 mM ADP at 2.0 min. Representative oxygen consumption traces for mitochondria isolated from mice fed control or high fat diet for 20 wk using A. pyruvate or D. palmitoylcarnitine. B. Percentage decrease in the rate of pyruvate-supported ADP-dependent (state 3) respiration relative to controls for diet durations indicated (n = 5, 11, and 9 for 1 d, 1wk, and 20 wk). C. Mass spectrometric quantification of the protein levels of each PDH subunit (n = 5). All data are presented as the mean ± SEM with p values: * < 0.05; ** < 0.01; and *** < 0.001.
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.
Figure 3.
High Dietary Fat Drives a Selective Increase in the Expression of PDK4 in the Heart.
mRNA and protein content of PDK and PDP isoforms were measured in hearts of mice fed either the control or high fat diet for 1 d or 1 wk. A. mRNA expression determined by qRT-PCR (n = 5 and 9 for 1 d and 1 wk). B. Protein content measured by quantitative mass spectrometry (n = 5 and 6 for 1 d and 1 wk). C. Expression of PDK4 in isolated cardiac mitochondria quantified by densitometric analysis of Western blots (n = 5 for 1 wk). All data are presented as the mean ± SEM with p values: ** < 0.01; and *** < 0.001.
Figure 4.
High Dietary Fat Alters the Kinetics of PDH Inhibition in Wild-type but not PDK4-/- Mice.
Cardiac mitochondria (0.25 mg/mL) from mice fed control or high fat diets for 1 wk 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. PDH activity was assayed at indicated time points for A. wild-type mice (n = 5) and B. PDK4-/- mice (n = 5). C. Oxygen consumption traces for mitochondria respiring with pyruvate were recorded. The rate of state 3 respiration was calculated for wild-type mice (n = 11) and PDK4-/- mice (n = 5). D. The relative levels of PDH E1α and Hsp60 as a loading control were assessed in wild-type and PDK4-/- mice fed control or high fat diet by Western blot analysis. All data are presented as the mean ± SEM with p values: * < 0.05 and ** < 0.01.
Figure 5.
PDH Inhibition is Relieved and PDK4 Content Reduced Upon Removal of Mice from the High Fat Diet.
C57BL/6 mice were fed either a control diet for 1 d, a high fat diet for 1 d, or a high fat diet for 1 d followed by a control diet for 1 d. A. Cardiac mitochondria were incubated with 100 µM pyruvate and 1.0 mM malate. State 3 respiration was initiated by the addition of ADP (0.25 mM). PDH activity was assayed during state 3 respiration (n = 3). B. Western blot analysis (representative of n = 3) and quantitative mass spectrometry (n = 3) were used to determine PDK4 protein levels in isolated cardiac mitochondria. All data are presented as the mean ± SEM with p values: * < 0.05; ** < 0.01; and *** < 0.001.
Figure 6.
Fasting Conceals Rapid Diet-Induced Inhibition of PDH.
Mice were fasted for 24 h or fed control or high fat diets for 1 d. Mitochondria were incubated with 100 µM pyruvate and 1.0 mM malate. State 3 respiration was initiated by addition of 0.25 mM ADP. A. PDK4 levels in isolated cardiac mitochondria assessed by Western blot analysis (representative of n = 5) and mass spectrometry (n = 5). B. PDH activity was assayed at 4.5 min during state 3 respiration with 100 µM pyruvate and 1.0 mM malate in cardiac mitochondria from mice fed the indicated diets (n = 5). C. Representative oxygen consumption traces for cardiac mitochondria from mice that were fasted or maintained on control or high fat diets for 1 d. D. Percent decrease in state 3 respiratory rate for each dietary condition relative to respective controls (n = 5). E. Mice were fed control or high fat diets for 2 d. All mice were fasted overnight (12 h) before sacrifice. PDH activity was assayed at 4.5 min during state 3 respiration with 100 µM pyruvate and 1.0 mM malate in the presence or absence of 25 µM palmitoylcarnitine. F. Rate of pyruvate supported state 3 respiration of cardiac mitochondria from mice fasted overnight (12 h) after indicated diets. All data are presented as the mean ± SEM with p values: * < 0.05 and *** < 0.001.
Figure 7.
PDK4-Upregulation Occurs Prior to Reductions in GLUT4 Expression and Insulin-Stimulated Akt Phosphorylation.
Mice were placed on a high fat or control diet for 3 d or 1 wk. Hearts were then processed and A. GLUT4 expression was determined by Western blot analysis and quantified by densitometric analysis (n = 5) and B. Cardiac insulin signaling was assessed as insulin-stimulated Akt phosphorylation, quantified by densitometric analysis (n = 5), and represented relative to total Akt. As indicated, wild type or PDK4 knockout mice were utilized. All data are presented as the mean ± SEM with p values: * < 0.05 and *** < 0.001.