Figure 4. CrAT regulates PDH activity.

Gastrocnemius muscles were harvested after a 4 h fast and lysates were prepared for immunoblot analysis of the mitochondrial marker protein, citrate synthase (CS), and electron transport chain complexes I, II and V. (panel A). Mitochondria were isolated from gastrocnemius muscles and a portion of the suspension was used to assess citrate synthase activity (panel B; n=12). State 3 and state 4 mitochondrial respiration rates were measured using BD Oxygen Biosensor plates after addition of 10 mM pyruvate and 5 mM malate ± 1 mM carnitine (panel C; n=6). RCR values were 7.2-7.6 for all groups. State 3 respiration was also measured with 10 mM pyruvate ± 1 mM carnitine (panel D; n=6). PDH activity was determined by measuring ¹⁴CO₂ produced from 1 mM [1-¹⁴C]pyruvate ± 5 mM carnitine in isolated muscle mitochondria from Crat^fl/fl and Crat^M-/- mice (panel E; n=6). The acute effect of 5 mM carnitine on PDH activity was evaluated in mitochondria from rat gastrocnemius muscle compared to rat liver (n=3) (panel F). Soleus muscles (n=6) were incubated 2 h with 10 mM [¹³C₆]glucose and 200 μM fatty acid ± 100 nM insulin, followed by LCMS-based mass analysis of [¹²C]acetylcarnitine (C2), [¹³C₂]acetylcarnitine ([¹³C₂]C2) and total acetylcarnitine (C2+([¹³C₂]C2) in the muscle tissue (panel G) and incubation buffer (panel H). * Genotype and # carnitine effects (p< 0.05) detected by Student's t-test.