Figure 4.

Cardiomyocyte-specific deletion of acetyl-CoA carboxylase reduces insulin-dependent increases in malonyl-CoA and pyruvate dehydrogenase activation. Control (Acc1^f/f/Acc2^f/f and αMHC-MerCreMer) and ACC-1/2 knockout mice, 3 days post-tamoxifen, received an intraperitoneal injection of saline or insulin (0.05 U/g body weight) at 8 AM. Hearts were excised 10 min post-injection, a piece of the left ventricle was frozen for metabolite analysis, and the remainder was homogenized and mitochondria isolated (+DCA and NaF). Frozen tissue from control and ACC knockout ± insulin was extracted, and metabolites resolved by reverse phase HPLC and (A) malonyl-CoA, (B) acetyl-CoA, and (C) CoASH quantified by UV/Vis spectroscopy. Isolated mitochondria were evaluated for (D) PDH activity as measured spectrophotometrically and (E) E1α subunit content and phosphorylation status of each site on the PDH E1α subunit (Ser²³², Ser²⁹³, and Ser³⁰⁰) by Western blot followed by densitometric analysis. Values are presented as the mean ± SD (n = 6) where significant differences (ANOVA with the Tukey test) are indicated by ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Each data point indicates a separate animal.