FIG. 2.

Sirt3 deletion induces synergistic switch from glucose oxidation toward fatty acid oxidation and suppresses PDH activity by inducing PDH E1α hyperacetylation and hyperphosphorylation in fed Sirt3 KO skeletal muscle. A: Basal and insulin-stimulated (1 mU/mL) glucose oxidation, glycogen synthesis, glycolysis, and palmitate oxidation were measured in EDL muscles isolated from randomly fed 8- to 16-week-old WT and Sirt3 KO mice (n = 9; §P < 0.05 vs. basal, #P < 0.05 vs. WT, ANOVA). B: Basal and insulin-stimulated (10 mU/mL) phosphorylation of insulin/IGF-1 receptor, Akt, and ERK in tissue lysates of EDL muscles from fed and fasted 8- to 16-week-old WT and Sirt3 KO mice. C: Insulin-stimulated (1 mU/mL) glucose and palmitate oxidation were measured as described in research design and methods in diaphragms isolated from randomly fed 8- to 16-week-old WT and Sirt3 KO mice (n = 9; *P < 0.05, Student t test). D: Mitochondrial lysates were subjected to Western blot analysis using antibodies against p-232, p-293, and p-300 serine phosphorylation sites on PDH E1α, total PDH E1α, and Sirt3 (densitometry in Supplementary Fig. 2F). E: PDH activity was measured in 20 mg mitochondrial lysate from hindlimb of fed WT and Sirt3 KO mice using a PDH activity microplate kit as described in research design and methods. The PDH activities were calculated by linear regression of the steady-state kinetics and quantified (n = 4–5, *P < 0.05, Student t test). F: Western blot analysis was performed on tissue lysates from gastrocnemius muscle from fed and fasted WT and Sirt3 KO for PDHK4, Sirt3, and glyceraldehyde-3-phosphate dehydrogenase. G: Native PDH activity (PDHa) was measured in gastrocnemius muscle homogenate from WT fed, Sirt3 KO fed, and fasted C57Bl/6 mice by collection of ¹⁴CO₂ release from ¹⁴C-pyruvate. Prior to PDH assay, aliquots of homogenates were incubated in the presence of 50 mmol/L NaF with or without deacetylase inhibitors (1 mmol/L nicotinamide and 1 mol/L trichostatin A) as described in research design and methods (n = 6–7; #P < 0.05 vs. WT plus deacetylase inhibitors, ANOVA). OD, optical density.