Figure 3. Gα₁₂ regulation of SIRT1-dependent mitochondrial respiration in the liver.

(A) SIRT1 inhibition by Gna12 KO. Immunoblottings for SIRT1, SIRT3, and SIRT5 were performed using liver homogenates from 14-week-old WT or Gna12-KO mice fed ND (upper panel). Lower panel shows quantification (n = 3/group). (B) Effects of Gα₁₂ modulation on SIRT1 levels. Immunoblottings for SIRT1 were performed (upper) and quantified (lower) using primary hepatocytes from WT or Gna12-KO mice (left, n = 3/group), HepG2 cells infected with Ad-Gα₁₂QL or control (Ad-Con) (middle, n = 4/group), or AML12 cells stably expressing sh-Gα₁₂ or control (sh-Luci) (right, n = 3/group). (C) Immunoblottings for CPT1 and PGC1α in liver or primary hepatocytes from WT or Gna12-KO mice (upper) and their respective quantifications (lower, n = 3/group each). (D) qRT-PCR assays for PPARα target genes responsible for FA oxidation in the liver or primary hepatocytes (n = 3–11/group). (E) OCR in mitochondria. OCR was measured using the mitochondrial fraction prepared from liver tissues of WT or Gna12-KO mice (n = 3/group). Analyzed OCR was normalized to the protein concentrations for each set of samples determined by the Bradford method. (F) Palmitate oxidation in primary hepatocytes. [³H]-palmitate oxidation rate was determined using primary hepatocytes from WT or Gna12-KO mice, and 5 × 10⁵ cells per well were cultured in 12-well plates. Data shown are from 1 representative experiment of 2 independent experiments (n = 3 mice/group). Each dot represents an individual pool of primary hepatocytes isolated from each mouse. Values represent mean ± SEM. Data were analyzed by 2-tailed Student’s t test (A–F). For A–C, the blots in each panel were run in parallel using the same samples, and β-actin was used as a normalization control for densitometric analysis. For D, box-and-whisker plots show median (horizontal lines within boxes), 5%–95% (ends of the boxes), and range of minimum to maximum.