(A) Immunoblotting of 20 μg of mouse tissue lysates after 7 days on HFD (HF) or DC₁₂ diets with a pan anti-succinyllysine (Suc-Lys) antibody, with Ponceau staining as loading control. Note: To visualize muscle succinylation, 40 μg protein and a longer exposure time were needed. (B) Anti-succinyllysine immunoblotting of BAT, epididymal WAT (eWAT), and inguinal WAT (iWAT). (C) Liver and kidney extracts from mice on DC₁₂ or HFD (n = 4) were used for quantitative site-level succinylomics by mass spectrometry. Peroxisomal and mitochondrial peptides were curated and plotted as log₂ fold-change (DC₁₂/HFD) to visualize the effects of DC₁₂ on succinylation in each compartment. Gold dots represent peptides with significantly increased succinylation, blue dots represent peptides with significantly decreased succinylation, and gray indicates statistical insignificance. (D) Pathway analysis of all succinylated peroxisomal proteins in liver reveals strong clustering to the fatty acid metabolism pathway; the most heavily succinylated peroxisomal proteins are depicted in E. See Supplemental Tables 7 and 8 for succinylome data sets and full protein names. (F and G) Mass spectrometry was used to measure succinate in urine and feces from male 129S1 mice during the initial 7 days of adaptation to DC₁₂ diet. (H) After chronic adaptation to the DC₁₂ diet or HFD (5 wk), mass spectrometry was used to quantify succinate in serum, urine, liver, muscle, brain, and heart. Succinate is presented as a ratio of DC₁₂: HFD, and the dashed line represents no change (ratio of 1.0). (I) The ratio of succinate to fumarate represents the substrate: product ratio for the enzyme succinate dehydrogenase, the entry point of succinate in the TCA cycle. Panel F was analyzed with 1-way ANOVA and Tukey-corrected multiple comparisons, and remaining panels were analyzed with 2-sided Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001. Ser, serum; Uri, urine; Liv, liver; Kid, kidney; Mus, muscle; Brn, brain; Hrt, heart.