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. Author manuscript; available in PMC: 2021 Jan 7.
Published in final edited form as: Cell Metab. 2019 Dec 5;31(1):131–147.e11. doi: 10.1016/j.cmet.2019.11.003

Figure 3. Hyperacetylation of mitochondrial proteins is accompanied by increased oxidation of long-chain fatty acid fuel in permeabilized myofibers.

Figure 3.

Mitochondrial oxygen consumption (JO2) was assayed in permeabilized fiber bundles from red gastrocnemius muscle from mice fed standard chow (A, C, and E) or a high fat diet (B, D, and F). Glutamate/Malate promotes electron flux through complex I, whereas succinate engages complex II. (G) Scheme depicting 13C-glucose labeling strategy and 13C metabolic flux analysis (MFA) in soleus muscles from DKO and DFC mice fed standard chow. Muscles were incubated with 10 mM [U-13C]glucose + 200 uM palmitate ±100 nM insulin. The citrate labeling pattern provides information on the relative contribution of pyruvate to the acetyl-CoA pool via PDH flux as compared to alternative routes of entry into the tricarboxylic acid cycle (TCAC). When [U-13C]pyruvate enters into the TCAC solely via PDH, the first condensation reaction produces M+2 citrate. By contrast, entry of [U-13C]pyruvate via malic enzyme (ME) or pyruvate carboxylase (PC) results in M+3 malate/oxaloacetate (OAA), which then forms M+3 or M+5 citrate upon condensation with unlabeled or M+2 acetyl-CoA, respectively. After the first spin of the TCAC, malate will be labeled M+2 from PDH flux or M+4 from combined PDH+ME/PC flux (see left text box*). Subsequent turns of the TCAC can produce more highly enriched citrate isotopomers (e.g. M+4, M+6). (H) Average percent 13C enrichment in TCAC metabolites after incubation with [U-13C]glucose without insulin. (I) Average percent 13C enrichment in TCAC metabolites after incubation with [U-13C]glucose plus 100 nM insulin. (J) Citrate mass isotopomer data (MID) from soleus muscles incubated with insulin. (K) The citrate M+5 to aspartate M+3 ratio provides insight into the enrichment of the acetyl-CoA pool because M+5 citrate is produced when M+3 OAA condenses with M+2 acetyl-CoA (see G). Aspartate M+3 was similar between genotypes and was used as a proxy for OAA M+3. Ratios were estimated by normalizing MID to the sum of the labeled isotopomers. Data represent mean ±SEM. (A–F) N=10–13 per group. (H–K) N=8 per group, and were analyzed by Student’s t-test. *P≤0.05, **P≤0.01, ***P≤0.001. N represents biological replicates.