Branched chain amino acid degradation induces lipid oxidation and TCA cycle metabolism in cultured myoblasts

Higher levels of circulating branched chain amino acids (BCAAs; Valine, Leucine, Isoleucine) and changes in their degradation rates are known to interact and alter lipid metabolism in insulin resistant muscle. However, the precise mechanisms by which BCAAs alter lipid metabolism and mitochondrial function remain unclear. We hypothesized that higher rates of BCAA degradation will result in a parallel induction of lipid oxidation and tricarboxylic acid (TCA) cycle metabolism via AMP-activated protein kinase (AMPK). Mouse C2C12 myoblasts were primed to induce BCAA degradation in DMEM: F12 media with increasing concentrations (0, 50, 100mM) of 3,6-dichloro-benzo[b]thiophene-2-carboxylic acid (BT2) for 3 days. Myoblasts were then seeded in 6-well plates at 500,000 cells/well in DMEM low glucose (5mM) media containing 250µM 2:1 13C18 Oleate: 13C16 Palmitate for 5-hrs. In a follow-up experiment, myoblasts primed with BT2 as above were treated with 2mM of 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, during the 5-hrs of incubation with 250µM 2:1 13C18 Oleate: 13C16 Palmitate. Cells were collected in 100µl of cold methanol and processed for gas chromatography- mass spectrometry analysis of 13C enrichment in TCA cycle metabolites. Intracellular BCAAs (µg/500,000 myoblasts ± SEM; Isoleucine; 0µM BT2; 0.61±0.06 vs. 100 µM BT2; 0.44±0.05 p = 0.06) and their corresponding ketoacids (ng/500,000 myoblasts ± SEM; α-keto-β-methylvalerate (KMV); 0µM BT2; 1.57±0.16 vs. 100 µM BT2; 1.20±0.08 p = 0.07) trended lower with increasing levels of BT2, indicating higher rates of BCAA degradation. Lower levels of intracellular branched chain keto-acids following BT2 treatment were significantly correlated with higher M+2 enrichment (APE; atom percent excess) in Citrate arising from 13C18 Oleate and 13C16 Palmitate (e.g., M+2 Citrate vs. KMV; r (21) = -0.67, p ≤ 0.001), suggesting that fatty acid oxidation is induced together with higher BCAA degradation. Induction of BCAA degradation also resulted in higher enrichments (APE) of M+1, M+2, and M+3 isotopomers of multiple TCA cycle intermediates pointing to a general induction of mitochondrial TCA cycle metabolism (α-ketoglutarate; M+1; 0µM BT2; 0.81±0.18 vs. 100µM BT2; 1.50±0.25; M+2; 0µM BT2; 5.76±0.58 vs. 100µM BT2; 8.36±0.87; M+3; 0µM BT2; 1.58±0.38 vs. 100µM BT2; 4.26±0.63, p ≤ 0.05). Interestingly, AMPK activation in myoblasts with AICAR resulted in a further enhancement of BT2-mediated induction of lipid oxidation and TCA cycle metabolism (Citrate M+2 (APE); 100µM BT2 + No AICAR; 30.81±1.19 vs. 100 µM BT2 + 2mM AICAR; 37.75±1.29 p ≤ 0.001). In conclusion, these results demonstrate that priming myoblasts to induce BCAA degradation resulted in higher oxidation of fatty acids through the TCA cycle, in an AMPK dependent manner. Biochemical modulation of BCAA degradation could be a strategy to enhance mitochondrial lipid oxidation and TCA cycle function in the myoblast. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.