(OR15-01-26) All-Trans Retinoic Acid Alters Fatty Acid Metabolism in Human Myotubes Through Modulation of Mitochondrial Activity

Objectives: Retinoid signaling in human skeletal muscle remains incompletely understood. We investigated how all-trans retinoic acid (ATRA), a bioactive metabolite of vitamin A, alters lipid metabolism, gene expression, and mitochondrial function in human myotubes.

Methods: Primary human myoblasts from multiple donors were differentiated into myotubes and treated with ATRA or vehicle control. Fatty acid uptake and oxidation were assessed using radiolabeled oleic acid. Incorporation into intracellular lipid pools was measured following incubation with radiolabeled oleic acid or acetoacetate, followed by filtration of cell lysates through hydrophobic MultiScreen HTS plates and scintillation counting of lipid-associated radioactivity. Mitochondrial function was evaluated by measuring oxidative and reserve capacity following mitochondrial uncoupling with FCCP. RNA was isolated for RT-qPCR analysis of selected genes, alongside global transcriptomic and proteomic profiling using RNA sequencing and untargeted proteomics, followed by differential expression and pathway enrichment analyses. Statistical analyses were performed in R using models accounting for donor variability and multiple testing; significance was defined as P < 0.05.

Results: ATRA treatment increased fatty acid uptake, oxidation, and incorporation into intracellular lipid pools in human myotubes. Mitochondrial maximum oxidative capacity and reserve capacity increased following FCCP uncoupling. RT-qPCR showed increased expression of PDK4 and PLIN2. Transcriptomic analyses revealed upregulation of lipid metabolic and mitochondrial remodeling genes (e.g., FASN, ACSL5, ACACA, MTFR2). Proteomics showed 78 upregulated mitochondrial proteins involved in oxidation, respiratory chain assembly, and nucleotide synthesis. Integrated analyses identified established retinoid-responsive genes including RARRES1 and CYP26B1 alongside regulators of lipid metabolism.

Conclusions: ATRA enhances fatty acid uptake, oxidation, and incorporation into intracellular lipid pools in human myotubes, partly through modulation of mitochondrial activity and metabolic gene networks. These findings support a role for retinoid signaling in regulating skeletal muscle lipid metabolism.

Funding Sources: University of Oslo and S. G. Sønneland Foundation.