Adaptation to glucose restriction and β-hydroxybutyrate supplementation is associated with metabolic flexibility in lung cancer cells

Metabolic reprogramming in tumor cells involves alterations in enzymatic machinery and metabolic profile reflect adaptive changes in malignant phenotypes. This study assessed the metabolic adaptability of A549 lung cancer cells under glucose restriction and β-hydroxybutyrate supplementation. Cells were cultured in media with varying glucose concentrations, with or without β-hydroxybutyrate. Targeted metabolomic analysis of conditioned media quantified 20 amino acids and 30 acylcarnitines using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Multivariate statistical analysis revealed distinct metabolic signatures and clear clustering of experimental groups based on extracellular metabolite profiles. The glucose-restricted, β-hydroxybutyrate–supplemented group exhibited a uniquely distinct pattern with specific alterations in extracellular amino acids (e.g., methionine, citrulline) and carnitine derivatives. Treated groups showed decreased tryptophan and increased arginine levels, suggesting altered tryptophan utilization coupled with coordinated shifts in arginine metabolism during metabolic stress. Notably, short-chain acylcarnitine species were elevated across all comparisons (Glucose-restricted- β-hydroxybutyrate-enriched group vs. control, Glucose-restricted group, and β-hydroxybutyrate-enriched group). These findings underscore the profound impact of substrate availability on cancer cell metabolism, with glucose limitation driving changes in amino acid pools consistent with heightened utilization. Extracellular amino acid and acylcarnitine profiles capture metabolite and pathway shifts linked to altered nutrient conditions; however, functional studies are needed to establish their biological relevance. Importantly, all reported changes reflect concentrations in the culture medium, indicating altered cellular uptake, secretion, or utilization rather than direct measurements of intracellular metabolite pools.