Beta-hydroxybutyrate alters one-carbon and branched-chain amino acid metabolism in glucose-restricted ER+ breast cancer cells

Ketogenic diet interventions show promise in breast cancer treatment, yet precise metabolic adaptations remain incompletely characterized. We investigated amino acid metabolic reprogramming in estrogen receptor-positive (ER+) breast cancer cells subjected to glucose restriction with beta-hydroxybutyrate (BHB) supplementation to model ketogenic conditions. MCF-7 and T47D cells were cultured in glucose-restricted medium (5% standard concentration) with graded BHB supplementation (2.5-15 mM) for four days. Metabolomic profiling was performed using comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) with Fisher ratio analysis. Targeted quantification of glycine and branched-chain amino acids (BCAAs) was conducted using colorimetric and fluorometric assays. SHMT1 protein expression was measured by ELISA. Bioinformatic analysis of TCGA Breast Invasive Carcinoma data (n=1,084) assessed co-expression patterns between one-carbon metabolism genes (SHMT1/2), BCAA metabolism (BCAT1), and ketone body catabolic enzymes (OXCT1, ACAT1, BDH1). Differential expression analysis was performed on paired ER+ tumor and adjacent normal tissue samples (n=20) from GSE58135. R-code for DEG analysis was partially created using Anthropic Claude (version Sonnet 4.5). Metabolomics analysis revealed significant perturbations in one-carbon and BCAA metabolism pathways. Direct quantification confirmed dose-dependent glycine accumulation, with peak elevation at 5 mM BHB in both MCF-7 (51.1±29.5 μM vs. 27.7±3.9 μM low glucose, p<0.05) and T47D cells (40.7±9.8 μM vs. 29.6±9.5 μM, p<0.05). Despite altered glycine levels, SHMT1 protein expression remained stable across treatments in both cell lines. TCGA analysis showed weak positive correlations between SHMT1 and ketone catabolic genes BDH1 (r=0.18, p=2.88×10⁻⁹) and ACAT1 (r=0.05, p=0.133), while BCAT1 showed moderate negative correlation with BDH1 (r=-0.36, p=3.69×10⁻³⁵). Differential expression analysis revealed significant downregulation of OXCT1 (log2FC=-1.41, p=5.75×10⁻⁷), SHMT1 (log2FC=-1.31, p=1.62×10⁻⁵), and ACAT1 (log2FC=-1.07, p=1.07×10⁻⁴) in ER+ tumors versus normal tissue, indicating selective suppression of ketone catabolism and cytoplasmic one-carbon metabolism. BHB supplementation under glucose restriction induces specific alterations in glycine homeostasis without corresponding SHMT1 changes, suggesting post-translational regulation or altered metabolic flux. Downregulation of ketone catabolic enzymes in ER+ tumors may represent a metabolic vulnerability exploitable through ketogenic interventions, warranting investigation of one-carbon metabolism as a therapeutic target in ER+ breast cancer.