Metabolic Adaptations to Long-Term Caloric Restriction: Principal Components Analysis of Mass-Spectrometry Metabolomics from the CALERIE™ Phase 2 Trial

ABSTRACT Background Caloric restriction (CR) improves markers of biological aging, yet long-term effects on the human metabolome remain unclear. Objective This study examined the effects of CR (2 years) in healthy adults without obesity on circulating metabolites linked to aging and metabolic adaptations. Methods Untargeted metabolomics was performed using fasted plasma samples collected at baseline, 12, and 24 months (BL, 12M, 24M) from CALERIE™ participants randomized to CR or ad libitum (AL) control. A total of 864 known metabolites were identified and grouped into nine biologically coherent super pathways to support pathway-level interpretation (amino acid, peptide, carbohydrate, energy, lipid, nucleotide, cofactors and vitamins, xenobiotics, and partially characterized molecules). Principal component analysis (PCA) summarized metabolite variation, and linear mixed models assessed intervention effects on each PC in group-by-time interactions. Results Three principal components showed significant group-by-time interactions: PC2 (carbohydrate), PC5 (partially characterized molecules), and PC4 (lipid). Carbohydrate (PC2) and partially characterized metabolites (PC5) decreased from baseline to 12M in both groups; from 12M to 24M, levels stabilized in CR but increased in AL for PC2, while PC5 continued to decline in CR and increased in AL. Lipid metabolites (PC4) decreased in CR and increased in AL at 12M, with the pattern reversing from 12M to 24M. Key contributors included malto-saccharides and related carbohydrate intermediates for PC2, glutamine degradants and lactone sulfates for PC5, and sphingolipids for PC4. Conclusion This study provided insights into metabolic changes during CR, particularly for carbohydrate and lipid metabolism. Carbohydrate and lipid metabolites that were reduced by CR during the weight loss phase (BL to 12M) followed by stabilization or compensatory responses during the weight maintenance phase (12M to 24M) may link CR-induced changes in metabolism to inflammation. Future research is needed to tease out CR adaptations versus diet related changes in metabolites and explore the functional significance of these metabolic changes during CR for aging and long-term metabolic health. Conclusion CR produced distinct, time-dependent shifts in carbohydrate and lipid pathways. Early reductions during weight loss followed by stabilization or compensatory responses during weight maintenance suggest dynamic metabolic remodeling that may relate to inflammation-linked mechanisms. Further work is needed to distinguish CR-specific adaptations from dietary influences and to clarify the functional significance of these metabolic changes for aging and long-term metabolic health.