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Abstract INTRODUCTION:
Aging is associated with widespread metabolic changes that impair energy homeostasis and increase disease risk. Adipose tissue and liver play central roles in lipid storage, thermogenesis, and metabolic regulation, making them particularly susceptible to age-related dysfunction. While previous studies have described shifts in lipid composition with aging, comprehensive molecular profiling across multiple tissues remains limited. In this study, we employed untargeted lipidomics using high-resolution LC/Q-TOF mass spectrometry to systematically investigate lipid remodeling in white adipose tissue (WAT), brown adipose tissue (BAT), and liver across the lifespan of mice. By obtaining broad lipid coverage, including low-abundant and isomeric lipids, we aimed to uncover age-dependent signatures and pathways underlying metabolic inflexibility.
METHODS:
Liver, WAT, and BAT were collected from female C57BL/6 mice across three ages groups (young, mature, and old; n = 8 per group). Lipids were extracted from 10 – 15 g of tissue using a modified methanol/MTBE water protocol, incorporating the EquiSPLASH internal standard mix for normalization. Samples were homogenized, phase-separated, and dried under nitrogen before reconstitution in 9:1 methanol/chloroform for LC-MS/MS analysis. Untargeted lipidomics was performed using a 16-minute reversed-phase method on a ZORBAX Eclipse Plus C18 column coupled to the Revident LC/Q-TOF system operating in both positive and negative ion modes. Pooled QC samples were used for system suitability and Iterative MS/MS acquisition to enhance lipid annotation. Lipid species were identified using MS-DIAL 5, Lipid Annotator, and MassHunter Explorer. Data was normalized to the corresponding internal standard and tissue weight and downstream visualization was conducted using the lipidR package.
RESULTS:
Lipidomic profiling across liver, WAT, and BAT revealed distinct tissue-specific alterations associated with aging. Principal component analysis (PCA) showed clear clustering by tissue type, underscoring unique lipidomic signatures. Differential abundance analysis demonstrated a dramatic increase in the number of significantly altered lipid species in old versus young mice, with the liver and BAT showing the largest shifts. Triacylglycerols, EtherTG and TG esters were consistently upregulated in aged tissues, while phospholipids (PC, PE) and sphingolipids (SM, Cer) were commonly downregulated. Chain length analysis indicated enrichment of long-chain lipids in older mice, particularly in BAT and liver, suggesting age-related shift in lipid biosynthesis or degradation pathways. Notably mature age groups displayed intermediate profiles, reinforcing a progressive pattern of lipid remodeling. These findings highlight a systemic loss of lipid homeostasis with age, marked by increased lipid storage and reduced membrane and mitochondrial lipid species.
CONCLUSION:
Untargeted lipidomics using the Revident LC/Q-TOF platform revealed that aging induces profound, tissue-specific remodeling in metabolically active organs such as liver, white adipose tissue, and brown adipose tissue. With over 650 lipids confidently identified, we observed consistent age-related accumulation of long-chain and neutral lipids, including triacylglycerols and ether triacylglycerols. These changes were most pronounced in older mice and were associated with disrupted lipid homeostasis, mitochondrial dysfunction, and membrane composition shifts. Collectively, our findings highlight how aging drives metabolic inflexibility across key energy-regulating tissues, offering insight into lipid-based mechanisms that may contribute to age-associated disease risk.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
