Gary A. Churchill

Background: The systemic biochemical diversity of circulating metabolites and lipids reflects the integrated effects of genetic variation and environmental exposure. Metabolite quantitative trait locus (mQTL) studies in humans have established gene-metabolite associations, but genetic contributions can be obscured by sex, diet, age, medication use, and environmental exposures. Genetically diverse model systems offer a powerful complementary strategy to isolate genetic contributions to the biochemical diversity of the circulating metabolome. Methodology/Principal findings: We applied mass spectrometry profiling to serum samples in 541 mice from the Diversity Outbred (DO) population and identified 1,933 mQTL across 240 metabolites, 561 lipids, 43 oxylipins, and 4,465 MS/MS features. QTL hotspots on chromosomes 8, 9, 10, and 17 implicated distinct loci in the regulation of serum lipids, metabolites, and peptide-like features. The chromosome 8 Ces1 and Ces2 gene clusters were associated with lipids, acylcarnitines, and acyl-CoAs, while chromosomes 9 and 10 loci included additional lipid and metabolite QTL. On chromosome 17, MHC and C3 regions were associated with untargeted peptide-like MS/MS features. Comparisons with matched red blood cell mQTL revealed widespread compartment-specific genetic control. Conclusions/Significance: Collectively, these findings provide a high-resolution map of the genetic regulation of the circulating metabolome, offering mechanistic insights that complement and extend human metabolic genetics.

Control cohorts anchor the interpretation of lifespan-extending interventions, yet their lifespans vary even under identical experimental conditions. Without quantifying this expected variation, it is difficult to distinguish unusually short- or long-lived cohorts arising by chance from those reflecting potential experimental anomalies, with important consequences for interpreting treatment effects. The National Institute on Aging Interventions Testing Program (ITP), which tests interventions in mice across three independent sites using standardized protocols, provides a unique opportunity to address this problem. Previously, unusually short-lived males at the University of Texas in 2017 and unusually long-lived females at The Jackson Laboratory in 2022 were identified as potentially anomalous, but no standardized framework existed to determine whether such deviations exceeded expected variation. Here, we analyzed 10,757 control mice spanning 18 years of the ITP to quantify historical variation within and between sites and develop a prediction-interval framework for prospectively identifying unusual cohorts. We found that year-to-year variation, particularly in males, contributed substantially more to variation among cohort means than expected from random sampling alone. Accounting for this variation, our predictive framework flagged the 2022 Jackson Laboratory females but not 2017 University of Texas males, while flagging a previously unrecognized male cohort. Excluding this newly flagged cohort from the original lifespan analysis changed the statistical significance of two rapamycin regimens. These findings highlight the importance of a standardized framework for outlier detection and demonstrate the importance of accounting for natural variability in aging studies.

Cells release membrane-bound extracellular vesicles into the bloodstream laden with proteins that may reflect their physiological state. How this circulating EV proteome changes across life remains poorly understood. Identifying molecular signatures of aging in accessible biofluids could facilitate earlier intervention and monitoring of age-related disease. Many circulating aging proteome studies rely on affinity-based platforms which suffer from poor cross-species translation, ambiguous signal attribution, and inconsistent agreement between platforms. Here, we present a characterization of the aging plasma EV proteome from a cross-sectional cohort of 86 male and female C57BL/6J mice (5-31 months). We leveraged a species-agnostic EV enrichment (Mag-Net) and mass spectrometry to detect 2,575 protein groups from 15,969 peptides. Protein abundance heterogeneity increased with age and the abundance of 272 proteins were significantly correlated with chronological age including established senescence and frailty markers. Proteins increasing with age were enriched in genome maintenance pathways, while those decreasing were associated with the extracellular matrix organization and lipid metabolism. Notably, several of the strongest age-increased proteins converged on Alzheimer's and Parkinson's disease pathology. We observed sexual divergence in the aging EV proteome not previously characterized at this resolution. A proteomic clock built from this data accurately predicts chronological age, and peptide-level analysis reveals aging signals invisible at protein-level. These findings demonstrate that EV-enriched plasma proteomics can identify known aging markers, reveal novel sex-specific age-related changes, and generate predictive models of chronological age. This study provides a species-agnostic foundation for proteomic clocks that complement epigenetic approaches to monitor aging and evaluate healthspan.

Background: The systemic biochemical diversity of circulating metabolites and lipids reflects the integrated effects of genetic variation and environmental exposure. Metabolite quantitative trait locus (mQTL) studies in humans have established gene-metabolite associations, but genetic contributions can be obscured by sex, diet, age, medication use, and environmental exposures. Genetically diverse model systems offer a powerful complementary strategy to isolate genetic contributions to the biochemical diversity of the circulating metabolome. Methodology/Principal Findings: We applied mass spectrometry profiling to serum samples in 541 mice from the Diversity Outbred (DO) population and identified 1,933 mQTL across 240 metabolites, 561 lipids, 43 oxylipins, and 4,465 MS/MS features. Co-mapping QTL, i.e., QTL hotspots, on chromosomes 8 and 17 implicated carboxyl esterase gene clusters ( Ces1 and Ces2 ) as major regulators of circulating lipid remodeling and demonstrated genetic control of circulating protein/peptide-like features at the major histocompatibility complex and complement C3 loci. QTL hotspots on chromosomes 9 and 10 revealed previously unknown genetic drivers of lipid and amino acid metabolism. Comparisons with matched red blood cell mQTL revealed widespread compartment-specific genetic control. Conclusions/Significance: Collectively, these findings provide a high-resolution map of the genetic regulation of the circulating metabolome, offering mechanistic insights that complement and extend human metabolic genetics.

Dietary restriction extends lifespan across model organisms, but the plasma molecular changes mediating this effect remain incompletely characterized. We present a longitudinal multiomic analysis of 2,234 plasma samples from 960 Diversity Outbred mice subjected to intermittent fasting or caloric restriction and followed to natural death. Using mass spectrometry, we quantified 1,512 metabolites, lipids, and proteins and mapped their associations with diet, age and longevity. DR-induced molecular changes scale with caloric intake and modulate inflammatory, lipid catabolism, and oxidative stress pathways. Aging showed a biphasic signature with sharp acceleration beyond 85% of lifespan, demarcating terminal decline. Mediation and survival modeling both identified superoxide dismutase (SODE) and vascular cell adhesion molecule (VCAM1) as top lifespan predictors. Genetic analysis revealed 9,599 QTL, nine of which coincided with previously identified lifespan QTLs, and were largely related to immune regulation. These findings provide a rich multiomic and genetic resource for the aging research community.

Aging reshapes the cellular and molecular landscape of mammalian tissues. These changes can be progressive, preceding linearly with age, or occur as abrupt transitions of the course of lifespan. To investigate the age-dependent cellular and molecular shifts we profiled matched proteomes and transcriptomes from male and female murine spleens across eight time points, from stable adults through late life. The spleen was chosen to integrate understanding of age-dependent changes associated with immune surveillance, inflammaging, and immune-related proteostasis. Male and female mice follow distinct aging trajectories particularly in protein–RNA correlation in late life, reflecting both compositional shifts and failure of post-transcriptional buffering. To investigate whether these changes could be attributed to specific cell-types within the spleen, we developed Celestial, a machine-learning framework to identify cell-type-specific changes in bulk tissue samples. We found that age-related bulk molecular changes could be attributed in part to compositional remodeling of cell-types—expansion of GZMK+ CD8+ T cells and C1Q+ macrophages alongside naive T cell and global B cell loss. These results demonstrate that cell-type-aware interpretation can inform bulk multi-omic data for accurate mechanistic inference in heterogeneous tissues undergoing complex molecular remodeling.

The systemic biochemical diversity of circulating metabolites and lipids reflects the integrated effects of genetic variation and environmental exposure. Metabolite quantitative trait locus (mQTL) studies in humans have established gene-metabolite associations, but genetic contributions can be obscured by sex, diet, age, medication use, and environmental exposures. Genetically diverse model systems offer a powerful complementary strategy to isolate genetic contributions to the biochemical diversity of the circulating metabolome. We applied mass spectrometry profiling to serum samples in 541 mice from the Diversity Outbred (DO) population and identified 1,933 mQTL across 240 metabolites, 561 lipids, 43 oxylipins, and 4,465 MS/MS features. Co-mapping QTL, i.e., QTL hotspots, on chromosomes 8, 9, and 17 implicated carboxyl esterase gene clusters (Ces1 and Ces2) as major regulators of circulating lipid remodeling, revealed Gk5 as a previously unrecognized determinant of systemic lipid metabolism, and demonstrated genetic control of circulating peptide-like features at the major histocompatibility complex and complement C3 loci. Comparisons with matched red blood cell mQTL revealed widespread compartment-specific genetic control. Collectively, these findings provide a high-resolution map of the genetic regulation of the circulating metabolome, offering mechanistic insights that complement and extend human metabolic genetics.

Dietary interventions such as caloric restriction and periodic fasting improve metabolic health and extend lifespan in preclinical models, yet individuals differ widely in their physiological responses—variation that remains poorly understood but is critical for safe and effective translation to humans. We applied a 2 days per week intermittent fasting (IF) regimen to 10 inbred strains from the Collaborative Cross (CC), a genetically diverse, reproducible panel ideal for dissecting genetic effects on intervention responses. Using longitudinal phenotyping, we measured hundreds of traits, including lifespan. Our results show that sex and genetic background shape physiological responses to IF across metabolic, hematologic, and immunologic domains. Lifespan effects were also sex specific and varied among strains. These findings demonstrate that IF response is genetically determined in a mammalian model with human relevant physiology. We further compared CC results with a parallel study in Diversity Outbred mice, identifying shared predictors of health and lifespan as well as key differences between inbred and outbred populations. Overall, our work highlights the central role of genetics in shaping dietary intervention outcomes and informs efforts to translate IF benefits to human health and longevity.

Dense temporal measurements of physiological health, using simple and consistent assays, are essential to characterize biological processes associated with aging and evaluate the effectiveness of interventions on these processes. We measured body weight in 960 genetically diverse female mice, every 7-10 days over the full course of their lifespan. We used a state space model to characterize the trajectories of body weight throughout life and derived novel traits capturing the dynamics of body weight, 10 of which were both heritable and associated with lifespan. Genetic mapping of these body weight-derived traits identified 5 genomic loci, none of which were previously mapped to body weight. We observed that the ability to maintain stable body weight, despite fluctuations in energy intake and expenditure, was positively associated with lifespan in an age-dependent manner and mapped to a genomic locus linked to energy homeostasis. Our results highlight how dense longitudinal measurements of physiological phenotypes offer new insights into the biology of aging.