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In-depth organic mass cytometry reveals differential contents of 3-hydroxybutanoic acid at the single-cell level

ABSTRACT: In-depth organic mass cytometry reveals differential contents of 3-hydroxybutanoic acid on single cell level. Single-cell transcriptome indicates the expression difference of BHB downstream anti-oxidative stress proteins such as MT2A while Fluorescence Activated Cell Sorting (FACS) assay validates positive relationship between BHB and target proteins, which suggests that contents heterogeneity of BHB may endow cancer cells with variant ability to resist surrounding oxidative stress. Our ID-organic cytoMS paves the way for deep single-cell metabolome profiling and the investigation of cancer physiological and pathological processes. Comprehensive single-cell metabolic profiling is critical for revealing phenotypic heterogeneity and elucidating molecular mechanisms of biological processes. However, single-cell metabolomics remains challenging because of the limited metabolites coverage and disability of isomer discrimination. Herein, we establish a novel single-cell metabolomics platform of in-depth organic mass cytometry (ID-organic cytoMS). Extended single-cell analysis time guarantees sufficient MS/MS acquisition for metabolites identification and the isomers discrimination with online high-throughput analysis, achieving the largest number of about 600 metabolites identified in single cells. Fine sub-typing of MCF-7 cells are first demonstrated by differential contents of 3-hydroxybutanoic acid (BHB) among clusters. Single-cell transcriptome indicates the expression difference of BHB downstream anti-oxidative stress proteins such as MT2A while Fluorescence Activated Cell Sorting (FACS) assay validates positive relationship between BHB and target proteins, which suggests that contents heterogeneity of BHB may endow cancer cells with variant ability to resist surrounding oxidative stress. Our ID-organic cytoMS paves the way for deep single-cell metabolome profiling and the investigation of cancer physiological and pathological processes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE262591 | GEO | 2024/03/31

REPOSITORIES: GEO

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Project description:Beta-hydroxybutyrate (BHB) is a ketone body synthesized during fasting or strenuous exercise. Our previous study demonstrated that a cyclic ketogenic diet (KD), which induces BHB levels similar to fasting every other week, reduces midlife mortality and improves memory in aging mice. BHB actively regulates gene expression and inflammatory activation through non-energetic signaling pathways. Neither of these activities has been well-characterized in the brain and they may represent mechanisms by which BHB affects brain function during aging. First, we analyzed hepatic gene expression in an aging KD-treated mouse cohort using bulk RNA-seq. In addition to the downregulation of TOR pathway activity, cyclic KD reduces inflammatory gene expression in the liver. We observed via flow cytometry that KD also modulates age-related systemic T cell functions. Next, we investigated whether BHB affects brain cells transcriptionallyin vitro. Gene expression analysis in primary human brain cells (microglia, astrocytes, neurons) using RNA-seq shows that BHB causes a mild level of inflammation in all three cell types. However, BHB inhibits the more pronounced LPS-induced inflammatory gene activation in microglia. Furthermore, we confirmed that BHB similarly reduces LPS-induced inflammation in primary mouse microglia and bone marrow-derived macrophages (BMDMs). BHB is recognized as an inhibitor of histone deacetylase (HDAC), an inhibitor of NLRP3 inflammasome, and an agonist of the GPCR Hcar2. Nevertheless, in microglia, BHB's anti-inflammatory effects are independent of these known mechanisms. Finally, we examined the brain gene expression of 12-month-old male mice fed with one-week and one-year cyclic KD. While a one-week KD increases inflammatory signaling, a one-year cyclic KD reduces neuroinflammation induced by aging. In summary, our findings demonstrate that BHB mitigates the microglial response to inflammatory stimuli, like LPS, possibly leading to decreased chronic inflammation in the brain after long-term KD treatment in aging mice.