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. 2022 Jun 21;13:3544. doi: 10.1038/s41467-022-31149-y

Fig. 3. Kir2.1-mediated Vm endows macrophages with the metabolic signature of inflammation.

Fig. 3

a Heatmap of metabolites identified by unbiased metabolomics in mouse peritoneal macrophages (n = 4). The second cluster represents metabolites upregulated in LPS-treated versus control groups and then downregulated in the in LPS plus ML133 group (highlighted). b KEGG pathway analysis of metabolites in the second cluster with MetaboAnalyst 5.0. c Metabolites of glycolysis, and the PPP and SSP pathways identified in unbiased metabolomics. L: LPS, LM: LPS + ML133. d Extracellular acidification rate (ECAR) analyzed by Seahorse. (n = 6, mean ± SD). e Quantification of total serine, glycine, and methionine in cells treated with 500 ng/ml LPS in the presence or absence of 25 μM ML133 for 6 h followed by LC–MS.(n = 3). f In vitro glucose uptake assays of mouse peritoneal macrophages. (n = 8, 8, 6, 6, 4, 4, 4 respectively for left panel; n = 4 for right panel; mean ± SD). g In vivo glucose uptake assays. Glucose uptake by inflammatory macrophages (CD45+ CD11b+ F4/80+) in PECs and monocytes (CD45+ CD11b+ Ly6Chigh) in PBMCs was measured as 2NBDG MFI by FACS. (n = 4, mean ± SEM). h, i Mass isotopomer distribution analysis of metabolites derived from glucose. (n = 3 or 4, respectively). j Mass isotopomer distribution analysis of metabolites derived from glucose in mouse peritoneal macrophages treated with 500 ng/ml LPS for 6 h in the presence of physiological or elevated [K+]e (50 mM) or gramicidin (1.25 μM) in medium containing U-[13C]-glucose (25 mM) (n = 5). k–m RNA-seq analysis of control and Lyz2-cre-Kcnj2f/f mouse BMDMs. GSEA analysis showing enrichment (k). The pathway enrichment analysis of overlapped downregulated (reference threshold: fold-change, <0.5; p value, <0.05) (l) and upregulated (reference threshold: fold-change, >1.5; p value, <0.05) (m) genes by both ML133 treatment and Kir2.1-deficiency in LPS-primed BMDMs. NES normalized enrichment score; (n = 3). Two-tailed unpaired Student’s t-test. Glucose uptake data are representative of three independent experiments. Gating strategies for FACS were shown in Supplementary Figs. 8 and 9. Source data are provided as a Source Data file.