Heme loading induces changes in metabolic gene expression to promote persistent PPP flux and macrophage redox homeostasis. (A) Fold change in individual genes involved in the glycolytic, pentose phosphate, or heme metabolism pathways, as measured by RT-qPCR of BMDMs treated with 1 µM, 5 µM, or 10 µM heme for 6 or 24 hours (n = 3). Data in a heat map show the fold change compared with vehicle-treated macrophages. Primer sequences are listed in supplemental Table 1. (B) Volcano plots representing gene upregulation in BMDMs treated with 10 µM heme for 6 or 24 hours (n = 3) with the horizontal cutoff for P < .05. PPP and related enzymes are highlighted in purple. (C) Pathway analysis summary of 10 µM heme-driven gene regulation in BMDMs. mRNA for each gene was measured after 6 hours (left box) and 24 hours (right box) of heme stimulation. Orange-shaded boxes indicate upregulation, and blue-shaded boxes indicate downregulation of the respective gene vs the corresponding vehicle control. (D) mRNA expression of G6pd and Pgd in BMDMs treated with 1 µM to 10 µM heme for 6 or 24 hours (n = 4). (E) G6PD activity measured as the rate of NADPH synthesis in BMDMs treated with vehicle or 10 µM heme for 6 or 24 hours (n = 4). (F) Intracellular concentrations of reduced NADPH of BMDMs treated with vehicle, 100 µM dehydroepiandrosterone (DHEA; an inhibitor of G6PD), and/or 10 µM heme (n = 4). (G) Levels of oxidized glutathione (GSSG) in macrophages treated with vehicle, 100 µM DHEA, and/or 10µM heme (n = 4). (H) Ratio of reduced glutathione (GSH) to GSSG in BMDMs treated with vehicle, 100 µM DHEA, and/or 10 µM heme (n = 4). Data are represented as mean ± SEM. Statistical significance between 2 groups was determined by Welch’s unpaired t test. Significance between more than 2 groups was determined by one-way ANOVA, with post hoc Tukey’s or Dunnett’s multiple comparison tests, as appropriate, to determine differences between specific groups. RLU, relative light unit; TCA, tricarboxylic acid [cycle]. *P < .05; **P < .01; ***P < .001. See also supplemental Figure 2 and supplemental Tables 1 and 2.