Figure 1.
Heme loading drives a bioenergetic shift in macrophages, reducing mitochondrial respiration and promoting glucose uptake. (A) BMDMs were treated with vehicle or heme (10 or 50 μM) for 6 hours, and intracellular heme content was normalized to protein (n = 3). (B) MST of BMDMs loaded with 10 µM vehicle or 50 µM heme for 6 hours (n = 3-4). The oxygen consumption rate (OCR) was measured initially (basal; blue) and after injection of 1 µM oligomycin, 2 µM BAM15 (maximal; green), and 10 µM antimycin A (AA) and 1 µM rotenone (Rot) (gray; non-mitochondrial). Basal and maximal OCR were calculated by subtracting the mean OCR of the first 3 (basal) or post-BAM15 (maximal) measurements from the mean OCR of the post-AA/Rot measurements. Reserve capacity is the difference in maximal and basal respiratory capacity. (C) Glucose uptake of BMDMs loaded with vehicle, 50 µM heme or lipopolysaccharide (LPS; 1 µg/mL) for 6 hours, measured by the accumulation of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) (n = 4). (D) Messenger RNA (mRNA) expression of Glut1 in BMDMs loaded with vehicle or 50 µM heme for 6 hours measured by real-time quantitative polymerase chain reaction (RT-qPCR) (n = 4). (E) Glycolytic stress test (GST) of BMDMs loaded with 10 µM vehicle or 50 µM heme for 6 hours (n = 4). The extracellular acidification rate (ECAR) was measured after injection of 20 mM glucose, 1 µM oligomycin, and 80 mM 2-deoxyglucose (2-DG) to produce the basal (blue), stressed (green), and background (gray) ECAR, respectively. Basal and stressed ECAR were calculated by subtracting the mean ECAR of the post-glucose (basal) or post-oligomycin (stressed) measurements from the mean ECAR of the post–2-DG measurements. (F) Bioenergetics plot of heme-loaded macrophages, based on glycolytic capacity (stressed ECAR) and maximal respiratory capacity (maximal OCR). (G) Intracellular ATP levels of BMDMs treated with 10 µM vehicle or 50 µM heme as measured by luminescent signal of the Cell-Titer Glo ATP Assay are presented as percent of initial vehicle control (n = 4). (H) Viability of RAW264.7 macrophages treated with 10 µM vehicle or with 50 µM or 100 µM heme was determined by percentage of cells that excluded trypan blue dye and is presented as percentage of initial vehicle control (n = 4). Data are represented as mean ± standard error of the 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 analysis of variance (ANOVA), with post hoc Tukey or Dunnett’s multiple comparison test to determine significance between groups. *P < .05; **P < .01; ***P < .001.

Heme loading drives a bioenergetic shift in macrophages, reducing mitochondrial respiration and promoting glucose uptake. (A) BMDMs were treated with vehicle or heme (10 or 50 μM) for 6 hours, and intracellular heme content was normalized to protein (n = 3). (B) MST of BMDMs loaded with 10 µM vehicle or 50 µM heme for 6 hours (n = 3-4). The oxygen consumption rate (OCR) was measured initially (basal; blue) and after injection of 1 µM oligomycin, 2 µM BAM15 (maximal; green), and 10 µM antimycin A (AA) and 1 µM rotenone (Rot) (gray; non-mitochondrial). Basal and maximal OCR were calculated by subtracting the mean OCR of the first 3 (basal) or post-BAM15 (maximal) measurements from the mean OCR of the post-AA/Rot measurements. Reserve capacity is the difference in maximal and basal respiratory capacity. (C) Glucose uptake of BMDMs loaded with vehicle, 50 µM heme or lipopolysaccharide (LPS; 1 µg/mL) for 6 hours, measured by the accumulation of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) (n = 4). (D) Messenger RNA (mRNA) expression of Glut1 in BMDMs loaded with vehicle or 50 µM heme for 6 hours measured by real-time quantitative polymerase chain reaction (RT-qPCR) (n = 4). (E) Glycolytic stress test (GST) of BMDMs loaded with 10 µM vehicle or 50 µM heme for 6 hours (n = 4). The extracellular acidification rate (ECAR) was measured after injection of 20 mM glucose, 1 µM oligomycin, and 80 mM 2-deoxyglucose (2-DG) to produce the basal (blue), stressed (green), and background (gray) ECAR, respectively. Basal and stressed ECAR were calculated by subtracting the mean ECAR of the post-glucose (basal) or post-oligomycin (stressed) measurements from the mean ECAR of the post–2-DG measurements. (F) Bioenergetics plot of heme-loaded macrophages, based on glycolytic capacity (stressed ECAR) and maximal respiratory capacity (maximal OCR). (G) Intracellular ATP levels of BMDMs treated with 10 µM vehicle or 50 µM heme as measured by luminescent signal of the Cell-Titer Glo ATP Assay are presented as percent of initial vehicle control (n = 4). (H) Viability of RAW264.7 macrophages treated with 10 µM vehicle or with 50 µM or 100 µM heme was determined by percentage of cells that excluded trypan blue dye and is presented as percentage of initial vehicle control (n = 4). Data are represented as mean ± standard error of the 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 analysis of variance (ANOVA), with post hoc Tukey or Dunnett’s multiple comparison test to determine significance between groups. *P < .05; **P < .01; ***P < .001.

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