78 Chapter 3 KDAC inhibition does not affect the levels of metabolites in alveolar-like macrophages Pro-inflammatory activation of many types of macrophages is not only characterized by a shift from oxidative phosphorylation to glycolysis, but also by interruptions in the TCA cycle (48). Breaks after the generation of citrate and after succinate (Figure 5) result in the accumulation of these two metabolites (48). To investigate these responses in alveolar-like macrophages in the context of LPS stimulation and KDAC inhibition, we investigated levels of the main metabolites of the TCA cycle and of the amino acids that are substrates of the TCA cycle. Stimulation of alveolar-like macrophages with LPS resulted in significantly more succinate but no changes in citrate compared to untreated controls (Figure 8A). However, we found significantly higher levels of α-ketoglutarate and malate after LPS stimulation, key TCA cycle metabolites that support immune responses (49,50). Pretreatment with either KDAC inhibitor prior to LPS stimulation did not change the levels of any of the measured metabolites or amino acids compared to LPS stimulation alone (Figure 8). Bar plots of individual metabolite levels can be found in Supplementary Figure 4 and Supplementary Figure 5. Figure 8: Effects of LPS and/or KDAC inhibitors MS275 or RGFP966 on TCA cycle metabolite and amino acid levels in alveolar-like macrophages. Alveolar-like macrophages were incubated with 1 ug/ml KDAC1/2/3 inhibitor MS275, KDAC3 inhibitor RGFP966, or vehicle for 16 h and then stimulated with 10 ng/ml LPS or vehicle for 4 h. Metabolite concentration levels were measured (A) with GC-MS or (B) with HPLC. Data are represented as a radar plot (as % of control, n=6). Levels of three metabolites were significantly higher in LPS-treated macrophages compared to control, respectively (C) malate, (D) succinate, and (E) α-ketoglutarate. Colored dots indicate the different independent replicates.
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