45 2 Macrophage Metabolic Reprogramming in Diabetes followed by adding isotopically labelled substrates. This study confirmed that LPS-stimulated macrophages use citrate to synthesize itaconate and transport it to the cytosol to produce lipids. The study established further that IL4-stimulated macrophages rely on oxidative metabolism as their main energy source (see Figure 3). Furthermore, the study showed that some metabolites, like itaconate, are only synthesized in mitochondria, while others were produced by both cytosolic and mitochondrial enzymes, depending on the polarization status of macrophages. The integration of different levels of biological information has been used by Jha et al. in a pipeline named “concordant metabolomics integration with transcription” (CoMBI-T) that integrates MS metabolomics data with RNA-seq results in order to characterize macrophage polarization (116). Polarization was first confirmed by flow cytometry and then metabolic profiles were acquired using non-targeted MS-analysis. Altered metabolic pathways were further studied by metabolite flux analysis using 13C labeled glucose and 13C- and 15N-labeled glutamine combined with a targeted LC-MS approach (selective reaction monitoring (SRM)). Using this pipeline they showed that glutamine is a major source of nitrogen in alternatively activated macrophages and that these cells have an augmented metabolism of amino sugars and nucleotide sugars like uridine diphosphate N-acetylglucosamine. This metabolite is known to link signaling and metabolism through glycosylation of proteins that are localized at the cell-surface, for example, various growth factor receptors (119). By using this combined approach a new metabolite was found, which plays a major role in alternatively activated macrophages (116). The authors also investigated breakpoints in the TCA cycle using metabolic flux analysis experiments and found that the aspartate-argininosuccinate shunt, a series of reactions that connect the TCA cycle and the urea cycle (120), plays a role in pro-inflammatory macrophages. To summarize, a number of analytical approaches can be used to study metabolic reprogramming in macrophages. Flow cytometry analysis allows to define the different phenotypes of macrophages on which subsequent comparative analyses are based. Extracellular flux analysis provides functional insight into macrophage metabolic reprogramming at the level of metabolic pathways that are altered but does not quantify individual metabolites. Enzymatic assays with luminescent, colorimetric or fluorimetric readouts quantify a number of key metabolites. These assays are relatively easy to perform and data analysis does not require specific expertise as is the case for MS analysis. MS in combination with GC or LC allows to cover a wider range of metabolites both known and unknown. It thus allows to follow known key metabolites as well as to potentially gain insights into new metabolites or metabolite patterns. MS analysis can be extended to comprise metabolic flux analysis in order to follow how metabolites are consumed and produced in cell
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