43 2 Macrophage Metabolic Reprogramming in Diabetes lactate production, it is critical to quantify lactate. In addition, since the TCA cycle is interrupted at two points, due to a reduced activity of isocitrate and succinate dehydrogenases, leading to accumulation of succinate and citrate, these two metabolites should be measured to assess the contribution of the TCA cycle to the overall conversion of glucose to ATP. Another reason for quantifying citrate is that it can be converted to acetyl-CoA, which serves as substrate for lysine acetyltransferases and is thus linked to protein acetylation and the regulation of gene expression (see paragraph “Lysine acetylation links metabolism and gene expression” for further details). Itaconate is another interesting metabolite to measure, since it plays a role in connecting the two breakpoints of the TCA cycle. To quantify lactate, an enzymatic assay can be used for in vitro studies (12). This assay is based on generating a luminescent signal that is proportional to the lactate concentration in the cell culture medium. Other enzymatic assays with colorimetric or fluorimetric readouts are available for most, but not for all of the metabolites of interest mentioned above (109,110). Moreover, these assays do not measure metabolite concentrations directly, but make use of enzymatic reactions that oxidize them, generating a product that reacts with a probe, producing a colorimetric or fluorimetric readout. Techniques that allow quantifying a wider range of metabolites simultaneously are therefore of increasing relevance to gain a more comprehensive view of metabolic changes in cells. Table 1. Metabolite analysis in macrophages metabolic reprogramming. Rows represent key metabolites or metabolic processes that can be measured in order to differentiate macrophage phenotypes. Columns present examples of references that use the different analytical techniques either alone or in combination. Extracellular flux analyzers Enzymatic essays Mass spectrometry Glycolytic activity (55,107,124–127) (55,128) OXPHOS activity (55,107,125–127,129) (55,128) Glucose (109,110,126) (55,109,110,128) Pyruvate (130) (55,125) Glucose-6-phosphate (55,125) Phosphoenolpyruvate (55) Lactate (12,127,131) (55,125,129,132) Succinate (109,110) (55,125,128,132–134) Citrate (127) (55,128,132–134) Acetyl-CoA (133) Itaconate (55,128,132,133)
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