12 Chapter 1 The metabolic reprogramming of macrophages has several consequences. First, there is a breakpoint in the tricarboxylic acid (TCA) cycle after citrate due to lower expression of isocitrate dehydrogenase, leading to citrate accumulation. Citrate can be transported to the cytosol (42) and converted into acetyl-CoA (43), which can be used for fatty acid synthesis or lysine acetylation of proteins. Second, there is a breakpoint after succinate caused by the inhibition of succinate dehydrogenase by itaconate, resulting in succinate accumulation. Succinate accumulation leads to the stabilization of the transcription factor HIF-1α (44), promoting the switch to glycolysis and inducing the expression of glycolytic enzymes. This metabolic shift also activates the pentose phosphate pathway, generating NADPH for ROS production (45). Additionally, HIF-1α promotes the expression of lactate dehydrogenase (46), which converts pyruvate to lactate, and pyruvate dehydrogenase kinase 1, inhibiting mitochondrial function further (47). In summary, macrophages undergo metabolic reprogramming to adapt to different activation states. The shift towards glycolysis and altered metabolism of amino acids and fatty acids provide the necessary energy and biosynthetic precursors for macrophage activation and function during inflammatory responses (48). ANALYSIS OF METABOLITES Flow cytometry is commonly used to characterize macrophage phenotypes but does not provide information on cellular metabolism (49). Recent efforts have utilized flow cytometry with antibodies against metabolic enzymes to investigate single-cell metabolism, although quantitative insight into metabolite production and enzyme activity is still lacking (50). Other techniques such as extracellular flux analysis, colorimetric/fluorometric enzyme activity assays, and mass spectrometry (MS)- based metabolomics and flux analysis are used to measure the metabolic status of cells. Extracellular flux analyzers provide a functional readout of glycolytic or mitochondrial activity but do not directly measure individual metabolites (51). MS, coupled with gas or liquid chromatography, allows for detecting and quantifying a wide range of metabolites (52–54). Stable isotope labeling combined with MS can be used to study metabolic flux and pathway analysis (55). Targeted and untargeted MS methods can provide quantitative information on known metabolites as well as reveal new metabolites (56). Combining multiple analytical approaches, including metabolomics, lipidomics, fluxomics, transcriptomics, and proteomics, can help gain a comprehensive understanding of the mechanisms involved in macrophage metabolic reprogramming. This integrated approach has the potential to discover mechanistic links between inflammation and metabolic disturbances in chronic diseases.
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