41 2 Macrophage Metabolic Reprogramming in Diabetes expressing mice had fewer macrophages in adipose tissue (94). Moreover, treatment of RAW264.7 macrophages and primary intraperitoneal macrophages with SIRT1 activators inhibited inflammatory responses to LPS (96). The same treatment in Zucker fatty rats induced a shift from pro- to anti-inflammatory behavior in adipose tissue macrophages, in addition to improved glucose tolerance (96). In addition to activating KDACs, deacetylase inhibitors have also been used in the context of chronic inflammatory diseases, like chronic obstructive pulmonary disease (97,98), rheumatoid arthritis (99), and cancer (100) with positive outcomes. This suggests that inhibiting certain deacetylases associated with chronic inflammation in diabetes and obesity may also have therapeutic potential. Inhibitors of Class I KDACs, particularly KDAC3 (101) have been used in vitro and in vivo in the context of diabetes and obesity (102–104). None of them have reached the stage of clinical trials yet, even though promising results have been achieved in glycemic control and reduction of obesity, highlighting their potential as therapeutic treatment for metabolic disorders. The above paragraphs have highlighted that macrophages, inflammation and metabolic changes are intimately connected in DMTII and obesity. However how metabolic pathways in macrophages are changed is still unclear and many knowledge gaps remain. To be able to address the lack of knowledge on which metabolic pathways are affected by reprogramming, different techniques must be used and the results integrated. In the following paragraph, the most common ones will be described along with advantages and disadvantages and suggestions which would be most appropriate to use when. ANALYTICAL METHODS TO CHARACTERIZE MACROPHAGE METABOLIC REPROGRAMMING The most widely used method to characterize macrophage polarization and the corresponding phenotypes is flow cytometry (105). This technique determines properties of single cells by assessing the presence of proteins on the surface of cells or intracellularly with fluorescently-labeled antibodies using laser-induced excitation of the fluorescent labels. However, this approach does not provide information on cellular metabolism. Recent work has tried to fill this gap by using flow cytometry to investigate single-cell metabolism using antibodies against key metabolic enzymes (106). Although this work is a major step forwards, it still does not provide quantitative insight into metabolite production and enzyme activity, which is why other analytical methods are needed to gain a better mechanistic understanding of macrophage metabolic reprogramming.
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