26 Chapter 2 single-cell RNA sequencing, and real-time extracellular flux analysis. Especially the latter has the potential to improve our understanding of how macrophages can switch between different types of responses. In DMTII and obesity, the changes in macrophage cellular metabolism coincide with profound changes in metabolism on a tissue and organism level, that probably interact and give rise to a specific DMTII-associated macrophage activation state (11). This review aims to summarize what is currently known about macrophage activation in DMTII-related meta- inflammation, how changes in intracellular metabolism are influenced by the changed presence in extracellular nutrients and metabolites, and how fluctuations in key metabolic intermediates could also play a role in cellular processes like gene expression. This overview emphasizes that profiling metabolites can help to characterize macrophages and their responses and to understand how changes in their intracellular metabolites affect DMTII progression. Therefore, we finish with a comparison between different approaches to metabolite analysis to provide an overview of the currently available methods and their pros and cons, highlighting metabolomics studies that have made use of these methods and have been central to characterizing macrophages. INSULIN RESISTANCE AND INFLAMMATION One of the key characteristics of DMTII is the altered insulin response. In healthy individuals, with a body mass index (BMI) in the normal range, pancreatic β cells produce insulin in response to circulating glucose levels. This will bind and activate insulin receptors on the cell membrane of different cell types, including macrophages, to lower blood glucose levels by enhancing its uptake by these cells. The binding of insulin to its receptor drives a cascade of events ultimately leading to uptake of glucose and further downstream effects (Figure 1). First glucose transporters, GLUT4 in most cell types and GLUT1 in macrophages, will either translocate from vesicles in the cytoplasm to the cell surface or their expression is upregulated, both increasing glucose influx into cells up to 10 times (12). Mammalian target of rapamycin (mTOR) will then be activated and protein synthesis will be induced. Furthermore, glycogen synthase kinase-3β (GSK3B) is inhibited allowing the activation of glycogen synthesis. When GSK3B is activated, it phosphorylates and inactivates glycogen synthase, decreasing glycogen synthesis, therefore GSK3B inhibition by Akt results in higher glycogen production. A change in gene transcription will also be initiated: expression of genes that favor either the synthesis of glycogen from glucose in the liver and muscles or of triglycerides from free fatty acids (FFA) in adipocytes will be induced and expression of genes that favor glycolysis will be transiently inhibited (13).
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