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  • br Acknowledgments We would like to thank the Bloomington St

    2019-10-31


    Acknowledgments We would like to thank the Bloomington Stock Center and the Vienna Drosophila RNAi Center for stocks used in this study. RMG was supported in part by a Professors grant to R. Losick from the HHMI and this work was supported by NSF grant 074578 to A.A.N.
    Introduction The catabolic reaction of long-chain fatty acids, i.e., β-oxidation, takes place in the mitochondrial matrix space. Because fatty acids cannot permeate into this space, they are transported across the mitochondrial inner membrane in the form of acylcarnitines. For this, fatty acids are first converted into acyl-CoA by acyl-CoA synthase. Then, the acyl-CoA is converted into acylcarnitine by carnitine palmitoyltransferase 1 (CPT1, EC 2.3.1.21) located on the outer mitochondrial membrane. The acylcarnitine thus formed is transported across the mitochondrial inner membrane by carnitine/acylcarnitine carriers; and carnitine palmitoyltransferase 2 (CPT2), located on the inner mitochondrial membrane, carries out the reverse reaction of CPT1 and regenerates acyl-CoAs for the process of β-oxidation. Of these processes, the process of the conversion of acyl-CoA into acylcarnitine is known as the rate-limiting step; and CPT1 is regarded as the rate-limiting enzyme of β-oxidation (for review, see Refs. [1], [2], [3], [4]). Three isozymes of CPT1, CPT1a, 1b, and 1c, are known to be expressed in mammals [5], [6], [7]. CPT1a is mainly expressed in the liver and kidney; CPT1b is expressed in heart, skeletal muscle, and brown adipose tissue; and CPT1c is mainly expressed in the GSK2194069 and testis. Functional characterization of these CPT1 isozymes is very important to understand how the catabolism of fatty acids is regulated in various tissues. However, the enzymatic characterization of CPT1 is difficult, because this enzyme is easily inactivated upon solubilization with detergents [8], [9]. For this reason, functional analysis of this enzyme is mainly carried out by using expression systems such as mammalian cells or yeast cells [10], [11], [12], [13]. However, the question as to how these two expression systems differ from each other is still uncertain; and even discrepant results have been obtained with these two experimental systems [14], [15]. Thus, to conduct effective studies on the functional properties of CPT1, characterization of these two expression systems is an utmost important issue to be clarified.
    Materials and methods
    Results and discussion
    Main Text Metabolic GSK2194069 rewiring is a key hallmark of immune cell activation. Inflammatory macrophages, effector T (Teff) cells, and activated dendritic cells require increased aerobic glycolysis to support their phenotype and to fulfill their functions. Conversely, anti-inflammatory M(IL-4) macrophages, memory T (Tmem) cells, and regulatory T (Treg) cells are characterized by increased mitochondrial oxidative phosphorylation (OXPHOS), which is thought to energetically support their long-term survival and functions (O’Neill et al., 2016). The reported increased long-chain fatty acid oxidation (LC-FAO) in those cells can certainly aid OXPHOS, but studies interrogating the functional importance of LC-FAO to support M(IL-4) phenotypes have yielded conflicting results (Van den Bossche et al., 2017). The need for increased LC-FAO in M(IL-4) macrophages was first suggested by Vats et al. (2006) in a study using 50 μM etomoxir, an inhibitor of carnitine palmitoyl transferase 1 (CPT1). This mitochondrial membrane enzyme, together with CPT2, facilitates the transport of LC-FAs into the mitochondrial matrix for subsequent oxidation (Figure 1). Follow-up studies with etomoxir concentrations ranging from 10 to 100 μM observed no effect on IL-4-induced activation of mouse and human macrophages, and thus the field awaited genetic models to resolve the debate surrounding whether LC-FAO is obligatory for alternative macrophage activation or merely associated with it (Van den Bossche et al., 2017). Nomura et al. (2016) took the first step and applied CPT2-deficient macrophages to demonstrate that M(IL-4) activation does not require LC-FAO. While this suggested off-target effects of etomoxir, these results could not rule out that CPT1 may have functions independent of LC-FAO. Therefore, experiments with CPT1-deficient macrophages were still needed to unequivocally clarify its role in M(IL-4) cells.