*3.2. NOX*

Various cellular oxidases such as NOX and XO can produce ROS by reducing electrons from O2. Endothelial cells, chondrocytes, fibroblasts, myocytes, and phagocytes are the sites of NOX that produce ROS, particularly O2 •− and H2O2 to regulate cellular responses [31]. NOX initially produces O2 •−, followed by produces H2O2 by the action of the antioxidant enzyme SOD. Scientists have confirmed that H2O2 at low concentrations can modulate the signaling pathway and metabolism and have a similar function to ATP and calcium (Ca2+). Because this radical crosses the cell membrane by aquaporins (AQPS) or proxy purines it can cause effects such as proliferation and recruitment of immune cells [32].

When germs attack these cells, NOX enzymes are activated during a respiratory burst. The enhanced products then absorb NADPH and O2. Thus, NADPH can act as an electron donor. This action starts the NOX enzyme complex in the plasma membrane by producing O2 •− from O2 molecules. In general, the production of O2 •− by NOX is related to the time when an electron is taken from NADPH in the cytoplasm and transferred to an O2 molecule [33].

NOX consists of a total of seven isoforms of catalytic subunits, including NOX 1-5 and dual oxidase 1 (Duox1) and dual oxidase 2 (Duox2). It should be noted that the main isoform of NOX in fat cells is NOX4. In response to the excessive consumption of glucose or palmitate, this isoform concentration in AT increases [21]. On the other hand, classical cytosolic subunits are not required for NOX4 activation, and only P22 phox is needed. Furthermore, the modulation of NOX4 activity is responsible for Polymerase deltainteracting protein 2 (Poldip2), which ultimately produces O2 •− and H2O2. NOX5 and Duoxs 1 and 2 do not require cytosolic subunits for activation. These three members of the NOX family must bind to intracellular N-terminal EF hand motifs via Ca2+ for activation. The EF hand has a helix-loop-helix structure, which is mainly found in calcium-bound proteins. This eventually leads to the production of O2 •− and H2O2, respectively [34–37]. In short, all NOX members except NOX5 need the P22 phox subunit to form. This subunit is usually regulated by the mineralocorticoid receptor (MR). It should be noted that all NOX components look at NADPH as an electron donor for the production of O2 •− and H2O2 [16]. NOX enzyme complexes play an important role in the production of O2 •− by transferring electrons from NADPH to O2. H2O2 is known as a highly absorbent radical in cell membranes. Finally, H2O2 is reduced to H2O and O2 by the enzyme CAT [34].

Mitochondria can produce ROS in both direct and indirect forms. Mitochondria can indirectly serve as a target for ROS production by the NOX enzyme complex, indicating a cross-link between NOX and mitochondria. In addition to acting as a potential source of ROS, mitochondria can also be responsible for NOX stimulation under certain conditions. This is especially important when ROS is neutralized by target mitochondrial antioxidant enzymes. By inhibiting ROS production, these enzymes can also partially alleviate NOX activity [33].

NADH and 1,5-dihydroflavin adenine dinucleotide (FADH2) are the products of glucose metabolism as electron donors in the tricarboxylic acid (TCA) cycle. This process eventually accelerates ROS production. On the other hand, the oxidation of free fatty acids (FFA) by mitochondria increases FFA intake. In this case, NADH and FADH2 are also produced by the oxidation of FFA-derived acetyl-CoA and the beta-oxidation of fatty acids (FAs) as electron donors. On the other hand, NOX is present on plasma membranes and can convert molecular O2 to O2 •−. NOX may be closely related to ROS production associated with nutrient overdose [38]. Excessive FFA accumulation in adipocytes increases ROS production. On the other hand, ROS overproduction is reversed by NOX inhibitors such as diphenyleneiodonium or apocynin. This indicates NOX's role in the production of ROS due to excessive consumption of fatty acids. Activation of NADPH oxidase by excessive consumption of fatty acids stimulates the synthesis of diacylglycerol and subsequent activation of protein kinase C (PKC) by FFA, especially palmitate [39]. FFA's molecular mechanism that activates the NOX enzyme complex is closely related to the stimulation of diacylglycerol synthesis and subsequently activated PKC [21].
