NOX5 belongs to the NADPH oxidase family, which has seven members in total - NOXs 1–5, DUOX1, and DUOX2. Of all the NOX isoforms found in mammals, NOX5 is the least explored. Indeed, the absence of the gene in rodents accounts in part for our ignorance of this NOX isoform [21]. Numerous tissues, including those in the testis, spleen, lymph nodes, vascular smooth muscle, bone marrow, pancreas, placenta, ovary, and stomach, express NOX5 [38–40]. The NOX5 structure serves as a transition between the DUOX enzymes and NOXs 1–4: Although NOX5 lacks the extra transmembrane domain and DUOX peroxidase homology, it does have a relatively long cytoplasmic N-terminus with Ca2+-binding motifs [41]. The six transmembrane domains, the two sets of heme-spanning histidines in transmembrane domains 3 and 5, the FAD-binding domain located in the proximal region of the cytoplasmic C-terminus, and the NADPH binding motif located in the distal region of the cytoplasmic C-terminus are all common structural electron transport elements of NOX enzymes that are well conserved in NOX5 [38, 42–44]. Along with these conserved components, the NOX5 sequence also has a number of distinctive features that contribute to some of its biological characteristics. Contrary to other NOX isoforms, NOX5 has an extended N-terminus with four EF-handmotifs [42] and a polybasic domain (PBR-N), and a polybasic domain (PBR-C), a serine-rich region with phosphorylation sites at Thr494 and Ser498 of NOX5v2 (ß), and a consensus signal for a calmodulin-binding domain [43]. In intact cells, Nox5 and the chaperone Hsp90 interact, as shown by proximity ligation assays: the C-terminal portion of Nox5 binds to the M domain of Hsp90, which promotes the effective generation of superoxide [44].
Reactive oxygen species production plays crucial physiological and pathological roles. The primary free radical species generated by the beta cells' mitochondrial respiratory chain and NOX enzymes is the superoxide anion (O2). Free radicals that can harm beta cells can also be produced by immunological and phagocytic cells [11, 45]. Through a number of molecular mechanisms, including an increase in cytosolic calcium and protein kinase activation, chronic hyperglycemia causes free radical production in islets [13–14, 46]. Due to the inadequate antioxidant defense system capacity of beta cells, oxidative stress in beta cells is common in T2D and significantly contributes to the loss of function in both T1D and T2D [47–49]. Beta-cell activity is compromised by oxidative stress in a number of different molecular ways. It significantly lowers the amount of insulin produced, hinders the insertion of proinsulin vesicles into the plasma membrane, and lessens their exocytosis in response to blood glucose levels. Additionally, it can cause pancreatic cells to undergo apoptosis, which results in cell death and beta cell loss [12, 50]. Furthermore, an excess of free radical species affects beta cells' metabolic pathways negatively and damages K-ATP channels, which reduces insulin release [51–52]. The covalent alteration of certain cysteine residues in redox-sensitive proteins mediates the unique signaling effects of ROS. These residues have unique features in that they contain a terminal thiol (-SH) functional group, which is electron-rich enabling different oxidation states, including S-nitrosylation (S-nitration; SNO), S-glutathionylation (RS-SG), sulfhydration (SSH), disulphide bonds (RS-SR'), sulfenylation (SOH), sulfinic acid (SO2H) and sulfonic acid (SO3H) [53]. Target protein structure and function are impacted by oxidative post-translational changes. Numerous proteins undergo several types of oxidative post-translational modifications, which have a variety of physiological effects. Actin, a crucial protein involved in cytoskeletal assembly in vascular cells, is one of the multiple proteins that experience oxidative alterations [54–55]. Due to its extreme redox sensitivity, actin is one of the most noticeable proteins to become oxidized in cells under oxidative stress [54–55]. By lowering fasting blood glucose levels and raising insulin levels, Lee E.S. and colleagues demonstrated that treatment of Nox5 transgenic mice with pan-NOX inhibitor APX-115 greatly improved pancreatic beta cell function [24].
The synthesis and release of islet insulin is significantly influenced by reactive oxygen species. Although NOX enzymes are crucial controllers of physiological insulin secretion, they can have negative effects if they are consistently overproduced [56–62]. Gene expression profiling revealed elevated NOX5 mRNA levels in the islets of Type 2 diabetic patients when compared to the islets of non-diabetic subjects (https://www.ncbi.nlm.nih.gov/geoprofiles/71220505 accessed 06.05.2023), suggesting a potential connection between NOX5 and islet function. NOX5 is a particularly fascinating molecule in islet function due to its chemical makeup and the features of its enzymatic activity. NOX5 activity, in contrast to other isoforms, is unaffected by cytosolic organizer or activator subunits [38], p22phox, or Rac. Calmodulin controls NOX5 activation, which is directly dependent on free intracellular calcium [42]. Near fact, calmodulin boosts NOX5's sensitivity to calcium by attaching to a calmodulin-binding domain near the C-terminus [63]. Additionally, NOX5 has two conserved polybasic domains in both the N- and C-termini [65] and a functionally important phosphorylation site in the C-terminus [64]. The intricate network of calcium signals, NADPH, and ROS regulates islet glucose sensing and insulin secretion [66]. Increased glucose levels cause cytosolic Ca2 + to build up, which in turn causes a biphasic wave of beta-cell insulin release. One "amplifying factor" supporting the second phase of insulin secretion is NADPH [67]. It was shown that a key second messenger boosting insulin secretion was a brief increase in cellular ROS [62, 68]. On the other hand, persistent ROS increase has been associated with decreased insulin release [69–70]. Data gathered by Bouzakri K. and colleagues suggested that NOX5 was a factor in Type 2 diabetes' overnutrition-induced beta-cell damage and eventual beta-cell failure [71].
Increased prevalence of numerous renal disorders has been linked to oxidative stress produced by NADPH oxidase, particularly in diabetic nephropathy (DNF) [72]. Notably, Holterman et al. discovered that podocyte NADPH oxidase 5, or NOX5, plays a critical part in both the decline of renal function and hypertension [22]. NOX5 is expressed in human renal proximal tubules to a greater level than the other Nox isoforms in hypertension as opposed to normotensive patients, according to research by Yu P. and colleagues [73]. Additionally, it has been demonstrated that NOX5 speeds up renal damage in DNF [23, 25]. Recently, it has been discovered that diabetic nephropathy patients express more NOX5 than other isoforms, and it has been proposed that NOX5 is important for the creation of therapeutic drugs [24]. In coronary lesions, hypertension, and stroke, NOX5 appears to be involved in the development of vascular oxidative stress [74, 75]. Using a mouse expressing human NOX5 in the endothelium, the researchers demonstrated that NOX5 was activated and played a detrimental role in promoting edema, infarction, and ultimately worsened neurological function after cerebral ischemia [76]. This was a previously unidentified role of NOX5 in cerebral infarction. Nox5 can have a significant role in the proliferation and fibrosis of human hepatic stellate cells, as demonstrated by Andueza A. and colleagues [77]. The NOX5-deficient rabbit model, however, suggested that NOX5 may also play a protective role. Significantly more plaques formed in the thoracic aortas of NOX5-deficient animals, indicating that NOX5 may act as a preventative in young male rabbits to prevent atherosclerosis [78].
Although all studied NOX5 gene variants are pathogenic according to VannoPortal (Suppl. Table 5), only minor allele rs35672233-T was associated with the T2D risk. SNP rs35672233 is also involved in epigenetic regulation (histone mark H3K4Me1) of the NOX5 gene enhancer in the pancreas (Haploreg v.4.1 data, Suppl. Table 5). Moreover, loci rs35672233 and rs438866 create transcription factor biding sites (SNP info web server). For instance, minor allele rs35672233-T forms binding site for transcription factors PAX6 and HNF4A responsible for type beta pancreatic cell differentiation (FDR = 0.00036) and negative regulation of cell population proliferation (FDR = 0.01). Transcription factors PAX4 and PAX6 that bind to DNA in the presence of minor allele rs438866-C are involved in endocrine pancreas development (FDR = 0.0381).
There is no data on the link between NOX5 polymorphic variants and T2D risk in the literature. The present study is the first to establish association of rs35672233 with the T2D risk and describe the fact that this association can be modified by such environmental factors as chronic psychological stress, sedentary lifestyle, and high-calorie diet. We also showed that T2D patients-carriers of haplotype rs35672233C-rs3743093A-rs2036343C-rs311886C-rs438866C containing reference allele rs35672233-C NOX5 had higher levels of total glutathione GSH/GSSG and uric acid, lower random glucose levels and lower concentration of epoxyeicosatrienoic acids in blood plasma. However, analysis of human genetic information linked to T2D and related traits stored in Type 2 Diabetes Knowledge Portal (https://t2d.hugeamp.org/) revealed that rs35672233 is associated with type 1 diabetes mellitus and diabetic retinopathy; rs3743093 is associated with diabetic kidney disease and random glucose; rs2036343 is linked to fasting blood glucose, two-hour glucose, glycated hemoglobin, insulin sensitivity, chronic kidney disease and stroke in T2D; rs311886 is associated with random glucose, neuropathy, peripheral artery disease and nephropathy in T2D, whereas rs438866 was linked to random glucose, insulin resistance, and diabetic nephropathy. Our study did not reveal associations of any of the studies NOX5 SNPs with the T2D complications.
Study limitations. Our research has some limitations. First, the power for subgroup analysis was reduced due to the relatively small patient sample size. Second, just a few NOX5 gene polymorphisms were included in the analysis.