Dysregulation of nitric oxide synthases during early and late pathophysiological conditions of diabetes mellitus leads to amassing of microvascular impedement

Graphical abstract

Diabetes is a major killer worldwide and its unprecedented rise poses a serious threat to mankind. According to recent estimation, 387 million people worldwide are affected from the disease with a prevalence rate of 8.3 and 46.3 % still remains undiagnosed. Important characteristics of diabetes are abnormalities of the physiological signalling functions of reactive oxygen species and reactive nitrogen species. Increased oxidative stress contributes to the activation of stress-sensitive intracellular signalling pathways and the development of gene products that trigger cellular damage and contribute to the vascular complications of diabetes. Growing evidence from studies into many diseases suggests that the pathogenesis of diabetes, obesity, cancer, ageing, inflammation, neurodegenerative disorders, hypertension, apoptosis, cardiovascular diseases, and heart failure are correlated with oxidative stress. This leads to cell metabolism and cell-cell homeostasis to be complexly dysregulated. This review focuses to investigate the status of oxidative stress, nitric oxide and reactive species in early and diabetes. Significance of nitric oxide synthases Evidences has accumulated indicating that the generation of reactive oxygen species (oxidative stress) may play an important role in the etiology of diabetic complications thus attention was given on the reactive oxygen and reactive nitrogen species and their potential role in pathogenesis. Additionally, the therapeutic advances in diabetes management are included. Nanotechnology, statins and stem cell technology are some techniques which can be considered to have a possible future in the treatment sector of diabetes.

Introduction

A metabolic disorder signalized by chronic hyperglycaemia is diabetes. The consequences of diabetes mellitus are failure of different organs, cellular damage and inflammation and they occur over a prolonged period. Micro -vascular risks include neuropathy, retinopathy and diabetic nephropathy [1]. Among all complications, in all diabetic patients the main concern is endothelial dysfunction. Endothelial cells secrete mediators such as endothelin-1 vasoconstrictors and nitric oxide vasodilators. Hyperglycemia and other metabolic parameters might cause disablement of nitric oxide (NO) production [2]. In T2DM patients impairment of endothelial function contributes to cardiovascular illnesses and therefore, in T2DM patients endothelial dysfunction can be an advanced attribute of cardiovascular tissue complications [3].

The substrate l-arginine is accountable for the production of nitric oxide (NO) in the vascular endothelium [4]. Nitric oxide discharge from the endothelium focus on the inhibition of both leukocyte and platelet aggregation and adhesion, regulation of vascular tone and suppression of proliferated cells. This point out that the degree of NO produced by endothelium shows a significant part in controlling vascular diseases. Examination utilizing mass spectrometry has disclosed that NOS delivers the NO from the terminal guanidino nitrogen. In this way, L-arginine use and NO conversion will create a regulatory position within the advancement of endothelial dysfunction [5]. All NOS contain haem and have calmodulin binding site. Expression of neuronal NOS is seen in many cells including peripheral and central neurons. Vasodilatation via peripheral nitrergic nerves and blood pressure regulation and relaxation of smooth muscle are some of the functions performed by neuronal NOS. The nNOS gene is located on chromosome 12 (12q24.2) and distributed in human genomic DNA over an area greater than 200 kb [6]. It has 1434 amino acids encoding 4299 nucleotides. nNOS can take both monomer and dimer forms but the dimer form is considered as the active form [7].

Inducible NOS (iNOS) which may have cytostatic effects on parasitic target cells may generate large amounts of NO. Pathophysiology of septic shock and inflammatory diseases can be caused by inducible NOS. Excess of iNOS induction typically takes place in oxidative condition and excess NO can react with superoxide, which give rise to the formation of by these properties, allowing it to participate in anti-tumor and anti-microbial activities as part of the oxidative burst of macrophages [8]. Endothelial NOS (eNOS, NOS III) which is another isoform of NO is usually demonstrated in endothelial cells, which helps in controlling blood pressure, keeps blood vessels dialated and has numerous other anti-atherosclerotic and vasoprotective roles. Many cardiovascular risk variables in the vasculature contribute to uncoupling of eNOS, dysfunction of endothelium and oxidative stress. Renin, statins, blockers of angiotensin receptor and enzyme-converting-inhibitors of angiotensin can be used to reduce vascular oxidative stress thus restore the functions of eNOS [9]. Among 135 million people worldwide impacted by type 2 diabetes mellitus (DM), cardiovascular complications is the primary reason of mortality and morbidity. Cardiovascular morbidity due to endothelial dysfunction is a risk factor in diabetic patients. Diabetes induced endothelial dysfunction is the reason for increased oxidative degradation of NO and the development of vasoconstrictor prostanoid. Due to NO deficiency, vascular resistance can occur. The low level of NO can also encourage atherogenesis [10, 11]. Platelet aggregation and impaired endothelium-dependent vasodilation in diabetes patients are due to decreased Vascular NO activity. Elevated cardiovascular morbidity is studied in individuals with diabetes due to the reduced NO activity and increase in levels of ADMA are predictive of carotid artery illness [12].

Neuronal nitric oxide synthase

In particular central nervous system is the region of expression of constitutive nNOS. Ca2 + and calmodulin regulate the function of enzymes. Functional diversity of nNOS is due to the different subcellular localization of the same. The domain present in nNOS help it to communicate with other protein containing the same domain. These interaction facilitates the subcellular distribution and enzyme activity are determined by these interactions [13]. Immunohistochemistry reports showed that nNOS is observed in the sympathetic ganglia ,spinal cord, adrenal glands, peripheral nitrergic nerves, epithelial cells of different organs, kidney cells, pancreatic cells, and vascular smooth muscle in addition to brain tissue. The skeletal muscle is the main source of nNOS in mammalians in terms of tissue mass [14, 15].

Inducible nitric oxide synthase

At resting state iNOS is not continually present in cells and is expressed only when the cell, is triggered by proinflammatory cytokines and/or bacterial lipopolysaccharides (LPS) [16, 17]. The production of large amount of NO by iNOS (micromolar range) will stay as such until the degradation of enzyme takes place. A significant concentration of NO can provide its effects in protecting from pathogens entering the body thus playing roles in inflammatory response and the innate immune system. In contrast high amount of this NO is injuries to the body, it can cause toxic effects and leads to several diseases including diabetes. The effects (beneficial vs. detrimental) shown by the NO is highly based on the concentration. Therefore regulation in the amount of NO produced is an important step in preventing the body from its dangerous effects [18].

Endothelial nitric oxide synthase

Endothelial nitric oxide syntahse can also be called as constitutive NOS or nitric oxide synthase 3 which is coded by the gene NOS3 which is located in chromosome 7 [19]. In the vascular endothelium, eNOS is mainly responsible for producing NO, [20]. The enzyme eNOS facilitates most of the important functions of endothelium. Their expression is more in large arteries compaired to small arteries and not expressed in capillary ECs [21]. The major roles played by endothelial NO is vascular sound regulation, cell proliferation, leukocyte adhesion, and platelet aggregation. For a healthy cardiovascular system, a functioning eNOS is therefore important.

Significant structural differences in the isoforms of NOS

All the three enzyme isoforms contain oxygenase domain and a reductase domain. The oxygenase domain contain the calmodulin binding region which act as a molecular switch in the transfer of electron from FMN to heme. The heme is located in the reductase domain. There is an ordered loop which will connect the oxygenase domain and reductase domain and will help in the transfer of electron from FMN in the oxygenase domain to heme which is in the reductase domain. These enzymes are stable in their dimeric form. When these enzymes bind with BH4, NO is released.

nNOS contain a loop which will help in easy adaptation to the suitable environment. This area remains disordered in all mammalian isoforms of nNOS and eNOS without such packaging. This loop is disordered in eNOS but it is not disordered in iNOS. The iNOS can bind strongly to the calmodulin binding region and have a deficiency in FMN-binding subdomain. Mass-spectrometric reports shows hydrogen-deuterium exchange shows interaction between the heme domain, the FMN subdomain and CaM with a few constructs of iNOS [22]. Most notably, for the first time, the contact surface was established between the heme domain and CaM. The disordered loop discovered in nNOS or eNOS is likely to be involved in CaM interactions in the proximity of this contact surface with CaM, specifically whenever the FMN sub-domain is able to transfer electrons via FMN to heme. Thus the small changes in the structure of different NOS isoforms can contribute to change in functions of the same.

Synthesis of nitric oxide

The isoforms of NOS use L-arginine as the substrate, and as co-substrates, it also uses reduced NADPH and molecular oxygen. The cofactors of all isoforms include, (6R-) 5,6,7,8-tetrahydro-L-biopterin (BH4), Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). Every isoforms are stable in their dimer form and not in monomer state. Functional NOS moves electrons from NADPH to haem in the amino-terminal oxygenase domain through the flavins FAS and FMN in the carboxy terminal reductase domain and this domain works by binding the cofactor BH4, the substrate L-arginine and molecular oxygen [23]. The electrons are utilized to activate and reduce O_2, it is also employed for the oxidation of L-arginine to NO and L-citrulline which takes place in the haem site. To the cysteine ligand of the heam there are Sequences which are involved in BH4 and l-arginine binding [24]. Mainly the NOS enzyme undergoes through following steps in order to synthesise NO, in the first step hydroxylation of L-arginine by NOS to N^ω-hydroxy-l-arginine takes place which greatly bond with the enzyme. In the next step, oxidation of N^ω-hydroxy-l-arginine by NOS to NO and l-citrulline occurs [25].

The NOS isoforms bind to calmodulin and the increase in intracellular Ca2 + in eNOS and nNOS is the reason behind the binding of calmodulin. There will be a flow of electrons from reductase domain through NADPH to oxygenase domain containing haem when there is an increase in affinity of calmodulin to haem. In inducible NOS, huge loss in intracellular Ca²⁺ binding rate of calmodulin is because of various amino acid structure of the calmodulin-binding site [26, 27]. Zinc performs structural functions in NOS rather than a catalytic role, and NOS generated by NO also can act as several target proteins and enzymes. The generation of cyclic GMP and the activation of soluble guanylyl cyclase are the most important NO signalling pathway [28].

As depicted in Fig. 1, in vascular homeostatic control, NO has an important role, partly by local effects on the diameter of the blood vessels and blood flow to the tissues.calmodulin binding activates the signalling pathway of nitric oxide synthases. Evidence is accumulating at the cellular level that an increase in intracellular Ca2 + is needed by agonists such as ACh, histamine, and bradykinin [29] for the induction of NO production. Recent reports have indicated that efficient NO development needs eNOS phosphorylation through the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway [30] in response to a variety of stimuli. PI3-K activation promotes phosphorylation of Akt at Ser473, which in turn phosphorylates eNOS at Ser1177, increasing the activity of eNOS and thus increasing the output of NO [31]. The relaxation induced by insulin and the 2-adrenoceptor agonist clonidine, but not that induced by ACh, was found to be regulated by the PI3-K / Akt signalling pathway in the mouse. It was further noted that the relaxation, NO development, and either clonidine or insulin induced AKT phosphorylation were all impaired in aortic rings from a mouse type 2 diabetic model, although those induced by ACh were not altered [32]. In addition, there is some evidence that one of the factors leading to vascular dysfunction in diabetes could be impaired regulation of the Akt pathway.

Fig. 1.

Production of reactive oxygen species due to oxidative stress. Hyperglycemia leads to the production of advanced glycation end products which triggers the oxidative stress pathway which results in release of reactive oxygen species. The reactive oxygen species react with nitric oxide radicle to form peroxynitrite which causes depletion of NO availability

However, while gender differences in some diabetes-related cardiovascular disorders are known to exist, but there is still no evidence reporting the differences in gender existing in diabetes depending on endothelial relaxations by different stimuli. When taking hypertension in to concern, there is a huge difference in the occurrence of disease based on gender. Males are found to develop more pathological conditions when compared to female in case of hypertension [33]. When compared to men, women has more amount of NO in the body [34], and NO pathway can lead to sexual dimorphisms in cardiovascular pathology [35]. Either insulin or clonidine induced endothelium-dependent relaxation of the aorta, depend on the sex of the animal in mouse model of type 2 diabetes mellitus. Insulin and clonidine responses are determined using Akt / eNOS thus proving their importance in the same using diabetic mice model was reported in several studies but this has a minor role in ACh responses which is shown in Fig. 1. Akt / eNOS activation to clonidine and insulin for the vascular response was carried out in mouse models and it concluded that the diabetic and non-diabetic female showed identical activation responses. Finally, in response to ACh, endothelium-dependent aorta is done by eNOS in female and male mice [36].

Formation of reactive oxygen species and reactive nitrogen species

Both ROS and RNS plays a role of key regulators in several phases including response to abiotic and biotic stresses, solute transport, metabolism, growth and development and autophagy and programmed cell death (PCD). ROS defines the chemical species as a whole which is an outcome of incomplete reduction of oxygen. However RNS corresponds to all reactive adducts and oxidation states of NOS products, from nitric oxide (NO) to nitroxyl (NO-), S-nitrosothiol (RSNO), and peroxynitrite (OONO-), as result of the interaction between NO and O2-. ROS and RNS undertake the major biological roles that are vital to normal physiology. Defected homeostasis and related pathology are the complications correspondent to deficiency or overproduction of RNS or ROS. Oxidants balanced by reductants (antioxidants) are commonly seen in healthy organism and it is seen that imbalances in the oxidative metabolism as “final common pathway” in pathogenetic mechanism [37].

Theory behind differential regulation of NOS in early and late pathophysiological conditions

The concentration of NO has a major role in development of many disorders. Taking diabetes in to account the concentration NOS differ in early and late phase. Initially at the early stage the amount of iNOS increases without significant decrease in eNOS. But at the late phase, the amount of iNOS increases with decrease in the amount of eNOS which can further lead to tissue damage and other complications.

In vascular endothelium, the presence of nNOS is seen, pointing out that this isoform is found in many peripheral nervous system cell types, including vasa nervorum [38]. Many neurodegenerative pathologies such as excitotoxicity, stroke, Alzheimer’s, Multiple sclerosis and Parkinson’s diseases are experienced due to the higher concentrations of NO [39]. Massive Ca2 + stimulates hyperactive nNOS associated in N-methyl-d-aspartate receptor-mediated neuronal death in cerebrovascular stroke [40]. Under these circumstances, NO causes excitotoxicity through peroxynitrite activation of mitochondrial permeability transition or activation of PARP. Elevated levels of NO leads to decrease in energy level leading to impairment of glycolysis and effects the mitochondrial respiration. Overproduction of NO by nNOS affects the tone of smooth muscles [41].

Neuronal NOS is known to be the significant source of nitric oxide for the production of peroxynitrite related with diabetes in dorsal root ganglion neurons (DRG), as it is used for maintaining the small sensory neurons and normal peripheral nerve function. This isoform doesn’t really contribute to the development of peroxynitrite in peripheral diabetic nerves. Intra-epidermal nerve fibre loss, sensory neuropathy and nerve conduction impairment are not prevented by nNOS insufficiency [42]. In both forms of diabetes, axonal degeneration, nerve ischemia and vasodialation inhibition are due to lack of nNOS. In type I Diabetes Mellitus, this process takes place as two stages, in the early phases of diabetes, due to the reduction in NO level in nitregic nerve cells and the axonal transport defect of NOS1 to axons from body cells diminishes. Insulin treatment can be taken to reverse the process. In parallel with advancement of diabetes in the late phase, NOS1 content is completely removed from the neurons and bear irreversible apoptotic degenerative alterations without reaction to treatment of insulin. In both cases, the release of NO rates from axons and then from neurons significantly alters nitrergic relaxation [43].

Activated macrophages produces high levels of NO which not only causes toxicity to parasites, tumour cells or undesired microbes, it can also harm the healthy cells when released at the wrong site. In vivo, the tissue or cell damage may be due to the production of NO or the formation of peroxynitrite (ONOO−) developed due to interaction between NO and O2. Multitude of activated neutrophils and macrophages characterise the majority of autoimmune and inflammatory lesions which can cause the damage of surrounding tissue due to the high amount of NO secreted by those cells [44, 45]. NO derived from iNOS is most probably gets involved in non-specific allograft rejection [46]. Numerous brain pathologies can be contributed by inflammatory neurodegeneration. Mechanisms by which activated microglia which is a type of neuroglia located throughout the brain and spinal cord, and astrocytes that kill neurons have been reported. The expression of iNOS in glia and stimulation of phagocyte NADPH oxidase in microglia are the mechanism associated with it and apoptosis can be produced by this combination through ONOO − production. NO prevents cytochrome oxidase which causes NO derived by iNOS to activate with hypoxia to stimulate neuronal death, resulting in the release and excitotoxicity of glutamate [47, 48].

In early insulin-influenced diabetes mellitus (IDDM), mediator of damage to beta cell is NO radical. iNOS is the most significant among all the isoforms for the beta cell damage mediated by immune system. Like the iNOS found in macrophages and other nucleated cells, the iNOS beta-cell is encoded on chromosome 17 by the same gene. iNOS expression feedback is dependent on iNOS regulation. Compared to that found in macrophages, iNOS regulation as well as other genes in beta cells are distinct and complex. Thus, there are variations within rodent as well as human pancreatic islets in the regulation of iNOS [49].

Lastly, iNOS produces NO which plays a crucial role in septic shock. Microvascular damage, hypotension and massive arteriolar. Prostanoids, thromboxane A2 and cytokines like interleukin-1, interferon-γ and tumour necrosis factor-alpha are mediators that are elevated in septic shock and have been involved in the pathophysiology of septic shock. Induced iNOS in the vascular wall produces an incredible number of NO, which is the causes declining blood pressure [50].

The failure in the production of bioactive NO, also known as endothelial dysfunction is evidenced in the patients having vascular diseases and cardiovascular risk factors. These risk factors are also connected with production of NO in a huge level. Production of ROS in the vessel wall can be done by many enzyme systems which include enzymes of mitochondrial respiratory chain, xanthine oxidase, NADPH oxidases and uncoupled eNOS out of which NADPH plays the major role in producing ROS. NADPH oxidases is generated by various O2-isoforms that reside in the vascular wall. They show their expression in adventitia, endothelial cells and in smooth muscle cells.

Figure 2 shows that, advanced glycation end products (AGEs) are produced due to high level of glucose which triggers the oxidative stress pathway such as PKC and hexosamine pathway which causes oxidative stress and realease of reactive oxygen species. The reactive oxygen species combine with nitric oxide radicle to form peroxynitrite which causes depletion of NO bio availability and tissue damage.

Fig. 2.

Signalling of reactive oxygen species in diabetes. Induction of reactive oxygen species and reactive nitrogen species through hyperglycemia and the major pathways and enzymes responsible for the production of ROS

Diabetes can cause oxidative stress which can damage DNA, lipids and proteins. Besides these damages they can also trigger the activation of different pathways such as PKC, JNK, and NFKB. When these molecules which has major regulatory effects in the body is dysregulated it can cause various complications related to diabetes which can be termed as diabetes vascular complications. When there is higher amount of reactive oxygen species in the body with minor concentration of anti-oxidant enzymes it can cause oxidative stress. Nitric oxide can easily get diffused through plasma membrane and cytoplasm. When there is high amount of glucose in the body or any occurrence of inflammatory state there will be production of superoxide and nitric oxide which can produce peroxynitrite and cause damage to DNA and lipid [51]. Many studies reported that the peroxynitrite creates major problems related to diabetes and thus higher concentrations of the same is found in patients with diabetes [52].

Nuclear enzyme poly (ADP-ribose) polymerases (PARP enzymes) is involved in the peroxynitrite-mediated vascular dysfunction. Nuclear receptor protein is polymerized by these activated enzymes by the cleavage of NAD + into nicotinamide and ADPribose. Peroxynitrite can cause cell dysfunction, apoptosis or necrosis [53]. Thus concluding that spontaneous EDRF released in diabetic rat aorta can be reduced by the treatment with superoxide dismutase (SOD). Several studies also reported that uncoupled NO can produce peroxynitrite which can act as a mediator of the cytotoxic effects of high glucose [54].

Because of the lack of proper antioxidant protection can lead to oxidative stress which can activate the stress signalling pathway and triggers the gene that can cause harm to the body and thus resulting in diabetes complications which is shown clearly in Fig. 3. Glucose and other glucogenic factors can contribute to negative regulation of insulin signalling pathway thus resulting in hyperglycemia. Several studies reported that diabetic subjects observed with vascular complications have defects in antioxidant responses thus fail to respond against oxidative stress which inturn can result in hyperglycemia.

Fig. 3.

Estimation of NO between male and female diabetic models. A comparison between the male and female diabetic model based on the production of nitric oxide in the subjects based on the differences in the gender

Microvascular complication due to nitric oxide level in patients with T1DM and T2DM diabetes

During any inflammatory state activation of iNOS takes place. NO produced by iNOS plays a major role in the pathogenesis and acts as a pleiotropic intracellular messenger [55]. Low NO levels are advantageous for many physiological and cellular functions, such as maintaining a healthy balance of vascular tone, coagulation and inflammation. Whereas high NO levels in Diabetes Mellitus can cause detrimental effects such as Peroxynitrite and Vascular Endothelial Dysfunction [56]. Depending upon its concentration, NO might have both useful and destructive effects [57]. NO induces blood vessel relaxation to decrease blood pressure, reduces platelet aggregation and adhesion, limits LDL cholesterol oxidation, inhibits smooth muscle cell proliferation, and lowers the release of atherogenesis-associated pro-inflammatory genes [51]. This stimulates the development of inflammatory mediators and lipid peroxidation, causing endothelial dysfunction to increase cell permeability [58, 59].The serum NOx levels of T2DM patients were positively correlated with fasting blood glucose and HbA1c levels. This induces inflammatory mediators and lipid peroxidation to be developed, causing cell permeability to be increased by endothelial dysfunction [60].

Involvement of iNOS during the formation of T1DM is seen in beta-cell death [61]. In beta-cells, studies shows evidences to support the role of eNOS and n-NOS. This entails control of the secretion of insulin and defence against apoptosis. In the growth of both T1DM and T2DM, both processes are involved [62]. In T2DM patients, NOx levels are seen higher. Whereas, in the presence of hypertension, the NOx levels were not increased in T2DM patients. An excessive concentration of this can cause eNOS uncoupling. Which can further reduce the bioavailability of NO and triggers O2 − production [51]. In diabetic patients, there are elevated numbers of NOx this may be due to the overproduction of nitric oxide by iNOS, which can be regulated by oxidative stress, inflammation and hypoxia caused by hyperglycemia [62].

Accordingly, hyperglycemia via iNOS up-regulation can enhance NO efficiency. T1DM and T2DM patients’ shows positive regulation to NOx levels with fasting blood glucose and HbA1c. An additional way by which an increase in NO level is observed in the body of diabetic patients can be by xanthine oxidoreductase (XOR) [57]. This enzyme will increase the quantity of NO through nitrite reduction [63, 64]. Thus, a combination of both iNOS and XOR can lift NO levels. The excess NO can be stored in the body in the form of nitrite and nitrate which can be observed in the serum or plasma of diabetic patients.

Nitric oxide-mediated vasodilation inhibited by hyperglycemia

When considering impaired glucose tolerance (IGT) in to consideration, most of the complications related to it are due to vascular endothlial and it occurs mainly as a result of acquired signalling nitric oxide (NO) defects. vasodilatory signaling and detoxification of ROS that may cause damage to oxidative tissue. Any failure in the NO dependent mechanism can cause increased rate of blood flow in response to methacholine or hyperemia in patients with insulin resistance. This blunted vasoreactivity in patients with IGT leads to increased hypertension [65]. Endothelial atherosclerosis and platelet aggregation are also hindered by the local NO. Whereas, endothelial free radicals are NOT needed to perform these vasoregulatory tasks. NO reacts with endothelial ROS and detoxifies them, thereby, large quantities of hyperglycemia-induced ROS act as a NO drain, making less NO usable for vasodilation.

Increased concentration of glucose in the body can give rise to ROS and stimulates multifactorial NO depletion. Hyperglycemia contributes to enzyme aldose reductase activation, which has the capability to produce sorbitol from excess glucose, which can then be metabolised to fructose (polyol pathway) by sorbitol dehydrogenase [66]. Further these fructose and sorbitol react together to produce advanced glycation end products (AGEs) through non enzymatic protein interactions which in turninduces the generation of ROS [67]. In hyperglycemia, abundant glucose and LDLs are responsible for the production of oxidative stress by dysregulation of transition metals which serve as auto-oxidation catalysts [68]. Finally, reducing efficiency of mitochondria by reperfusion damage or ischemia itself accelerates ROS development. The consequences faced are impaired vasodilation due to high concentration of NO.

Figure 4 shows that, vascular NO signalling disturbance and direct metabolic damage lead to complications of IGT. The eNOS-synthesized NO mediates vasodilation of large and small vessels and facilitates endothelial thrombolysis. NADPH is depleted by acute or prolonged hyperglycemia and increases ROS formation. ROS can damage organ cells and endothelial cells directly and transfers the usage of NO to ROS detoxification from vasoregulatory tasks. Whereas, metaolic injury can be observed due to AGEs due to the formation of ROS. This can further lead to many diabetic impediments such as atherogenesis and thrombogenesis which can be otherwise known as glucose tolerance impairment complications which is depicted in Fig. 4.

Fig. 4.

Microvascular complications of impaired glucose tolerance. Vascular NO signalling disturbance and direct metabolic damages can lead to complications of IGT. Depletion of NADPH by acute or prolonged hyperglycemia increases ROS formation. AGEs improve the formation of ROS and leads to direct metabolic injury

Correlation between ROS, RNS and diabetes mellitus

Extracellular and intra cellular stimuli is the cause for the release of RNS and ROS. Growth factors including PDGF, EGF, and TNF-α, hormones, and insulin are included in extracellular stimuli which acts through plasma membrane receptors [69]. Nutrients, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [70] NOS [51] and mitochondrial electron transfer [71] are included in intracellular stimuli [72]. It is possible to create reversible or irreversible oxidative changes that respond with lipids, Proteins and nucleic acids. The MAP kinase family and JNK are some of the RS-activated signalling cascades assigned to detect and respond to stress [73]. When strictly monitored development in a tight spatial-temporal way requires RS participation in normal physiological processes, causing oxidative modifications which are reversible. RS-mediated pathophysiological processes include irreversible cellular component modifications. The increased production of ROS can be related to cardiovascular risks. Variation between endogenous oxidants and antioxidants can be the reason behind occurrence of oxidative stress. This condition can result in weakened vascular dysfunction and NO bioavailability. Diabetes, atherosclerosis and hypertension which comes under the category of cardiovascular diseases manifest an expression of eNOS which underwent alteration.

According to numerous studies, the pathways of diabetic complexity have a correlation with oxidative stress and ROS plays a vital part in the pathogenesis of vascular dysfunction. Specifically, increased mitochondrial formation of ROS can be caused due to increased glucose production. When NO react with, superoxide which is a ROS can produce peroxynitrate which can cause damage to beta cells. DNA strand breakage, polymerase (PARP) nuclear enzyme poly (ADP-ribose) activation, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition can also takes place. The pathways related to diabetic complications include polyol pathway, advanced glycation end products (AGE) pathway, protein kinase C (PKC) pathway, and the hexosamine pathway. The upregulation of proinflammatory pathways can be caused due to PARP activation which further leads to pathological alteration in expression of adhesion molecule and angiogenesis [74].

Glucose-induced oxidative stress can be found related to “glucose variability” and “glucose memory”. According to the studies related to glucose variability, the irregular low and elevated glucose conditions cause damage cell function of endothelium than a steady, constant rise in level of glucose. This propels the endothelial cells goes into a proinflammatory state and causes oxidative stress. This inturn is connected to upregulation of several adhesion molecules and proinflammatory cytokines. Activation of PKC, NADPH oxidases, and mitochondrial oxidants are some of the pathways exhibited as a result of this aggravated cellular response. Regarding glucose memory, long after the normalization elevated level of glucose in the endothelial cells of diabetic rats and culture, a existance or ‘memory’ of induced basement membrane mRNA is expressed. This demonstrates that glucose causes dangerous long-term effects beyond the hyperglycemia period [75].

Oxidative stress related damaged caused to beta cells

Oxidative stress give rise to glucotoxicity and lipotoxicity which are phenomenas in relation with diabetes. The pathogenesis of beta-cell dysfunction is an implication of this stress [76]. Following the pathogenesis of diabetes, hyperglycemia and hyperlipidemia exerts a supplementary toxic effect on the beta-cells. From in vivo and in vitro studies, it is evident that lipids and glucose are detrimental to beta-cells. According to some studies, lipotoxicity transpire in the existence of concomitant raise in glucose levels [77, 78]. Hyperglycemia can be considered as a requirement for the destructive effects of lipotoxicity. Thus glucolipotoxicity can be considered as a substitute for lipotoxicity which relates the detrimental correlation between lipids and beta-cell function.

High concentration of glucose can lead to oxidative stress which can negatively regulate insulin signalling pathway and lead to insulin resistance and beta cell damage. Beta cells are sensitive towards ROS because they lack antioxidants enzymes. Thus the oxidative stress can cause damages to mitochondria and blunt insulin production. Thus showing evidences that lacking of anti-oxidants enzyme is a major reason for beta cell damage and thus the treatment with antioxidant enzymes can help in reducing oxidative stress [79, 80].

Utilizing aminoguanidine or N-acetylcysteine, under high glucose conditions, insulin mRNA levels and insulin promoter activity of antioxidant drug concentration-related preservation can be observed. Insulin-secreting cells shown high fatty acid levels due to impairement in insulin gene expression, disrupted glucose-induced insulin secretion and initiation of cell death by apoptosis. Glucotoxicity that is the structural or functional changes in the beta cells can be due to oxidative stress production and inflammation [81].

Diabetes related impediments caused by nitric oxide

Vascular dysfunction

Vascular dysfunction can be caused by impaired NO availability attenuation in conduit and resistance vessels of NO-dependent flow-induced vasodilation caused by decreased NO bioavailability, which can be described as endothelial dysfunction. Deficient bioavailability of NOs and endothelial dysfunction regulates the first stage of development from natural vascular function to vasoconstriction, inflammation, atherogenesis, atherosclerosis and thrombosis.The irregular brachial or coronary vasodilatory response of the endothelium to elevated level of blood flow is the main independent indicator of the development of atheromatous disease and cardiovascular events [82].

Inflammation

Inflammation can be triggered by impaired availability of NO. Upregulation and nuclear factor activation – kB and protein activator-1 are triggered and upregulated to start the release of inflammatory cytokines such as TNF-α and interleukin-1. In the recruitment of VSMCs and monocytes, which activates atherogenesis, the migration of T lymphocytes to the vascular intima generates more cyto and chemotactic factors and adhesion molecules [82].

Atherogenesis

The progression of atheromatous vascular disease and NO availability is inversely related [83]. Vascular remodelling can be promoted by endothelium dysfunction. Myointimal hyperplasia, medial thickening and high conduit vessel stiffness may be triggered by depletion in NO action [84]. T cell and monocyte adhesion, development of foam cells, digestion of extracellular matrix, migration and proliferation of VSMC, triggering and promoting the development of atherosclerotic plaque, facilitated by proinflammatory factors in the absence of NO.

Atherombosis

The activity of defective vascular NO triggers the degradation of antithrombotic factors. The attenuation of anticoagulation is attributed to the reduced expression of thrombomodulin dependent on the cell surface. Events like enhanced platelet activation and aggregation are found to be reduced NO and prostacyclin [82].

There is an increase in prothrombotic factors when there is a deficiency in NO. The development of the plasminogen activator inhibitor-1 fibrinolytic antagonist is therefore enhanced [82]. Dysfunctional endothelial cells contain a potent coagulant tissue factor. Thereby, increasing the risk of atherothrombosis, particularly in the later stages of the disease, resulting in plaque rupture, thrombus formation and acute ischemic syndrome [83, 84]. In patients with extreme coronary endothelial dysfunction, the cardiovascular injury level is 14 % over 2 years of follow-up and nearly 20 % over 8 years, compared with < 5 % for people with normal endothelial function [85].

Hypertension

Inhibition of both acute and chronic NO synthase (NOS) with arginine derivative causes a significant rise in blood pressure, indicating that NO function controls basal vascular tone. Cardiac hypertrophy and renal insufficiency are associated with a NOS inhibitor that causes chronic hypertension. The development of hypertension is illustrated by sodium retention via the transient system and the plasma and tissue renin / angiotensin system due to lower NO activity. Blockades of the renin/angiotensin system or co-administration of L-arginine can be used to reverse the hypertension and the associated target organ failure. The endothelial dysfunction, is a secondary cause of hypertension which could be altered by blood pressure correction [86].

Parkinson’s disease

NO can disperse and cause destruction to lipids, proteins and nucleic acids easily across the neuronal membranes. In the case of proteins, peroxynitrite often interacts with the tyrosine phenolic ring that forms nitro-tyrosines, which significantly affects the protein’s physiological functions. Protein nitrotyrosination is an inevitable mechanism that often results in the accumulation of transformed proteins that lead to neurodegenerative processes such as Alzheimer’s or Parkinson’s disease in the beginning and progressing state [87].

Therapeutic approaches for diabetes management

Diabetics has now became a killer disease worldwide with a prevalence rate of 422 million people in 2014 with a mortality rate of 5 % every year, and 8.3 and 46.3 % still remains undiagnosed [88] Which method to be opted to treat the disease depend on many factors which include the complication of the disease, level of glucose, body mass index, cost of treatment, risk of treatment [89]. Insulin injections can be done to keep the blood glucose level in the body, but it is not considered as a permanent treatment [90]. Treatment with metformin and many other combinational therapy is available in the treatment sector [91].

The conventional methods are considered to be painful and they also possess numerous side effects [92]. The drugs for treatment which are available now in market which is used to treat diabetes type 2 has limitations in the sense they possess different side effects. Combinational therapy was then introduced in which insulin will be treated along with sulfonylureas which can lower the daily need of insulin [89]. In the same way metformin can be used to replace sulfonylureas which can reduce the weight gain during the course of therapy [93]. Extensive research aimed at the detection of natural products and their potential in treating the disease. This has highlighted the urgent need for emerging trends in diabetes therapeutics and management.

Use of nanotechnology in diabetes

The nanotechnology has extended their helping hands in measuring levels of glucose and insulin delivery in the field of diabetes treatment. Researchers showcased the advancement of using this technique for treatment in diabetes patients as glucose sensors and in closed loop insulin delivery systems [94].

This biomedical device consist of glucose sensor that can be used to sense the level of glucose in the body and then the delivery of insulin can be done. It consist of microcapsule with small pores that allow the passage of insulin. The replacement technique works like, beta cells that are often extracted from pigs are carefully placed beneath the skin of patients suffering from diabetes. This can thus maintain the glucose level in the patient’s body and thus saving them from the serious risk of infection [95].

There are many advantages for nanoparticle mediated drug delivery and the major advantage is the increased bioavailability of drug, the other merit of this treatment is that large amount of drug can be administered and also the drug can be delivered correctly to the site where their action is required. And the main challenge include scalability of a nanoparticle and the exposure to nanoparticles might be toxic or hazardous [96].

Both the type1 and type 2 diabetic patients are in need of insulin. Infections, painful administration, and poor patient compliance have been experienced by conventional insulin delivery systems. However, this method with their promising advantages can be helpful in the treatment when compared to the painful conventional methods [97].

Statins in diabetes

Statins can be defined as inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A and it can reduces the cholesterol level in liver [98]. The major complication faced by the diabetic patients is high level of cholesterol accumulation in liver thus treatment with statin can reduce this complication [99]. This can be made possible by binding to the HMG receptor. Statin inhibit HMG receptor by binding to the active site of enzyme therefore it prevents the substrate from binding. Statin treatment can thus be effective in reducing the LDL cholesterol level in diabetic patients and thus reducing the diabetic complications [100].

Stem cell technology in diabetes

Different emerging scientific fields of study have ultimately addressed the interest in discovering a potential therapeutic for diabetes and stem cell technology can be one among them. Insulin resistance and beta cell damage can be the reasons behind hyperglycemia. Thus this technique look forward for a replacement method for beta cells, while existing islet cell and pancreas transplantation strategies are limited due to donor organ shortages [101].

Due to the immunosuppressive nature, mesenchymal stem cell (MSC) treatment can be used to treat type 1 diabetic patients. Due to direct contact and development of soluble markers, immunomodulatory effects was observed in MSCs in invivo and invitro studies [102]. MSCs has been selected because it has the potential differentiate into a variety of mesenchymal cells lineages. A type of multipotent stem cells is hematopoietic stem cells that can give rise to all types of cells in the blood and have immunomodulatory effects as well. Therefore, hematopoietic stem cell transplantation have proven their potential in successful therapy, thus type 1 diabetic patients were diagnosed and found improvement in β-cell function [103].

Conclusions

Glucose and other diabetogenic factors can contribute to oxidative stress which can inturn negatively regulate the insulin signaling pathway and cause insulin resistance and beta cell dysfunction. This can then create a condition which can be termed as oxidative stress. Oxidative stress can increase the reactive oxygen concentration which produces nitric oxides and superoxides and combine together to produce peroxynitrate which results in endothelial dysfunctions due to the high amount of inducible nitric oxide synthase and reduction in endothelial nitric oxide synthase. Oxidative stress can reduce the antioxidant enzymes thus damaging beta cells. It can also be a reason behind the occurrence of diabetic impediments.

As a result, NO’s bioactivity reduces platelet function, inhibits leukocyte adhesion, and decreases intima proliferation. The reduction in vascular NO bioavailability is linked to rapid oxidative inactivation by ROS. The present oxidative stress, triggers eNOS dysfunction leading to which O2- is generated instead of NO as a consequence. Therefore, epigenetic variations that persist during normoglycemia can be key event for redox imbalance. The treatment of antioxidants can be relief in the early phase of diabetes. Portraying the importance of gaining knowledge on hyperglycaemia-induced oxidative damage. Additionally, the therapeutic advances in diabetes management are included. Nanotechnology, statins and stem cell technology are some techniques which can be considered to have a possible future in the treatment sector of diabetes (Table 1).

Table 1.

Comparison between different nitric oxide synthases

Nitric oxide synthases	Significance of calmodulin binding	Significance of BH4	Sifnificance of FMN
Neuronal nitric oxide synthase (nNOS) NOS1	Calmodulin functions as a allosteric activator of nNOS. The calmodulin remains tightly bound to iNOS even at low intracellular ca.2+ activity. Calmodulin binding initiate electron transfer from flavin to heme domain.	Binding of BH4 makes nNOS active. The oxygenase domain which binds the substrate L-arginine contain BH4 which facilitate nNOS dimerization. BH4 helps in making nNOS a stable dimer.	Reductase domain which binds to substrate NADPH contains a binding site for FMN. FMN contain the auto inhibitory loop which controls nNOS activity. FMN help in transfer of electron from the reductase domain to oxygenase domain.
Inducible nitric oxide synthase (iNOS) NOS 2	The binding of oxygenase and reductase domain by calmodulin mediates the production of NO by iNOS. The tight interaction of calmodulin and iNOS allow their co-crystallization and can be described as calcium insensitive. Calcium binding is important for proper alignment of FMN and heme domain in iNOS.	Dimerization of iNOS to active form is done by BH4. Not only for the activity, regulation of iNOS expression also requires sufficient concentration of BH4. BH4 availability has been demonstrated to be a limiting factor for iNOS activity.	The iNOS reductase domain belongs to dual flavin reductase that contain N-terminal FMN containing module. Mobility of FMN region during NO synthases entrails the interaction between FMN molecule and oxygenase domain. The short insert that connect the FMN and FAD called the AR insert is not found in iNOS which repress electron flow.
Endothelial nitric oxide synthase (eNOS) NOS3	Calmodulin binding act as a link between oxidase domain and reductase domain. eNOS is a calmodulin dependent enzyme thus it is mediated by calmodulin protein. Binding of calmodulin to calcium exposes hydrophobic regions on the surface which are important for mediating interactions with its target.	Binding of cofactor BH4 is essential for eNOS to efficiently generate NO. In the absence of BH4, eNOS shift from dimeric to monomeric form. Thus sufficient concentration of BH4 is important for normal functioning of eNOS. BH4 act as a molecular target for oxidative stress and can cause uncoupling of eNOS.	Conformational changes of the FMN domain from its shielded electron accepting state activates eNOS to synthesize NO. Rate of interflavin electron transfer and FMN domain conformational motion are balanced to limit electron flux through eNOS. The hinge region which is between the FMN and connecting domain constrain the activity of eNOS.

The comparison between different isoforms of enzymes is reported based on the significance of calmodulin binding, BH4, and FMN and how these effects in the functioning of the enzymes in the production of nitric oxide

Declarations

Conflict of interest

No potential conflict of interest.