Tissue-specific role of Nrf2 in the treatment of diabetic foot ulcers during hyperbaric oxygen therapy.

Hyperbaric oxygen (HBO) therapy is proven to be very successful for diabetic foot ulcer (DFU) treatment due to its antimicrobial effect, increased angiogenesis and enhanced collagen synthesis. The molecular mechanism underlying HBO therapy particularly the involvement of Nrf2 in the wound healing process was investigated in the present study. In addition, we have studied the levels of angiogenic markers in ulcer tissues and their correlation with Nrf2 during HBO therapy compared with standard therapy (Non-HBO) for DFU. A total of 32 Patients were recruited and randomized to standard wound care procedure alone (n = 17) or HBO therapy in combination with standard wound care procedure (n = 15) for 20 days. Our results showed that the tissue levels of Nrf2 along with its downstream targets were significantly increased in patients who underwent HBO therapy when compared to Non-HBO therapy. Further, HBO therapy induced angiogenesis as assessed by increased levels of angiogenesis markers such as EGF, VEGF, PDGF, FGF-2 and CXCL10 in the tissue samples. The expressions of eNOS and nitrite concentrations were also significantly increased in HBO therapy when compared to Non-HBO therapy subjects. Moreover, HBO therapy sensitises the macrophages to release FGF-2 and EGF thereby promotes angiogenesis. Further, it increased the levels of neutrophil attractant CXCL-8 thereby promotes the release of chemokine CCL2, a well-known mediator of neovascularization. The Pearson correlation showed that Nrf2 has a positive correlation with EGF, VEGF and PDGF. In conclusion, the findings of the present study suggest that HBO therapy promotes wound healing by increasing oxygen supply and distribution to damaged tissues, stimulating angiogenesis, decreasing inflammation, and increasing the nitrite levels. Increased levels of Nrf2 transiently regulate the expression of angiogenic genes in wound biopsies, which may result in accelerated healing of chronic wounds.

Association of single-nucleotide polymorphisms of the KEAP1 gene with the risk of various human diseases and its functional impact using in silico analysis.

Keap1, Kelch-like erythroid derived Cap 'n' collar homology (ECH) associated protein 1 is a highly redox-sensitive member of the BTB-Kelch substrate adaptor protein which acts as a major upstream regulator of Nrf2 (Nuclear factor erythroid 2-related factor 2) by Cul3 ubiquitin E3 ligase complex, leading to its proteasomal degradation. Oxidative and electrophilic stresses impair the structural integrity of Keap1-Cul3 ubiquitin E3 ligase complex resulting in the dissociation of Nrf2-Keap1 binding and nuclear accumulation of Nrf2. Studies on tissue-specific Keap1 null mutation have demonstrated the important roles of Keap1 mediated Nrf2 degradation. An increasing body of evidence suggests that loss of functional mutation in Keap1 arbitrates constitutive activation and expression of Nrf2 which in turn provokes the chemotherapeutic resistance in various diseases. The current review addresses the genetic aspects of KEAP1 including somatic mutations and in silico functional profiling of human disease-associated and polymorphic amino acid substitutions.

Association of NF-E2 Related Factor 2 (Nrf2) and inflammatory cytokines in recent onset Type 2 Diabetes Mellitus.

We investigated the association of redox regulator Nuclear factor erythroid 2-related factor 2 (Nrf2) and inflammatory cytokines as well as clinical remission in patients with recent onset type 2 diabetes (DM). Blood was collected from 180 DM patients (105 males/75 females) and 150 control subjects (86 males/64 females). Blood glucose, HbA1c, lipid profile and Nrf2 levels were determined along with circulatory cytokines in study subjects. The data were adjusted with confounding factors such as age and sex using multiple logistic regression analysis. We found that Th1/Th2 and oxidative stress markers were significantly elevated, whereas Nrf2 and its downstream targets were decreased in peripheral blood mononuclear cells (PBMCs) of DM subjects when compared with control. The circulatory levels of Nrf2 showed a positive correlation with Th2 cytokines and negative correlation to Th1 cytokines. Further, the impaired insulin secretion in pancreatic ß-cells observed due to cytokine stress has been restored by activation of Nrf2 as assessed by glucose-stimulated insulin secretion (GSIS). This study identifies Nrf2 plays a central role in skewing Th1 and Th2 dominance in the progression of diabetes.

Establishment of pancreatic microenvironment model of ER stress: Quercetin attenuates ß-cell apoptosis by invoking nitric oxide-cGMP signaling in endothelial cells.

The involvement of endoplasmic reticulum (ER) stress in endothelial dysfunction and diabetes-associated complications has been well documented. Inhibition of ER stress represents a promising therapeutic strategy to attenuate endothelial dysfunction in diabetes. Recent attention has focused on the development of small molecule inhibitors of ER stress to maintain endothelial homeostasis in diabetes. Here we have developed a reliable, robust co-culture system that allows a study on the endothelial cells and pancreatic ß-cells crosstalk under ER stress and validated using a known ER stress modulator, quercetin. Furthermore, sensitizing of endothelial cells by quercetin (25 µM) confers protection of pancreatic ß-cells against ER stress through nitric oxide (NO∙) signaling. In addition, increased intracellular insulin and NO∙-mediated cyclic 3',5'-guanosine monophosphate (cGMP) levels in pancreatic ß-cells further confirmed the mechanism of protection under co-culture system. In addition, the potential protein targets of quercetin against ER stress in the endothelial cells were investigated through proteomic profiling and its phosphoprotein targets through Bioplex analysis. On the whole, the developed in vitro co-culture set up can serve as a platform to study the signaling network between the endothelial and pancreatic ß-cells as well as provides a mechanistic insight for the validation of novel ER stress modulators.

Treatment With Naringenin Elevates the Activity of Transcription Factor Nrf2 to Protect Pancreatic ß-Cells From Streptozotocin-Induced Diabetes in vitro and in vivo.

Chronic hyperglycemia and unusually high oxidative stress are the key contributors for diabetes in humans. Since nuclear factor E2-related factor 2 (Nrf2) controls the expression of antioxidant- and detoxification genes, it is hypothesized that targeted activation of Nrf2 using phytochemicals is likely to protect pancreatic ß-cells, from oxidative damage, thereby mitigates the complications of diabetes. Naringenin is one such activator of Nrf2. However, it is currently not known whether the protective effect of naringenin against streptozotocin (STZ) induced damage is mediated by Nrf2 activation. Hence, the potential of naringenin to activate Nrf2 and protect pancreatic ß-cells from STZ-induced damage in MIN6 cells is studied. In MIN6 cells, naringenin could activate Nrf2 and its target genes GST and NQO1, thereby inhibit cellular apoptosis. In animals, administration of 50 mg/kg body weight naringenin, for 45 days, significantly decreased STZ-induced blood glucose levels, normalized the lipid profile, and augmented the levels of antioxidants in pancreatic tissues. Immunohistochemical analysis measuring the number of insulin-positive cells in pancreas showed restoration of insulin expression similar to control animals. Furthermore, naringenin promoted glycolysis while inhibiting gluconeogenesis. In conclusion, naringenin could be a good anti-diabetic agent, which works by promoting Nrf2 levels and by decreasing cellular oxidative stress.

Association of A1538G and C2437T single nucleotide polymorphisms in heat shock protein-70 genes with diabetic nephropathy among South Indian population.

Diabetic Nephropathy (DN) is the leading cause of end-stage renal disease, characterized by progressive albuminuria and conferring additional risk of cardiovascular disease (CVD) and mortality. The crucial role of heat-shock proteins (HSPs) on renal function in patients with DN has been well documented. The present study was aimed to understand the association of HSP-70 gene variants on the susceptibility of Type 2 Diabetes Mellitus (T2DM) and DN. A total of 946 subjects (549 Males; 397 Females) were recruited and divided into four groups according to the levels of urinary albumin excretion (UAE): those with normoalbuminuria (UAE <30 mg/24 h; n=230), those with microalbuminuria (30≤ UAE ≤300 mg/24 h; n=230), and those with macroalbuminuria (UAE> 300 mg/24 h; n=230). The control group randomly enrolled a consecutive population of 256 healthy subjects who had a routine medical check-up in our hospital. Those subjects had no history or clinical symptoms of diabetes. Subjects were genotyped for HSP70-2 (+1538 A/G; rs2763979) and HSP70-hom (+2437 C/T; rs2227956) by PCR-restriction fragment length polymorphism (RFLP). The 'G' allele of HSP70-2 (+1538 A/G) single nucleotide polymorphism (SNP) showed relative risk for normoalbuminuria, microalbuminuria and macroalbuminuria subjects whereas the 'T' allele of HSP70-hom (+2437 C/T) SNP showed significant protection against macroalbuminuria subjects. In conclusion, our results indicate that the HSP70-2 (+1538 A/G) and HSP70-hom (+2437 C/T) SNPs are highly associated with renal complications in T2DM among the South Indian population.

Role of pterostilbene in attenuating immune mediated devastation of pancreatic beta cells via Nrf2 signaling cascade.

Nrf2 (nuclear factor erythroid 2-related factor-2) is a transcription factor that regulates oxidative/xenobiotic stress response and also suppress inflammation. Nrf2 signaling is associated with an increased susceptibility to various kinds of stress. Nrf2 has been shown as a promising therapeutic target in various human diseases including diabetes. Our earlier studies showed Pterostilbene (PTS) as a potent Nrf2 activator, and it protects the pancreatic ß-cells against oxidative stress. In this study, we investigated PTS confer protection against cytokine-induced ß-cell apoptosis and its role on insulin secretion in streptozotocin (STZ)-induced diabetic mice. The Nrf2 activation potential of PTS was assessed by dissociation of the Nrf2-Keap1 complex and by expression of ARE-driven downstream target genes in MIN6 cells. Further, the nuclear Nrf2 translocation and blockage of apoptotic signaling as demonstrated by the reduction of BAX/Bcl-2 ratio, Annexin-V positive cells and caspase-3 activity conferred the cyto-protection of PTS against cytokine-induced cellular damage. In addition, PTS treatment markedly improved glucose homeostasis and abated inflammatory response evidenced by the reduction of proinflammatory cytokines in diabetic mice. The inhibition of ß-cell apoptosis by PTS as assessed by BAX/Bcl-2 ratio and caspase-3 activity in the pancreas was associated with the activation of Nrf2 and the expression of its downstream target genes. PTS also inhibited the activation of iNOS and decreased nitric oxide (NO) formation in the pancreas of diabetic animals. The results obtained from both in vitro and in vivo experiments showed that PTS improves ß-cell function and survival against cytokine stress and also prevents STZ-induced diabetes.

Unraveling the role of ER stress inhibitors in the context of metabolic diseases.

ER stress is provoked by the accumulation of unfolded and misfolded proteins in the ER lumen leading to perturbations in ER homeostasis. ER stress activates a signaling cascade called the Unfolded Protein Response (UPR) which triggers a set of transcriptional and translational events that restore ER homeostasis, promoting cell survival and adaptation. If this adaptive response fails, a terminal UPR program commits such cells to apoptosis. Existing preclinical and clinical evidence testify that prolonged ER stress escalates the risk of several metabolic disorders including diabetes, obesity and dyslipidemia. There have been considerable efforts to develop small molecules that are capable of ameliorating ER stress. Few naturally occurring and synthetic molecules have already been demonstrated for their efficacy in abrogating ER stress in both in vitro and in vivo models of metabolic disorders. This review provides a broad overview of the molecular mechanisms of inhibition of ER stress and its association with various metabolic diseases.

Pterostilbene-mediated Nrf2 activation: Mechanistic insights on Keap1:Nrf2 interface.

The discovery of Keap1-Nrf2 protein-protein interaction (PPI) inhibitors has become a promising strategy to develop novel lead molecules against variety of stress. Hence, Keap1-Nrf2 system plays an important role in oxidative/electrophilic stress associated disorders. Our earlier studies identified pterostilbene (PTS), a natural analogue of resveratrol, as a potent Nrf2 activator and Keap1-Nrf2 PPI inhibitor as assessed by luciferase complementation assay. In this study, we further identified the potential of PTS in Nrf2 activation and ARE-driven downstream target genes expression by nuclear translocation experiments and ARE-luciferase reporter assay, respectively. Further, the luciferase complementation assay identified that PTS inhibits Keap1-Nrf2 PPI in both dose and time-dependent manner. Computational studies using molecular docking and dynamic simulation revealed that PTS directly interacts with the basic amino acids of kelch domain of Keap1 and perturb Keap1-Nrf2 interaction pattern. This manuscript not only shows the binding determinants of Keap1-Nrf2 proteins but also provides mechanistic insights on Nrf2 activation potential of PTS.

Anti-hyperlipidemic and anti-peroxidative role of pterostilbene via Nrf2 signaling in experimental diabetes.

Nuclear factor erythroid 2-related factor (Nrf2), a key transcription factor triggers the expression of antioxidant and detoxification genes thereby providing cellular protective functions against oxidative stress-mediated disorders. Recent research has identified that pharmacological activation of Nrf2 also regulates the largest cluster of genes associated with lipid metabolism. With this background, this paper highlights the anti-hyperlipidemic and anti-peroxidative role of pterostilbene (PTS), an Nrf2 activator, in streptozotocin (STZ)-induced diabetic model. PTS administration to diabetic mice for 5 weeks significantly regulated blood glucose levels through the elevation of insulin secretion. The circulatory and liver lipid profiles of total cholesterol (TC), triglycerides (TG) and non-esterified fatty acids (NEFA) were maintained to normal levels upon PTS treatment. Moreover, PTS administration also normalized the circulatory levels of very low-, low- and high density lipoprotein cholesterols (VLDL-, LDL-, HDL-C) and also reduced lipid peroxidation in STZ-induced diabetic mice. In addition, Nrf2 and its downstream targets, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) enzyme activities and glutathione (GSH) levels were significantly elevated in liver tissues of diabetic mice upon PTS administration. Further, H&E staining of diabetic mouse liver showed collapse in hepatic microvesicles due to altered lipid metabolism. Both structural and functional alterations were attenuated by PTS indicating its role in diabetic dyslipidemia through Nrf2-mediated mechanism that could be considered as a promising therapeutic agent.

Carvacrol induces mitochondria-mediated apoptosis in HL-60 promyelocytic and Jurkat T lymphoma cells.

The aim of the present study was to investigate the effect of carvacrol, a phenolic monoterpenoid on the induction of apoptosis in HL-60 (Human acute promyelocytic leukemia cells) and Jurkat (human T lymphocyte cells) cells. Carvacrol showed a potent cytotoxic effect on both cells with dose-dependent increase in the level of free radical formation as measured by an oxidation sensitive fluorescent dye, 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) levels. The reduction in the level of antioxidants such as catalase (CAT) and superoxide dismutase (SOD) (P<0.05) was observed in carvacrol-treated cells. The major cytotoxic effect appears to be intervened by the induction of apoptotic cell death as assessed by annexin-V labeling assay using flow cytometry. Western blot analysis showed that Bax expression was increased, whereas Bcl-2 expression was significantly decreased in carvacrol exposed HL-60 cells and Jurkat cells. Further studies revealed that the dissipation of mitochondrial membrane potential of intact cells was accompanied by the activation of caspase-3. Our results found that the potential mechanism of cellular apoptosis induced by carvacrol is mediated by caspase-3 and is associated with the collapse of mitochondrial membrane potential, generation of free radicals, and depletion of the intracellular antioxidant pool.

The emerging role of redox-sensitive Nrf2-Keap1 pathway in diabetes.

The pathogenic processes involving in the development of diabetes range from autoimmune destruction of pancreatic ß-cells with consequent insulin deficiency to abnormalities that result in resistance to insulin action. The major contributing factor for excessive ß-cell death includes oxidative stress-mediated mitochondrial damage, which creates an imbalance in redox homeostasis. Yet, ß-cells have evolved adaptive mechanisms to endure a wide range of stress conditions to safeguard its potential functions. These include 'Nrf2/Keap1' pathway, a key cellular defense mechanism, to combat oxidative stress by regulating phase II detoxifying and antioxidant genes. During diabetes, redox imbalance provokes defective Nrf2-dependent signaling and compromise antioxidant capacity of the pancreas which turnout ß-cells to become highly vulnerable against various insults. Hence, identification of small molecule activators of Nrf2/Keap1 pathway remains significant to enhance cellular defense to overcome the burden of oxidative stress related disturbances. This review summarizes the molecular mechanism behind Nrf2 activation and the impact of Nrf2 activators in diabetes and its complications.

Targeting SUMOylation cascade for diabetes management.

Post-translational modifications (PTMs) play important roles in regulating protein stability, trafficking, folding conformation, and functional activity. Small ubiquitin-like modifier (SUMO) protein mediates a distinct type of PTM called SUMOylation in which the SUMO protein is covalently ligated to the target protein and modifies its activities through a series of enzymatically-catalyzed reactions. SUMOylation regulates many cellular processes like transcription, the maintenance of the ion gradient across the cell membrane, stress response, autoimmunity, etc. Several target proteins of SUMOylation are involved in the biological pathways related to various human diseases, including cardiovascular diseases, diabetes, cancer, and neurodegenerative disorders. This review focuses on the SUMOylation process, regulatory roles of SUMOylation in diabetes, and prospects of developing novel anti-diabetic drugs targeting the SUMOylation process.