Naturally Occurring Nrf2 Activators in the Management of Diabetes

Nuclear factor erythroid-2 (NFE-2)-related factor-2 (Nrf2), a key leucine zipper transcription factor that regulate the expression of antioxidant enzymes, is an important target for mitigating the complications of diabetes. Several lines of evidences have concluded that targeted activation of Nrf2 using phytochemicals helps to protect insulin secreting pancreatic β-cells thereby reduce hyperglycemia induced changes such as retinopathy, neuropathy, nephropathy, cardiomyopathy, and many other complications. Mechanistically, Nrf2 activators promote the release of Nrf2 from Keap1 Nrf2 complex by disrupting the protein protein interactions or by promoting the degradation of Keap1 in cells. Once released, the Nrf2 translocates in to nucleus thereby trigger the transcription of genes such as NQO1, GST, HO1, HIF1 α, and many more, which are involved in controlling oxidative stress. Therefore, identification of naturally occurring Nrf2 activators and the use of diets rich in these activators is a potential strategy to control diabetes. However, further studies are warranted to test the safety and efficacy of Nrf2 activators for treating diabetes in clinical trials. In this mini-review, we have summarized the recent findings of cell-based and preclinical studies evaluating the role of Nrf2 in preventing and treating diabetes. In addition, the details of preclinical studies that have tested the Nrf2 activators are tabulated. For additional and more detailed information the readers can refer the publications listed in the references section.


Introduction
Despite various cost-effective treatment strategies and public health campaigns highlighting the key risk factors, the incidence and burden due to diabetes is increasing at an alarming rate globally with an estimated 422 million individuals currently suffering from this disease. Recent predictions by IDF projected that the number of diabetics is likely to increase to 642 million by 2040 [1]. Etiologically, Type-1 and Type-2 diabetes have originated due to a significant decrease in the number of insulinproducing β-cells [2]. As a result, diabetics experience chronic hyperglycemia [3,4] and several secondary abnormalities that include loss of vision, malfunction of kidneys and ultimately coma and death [5].
Therefore, coordinated up-regulation of genes coding for detoxifying and antioxidant enzymes has been shown to be a potential therapeutic strategy against oxidative stress-induced pancreatic β-cell damage [9,10]. Use of naturally occurring photochemical is one such strategy to mitigate the toxic effects, and protect pancreatic β-cells [11]. Several recent clinical trials have also confirmed the advantages of using bioactive compounds derived from natural sources for diabetes management [12][13][14].
Among various phytochemicals, polyphenolic compounds had shown potent anti-diabetic properties [15]. Polyphenols can A. Activate key transcription factors such as nuclear factor erythroid-2 (NFE-2)-related factor-2 (Nrf2), a known master regulator of the antioxidant response-and phase-II detoxifying enzymes; and B.
Reduce toxic reactive oxygen species in to less toxic hydroxyl radical and hydrogen peroxide [16].
Hence, in this review, we have summarized the key findings of various research studies demonstrating the anti-diabetic properties of naturally occurring Nrf2 activators. Interested readers can refer recent review articles published for more detailed information.

Role of Nrf2 in Diabetes
Several studies have demonstrated the role of Nrf2 in mitigating the complication of diabetes using cell-based and animal model systems [17,18]. In vitro studies using human and animal cells indicated that activation of Nrf2 depends on cell type and glucose concentration [19,20]. Moreover, many studies have also demonstrated that mice lacking Nrf2 (Nrf2knockout mice) or Keap1 (Keap1-knockout mice) failed to reduce the complications of insulin resistance as they could not activate Nrf2 and its target genes, indicating a key role of Nrf2 in preventing diabetic complications [21].
Additionally, recent clinical findings have also shown that Nrf2 function has significantly decreased in subjects with diabetes [22]. Supporting this, analysis of peripheral blood mononuclear cells (PBMC) from prediabetic and diabetic subjects showed decreased Nrf2 and HO-1 levels [23]. Likewise, a separate study reported that diabetic skin tissue showed down regulation of Nrf2 and its target genes NQO1 and HO-1, at both the mRNA and protein levels compared with non-diabetic tissue. Many other studies similarly have shown changes in the expression of Nrf2 in diabetes compared to non-diabetic individuals highlighting its role as a key regulator in diabetes.

Nrf2 Modulates Metabolic Pathways to Control Hyperglycemia-induced Aberration in Diabetes
Hyperglycemia arising from uncontrolled glucose regulation causes tissue damage through increased polyol pathway producing excessive sorbitol by aldose reductase activity [24,25].
Elevated sorbitol induces TGFβ1 and inhibits Nrf2 expression resulting in elevated ROS levels [26]. Excessive cellular ROS in turn activate the TGFβ1 pathway in feed-forward mechanisms, leading to increased AR expression. Therefore, restoring Nrf2 activity using naturally occurring Nrf2 activators helps to down-regulate sorbitol-mediated ROS induction. In support of this, a separate study recently showed that activation of Nrf2 inhibits the function of TGFβ1 [27]. Formation of toxic advanced glycation end (AGE) products methylglyoxal (MG), glyoxal and carboxymethyl lysine is another characteristic feature of diabetes [28]. AGEs that are produced as a result of non-enzymatic binding of reducing sugars with the amino groups in proteins, lipids or nucleic acids damage target cells by directly disrupting matrix-matrix and matrix-cell interactions through excessive cross-linking of proteins [29]. One way to overcome AGEs-mediated toxic effects is to increase the expression of glyoxalase-1 (Glo-1), a key enzyme which catalyzes the conversion of MG to lactic acid [30].
Interestingly, Glo-1 is a direct target of Nrf2 [31]. A study by Chang wc et al. [32] showed induction of hepatic glyoxalase mRNA and glutathione (GSH) by elevated Nrf2, which helped in reducing the serum and hepatic AGEs as well as inflammatory factors in MG-treated rats. In a separate study it has been demonstrated that Nrf2 activation prevented the AGEs-induced ROS formation in LX-2 and human stellate cells through up regulation of γ-glutamyl cysteine synthetase and glutathione synthesis [33,34].
Elevated diacylglycerol (DAG) levels are another characteristic feature of diabetes [35]. DAG is usually produced when the glycolytic intermediate dihydroxy acetone phosphate (DHAP) gets reduced in a series of reduction reactions first producing glycerol-3-phosphate followed by DAG [36]. DAG thus produced activates protein kinase C (PKC), a serine/threonine kinases responsible for various structural and functional changes that include A. Cellular permeability;
In summary, PKC is a key regulator of various processes that lead to the a) Overproduction of ROS; b) Development of insulin resistance; c) Impaired glucose tolerance and d) β-cell dysfunction [38].
Nrf2 plays an important role in modulating these metabolic aberrations during hyperglycemias by regulating PKC and the enzymes involved in glucose metabolism [39]. Mechanistically, Nrf2 up regulate Slc2a1, Hk2 and Pkm2 expression in liver, brown adipose tissue, brain and kidney, while down-modulating G6p expression, cAMP-CREB signalling pathways. Therefore, activation of Nrf2 using phytochemicals is a better strategy to inhibit diabetes-related complications.
Hence, several researchers have developed screening and validation methods to identify potent Nrf2 activators from plant sources. One widely used screening method for identifying Nrf2 activators is complementation assay. Complementation screening system works on the principle that natural products activating Nrf2 prevents the expression of luciferase by interfering with Nrf2 binding to Keap1 protein [40]. Therefore, a decrease in luciferase signal is an indicator of potent activator of Nrf2. Table 1 shows various plant products known to activate Nrf2 . Inhibition of TNF-α in the serum Induced the expression of Nrf2-target gene HO-1 in aorta [55] Nutrition & Food Science International Journal

Future Direction
Even though several studies have demonstrated the vital role played by Nrf2 in controlling diabetes related complications, and identified potential activators of Nrf2, not many studies have evaluated Nrf2 activators in clinical trials. Therefore, future studies should investigate the potential of naturally occurring Nrf2 activators for preventing / treating diabetes in clinical trials. In addition, strategies such as use of nano-formulations should be adopted to deliver poorly bioavailable Nrf2 activators. Schematic representation showing the mechanism of cytoprotection by Nrf2 activators: Schematic representation demonstrating various signaling cascades modulated through Nrf2 pathway in diabetes. Nrf2-activators reduce diabetes by upregulating glycolytic enzymes hexokinase (HK), phosphofructokinase-1 (PFK1) and glyceraldehyde 3-phoshate dehydrogenase (G3PD), and enzymes of hexose monophosphate shunt pathway in particular glucose-6-phosphate dehydrogenase (G6PD). In addition, Nrf2-activators inhibit the enzymes of gluconeogenesis Glucose 6 Phosphatase (G6P) and F1BPase. Furthermore, Nrf2 target genes protect pancreatic β-cells from ROS induced damage by up regulating NQO1 and SOD levels. Additionally, Nrf2 controls cell proliferation, apoptosis, autophagy and angiogenesis in diabetes (Figure 1).