Reactive Oxygen Comes of Age: Mechanism-Based Therapy of Diabetic End-Organ Damage
Authors: Mahmoud H. Elbatreek, Mayra P. Pachado, Antonio Cuadrado, Karin Jandeleit-Dahm, Harald H.H.W. Schmidt
Journal: Trends in Endocrinology and Metabolism (2019)

Lack of comprehensive understanding of DM and its complications, together with its increasing prevalence, represent a major unmet medical need. There is quite a lot known about its mechanisms, but more is needed. Reactive oxygen species (ROS) may hold the answer to this need, but long-standing misconceptions about oxidative stress and the inefficacy of antioxidants have prevented clinical breakthroughs. A new understanding of ROS as both essential signaling molecules and pathomechanism have led to the identification, at different stages of diabetes, of different therapeutically relevant sources and targets of ROS. Network pharmacology and precision diagnostics, currently in advanced stages of clinical development, in conjunction with essential lifestyle changes, will revolutionize the therapy and prevention of diabetes and its end-organ damage. Reactive oxygen species (ROS) have been mainly viewed as unwanted by-products of cellular metabolism, oxidative stress, a sign of a cellular redox imbalance, and potential disease mechanisms, such as in diabetes mellitus (DM). Antioxidant therapies, however, have failed to provide clinical benefit. This paradox can be explained by recent discoveries that ROS have mainly essential signaling and metabolic functions and evolutionally conserved physiological enzymatic sources. Disease can occur when ROS accumulate in nonphysiological concentrations, locations, or forms. By focusing on disease-relevant sources and targets of ROS, and leaving ROS physiology intact, precise therapeutic interventions are now possible and are entering clinical trials. Their outcomes are likely to profoundly change our concepts of ROS in DM and in medicine in general. Reactive oxygen species (ROS) have been mainly viewed as unwanted by-products of cellular metabolism, oxidative stress, a sign of a cellular redox imbalance, and potential disease mechanisms, such as in diabetes mellitus (DM). Antioxidant therapies, however, have failed to provide clinical benefit. This paradox can be explained by recent discoveries that ROS have mainly essential signaling and metabolic functions and evolutionally conserved physiological enzymatic sources. Disease can occur when ROS accumulate in nonphysiological concentrations, locations, or forms. By focusing on disease-relevant sources and targets of ROS, and leaving ROS physiology intact, precise therapeutic interventions are now possible and are entering clinical trials. Their outcomes are likely to profoundly change our concepts of ROS in DM and in medicine in general. Diabetes mellitus (DM) and its related end-organ damage, such as diabetic nephropathy, neuropathy, retinopathy, and cardiomyopathy, are major causes of death and long-term disability. Their underlying mechanisms are incompletely understood, which is why none of the current antidiabetic therapies target the underlying causes or are curative, but focus instead on normalizing surrogate parameters or risk factors such as blood glucose or hypertension [1NCD Risk Factor Collaboration Worldwide trends in diabetes since 1980: a pooled analysis of 751 population-based studies with 4.4 million participants.Lancet. 2016; 387: 1513-1530Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar]. Hence, our lack of mechanistic understanding of lifestyle change-resistant diabetic end-organ damage, together with the increasing prevalence of DM, represent a significant major unmet medical need. One mechanism that has been suggested for decades to cause pancreatic β cell dysfunction and diabetic end-organ damage is 'oxidative stress' (see Glossary), originally defined as an overproduction of reactive oxygen species (ROS). Antioxidants were considered the obvious therapeutic countermeasure but, clinically, have consistently disappointed [2Schmidt H.H. et al.Antioxidants in translational medicine.Antioxid. Redox Signal. 2015; 23: 1130-1143Crossref PubMed Scopus (81) Google Scholar]. Even worse, meta-analyses of clinical trials show that antioxidants may not only be ineffective, but harmful, and even increase mortality [2Schmidt H.H. et al.Antioxidants in translational medicine.Antioxid. Redox Signal. 2015; 23: 1130-1143Crossref PubMed Scopus (81) Google Scholar]. Recently, however, important conceptual breakthroughs in our understanding of ROS in general and DM in particular explain the failure of antioxidants and point towards entirely different mechanism-based and possibly curative therapeutic approaches. Our new understanding of ROS requires that many long-held misconceptions, such as the 'redox balance hypothesis' and the view that ROS are primarily stressors, disease triggers, and metabolic waste products, must be overcome. Instead, the many physiological roles of ROS and the existence of at least seven evolutionarily conserved ROS-producing enzymes (NOX1–5, DUOX1–2), in addition to their alternatively spliced variants, should be recognized. In particular, the discovery of NADPH oxidase isoforms, whose only known function is to produce ROS [i.e., NOX1, NOX2 (aka gp91phox) NOX3, NOX4, and NOX5], made it clear that ROS are not merely waste or toxic by-products that need to be removed in order to prevent cellular damage (Box 1).Box 1NADPH Oxidase FamilyUnlike several ROS sources (e.g., mitochondria, xanthine oxidase, cytochrome P450 enzymes, and uncoupled NOS), NADPH oxidases are the only enzyme family known to produce ROS as their primary and sole function [5Casas A.I. et al.Reactive oxygen-related diseases: therapeutic targets and emerging clinical indications.Antioxid. Redox Signal. 2015; 23: 1171-1185Crossref PubMed Scopus (0) Google Scholar]. NADPH oxidases are enzyme complexes with a membrane spanning catalytic NOX subunit in addition to other membrane and cytosolic proteins. There are seven identified members of the NADPH oxidase family, NOX1–5, and two dual oxidases (DUOX), DUOX1 and DUOX2. In humans, all seven enzymes are expressed and each NOX isoform has specific tissue expression, regulation, and type of ROS produced [3Dao V.T. et al.Pharmacology and clinical drug candidates in redox medicine.Antioxid. Redox Signal. 2015; 23: 1113-1129Crossref PubMed Scopus (3) Google Scholar, 5Casas A.I. et al.Reactive oxygen-related diseases: therapeutic targets and emerging clinical indications.Antioxid. Redox Signal. 2015; 23: 1171-1185Crossref PubMed Scopus (0) Google Scholar].The main NOX isoforms to be considered in pathophysiology of DM are NOX1 and NOX5, which produce superoxide, and NOX4, which produces H2O2. NOX3 and the DUOXs have very limited roles in the inner ear and in the synthesis of thyroid hormone, respectively [87Weaver J.R. et al.Role of NADPH oxidase in beta cell dysfunction.in: Islam M.S. Islets of Langerhans. 2nd edn. Springer, 2014Crossref Google Scholar]. NOX2 is a key enzyme of the innate and inflammatory response and its inhibition or genetic defects are associated with immune deficiency and increased risk of infection, in particular in DM [13Gray S.P. et al.NADPH oxidase 1 plays a key role in diabetes mellitus-accelerated atherosclerosis.Circulation. 2013; 127: 1888-1902Crossref PubMed Scopus (183) Google Scholar]. In addition, NOX2 has been suggested to be involved in an excessive and unlikely number of disease models [5Casas A.I. et al.Reactive oxygen-related diseases: therapeutic targets and emerging clinical indications.Antioxid. Redox Signal. 2015; 23: 1171-1185Crossref PubMed Scopus (0) Google Scholar], which indicates a possible positive publication bias, as shown by a meta-analysis of NOX2 studies in stroke [88Kleikers P.W. et al.A combined pre-clinical meta-analysis and randomized confirmatory trial approach to improve data validity for therapeutic target validation.Sci. Rep. 2015; 513428Crossref PubMed Google Scholar], or an epiphenomena without therapeutic relevance. Unlike several ROS sources (e.g., mitochondria, xanthine oxidase, cytochrome P450 enzymes, and uncoupled NOS), NADPH oxidases are the only enzyme family known to produce ROS as their primary and sole function [5Casas A.I. et al.Reactive oxygen-related diseases: therapeutic targets and emerging clinical indications.Antioxid. Redox Signal. 2015; 23: 1171-1185Crossref PubMed Scopus (0) Google Scholar]. NADPH oxidases are enzyme complexes with a membrane spanning catalytic NOX subunit in addition to other membrane and cytosolic proteins. There are seven identified members of the NADPH oxidase family, NOX1–5, and two dual oxidases (DUOX), DUOX1 and DUOX2. In humans, all seven enzymes are expressed and each NOX isoform has specific tissue expression, regulation, and type of ROS produced [3Dao V.T. et al.Pharmacology and clinical drug candidates in redox medicine.Antioxid. Redox Signal. 2015; 23: 1113-1129Crossref PubMed Scopus (3) Google Scholar, 5Casas A.I. et al.Reactive oxygen-related diseases: therapeutic targets and emerging clinical indications.Antioxid. Redox Signal. 2015; 23: 1171-1185Crossref PubMed Scopus (0) Google Scholar]. The main NOX isoforms to be considered in pathophysiology of DM are NOX1 and NOX5, which produce superoxide, and NOX4, which produces H2O2. NOX3 and the DUOXs have very limited roles in the inner ear and in the synthesis of thyroid hormone, respectively [87Weaver J.R. et al.Role of NADPH oxidase in beta cell dysfunction.in: Islam M.S. Islets of Langerhans. 2nd edn. Springer, 2014Crossref Google Scholar]. NOX2 is a key enzyme of the innate and inflammatory response and its inhibition or genetic defects are associated with immune deficiency and increased risk of infection, in particular in DM [13Gray S.P. et al.NADPH oxidase 1 plays a key role in diabetes mellitus-accelerated atherosclerosis.Circulation. 2013; 127: 1888-1902Crossref PubMed Scopus (183) Google Scholar]. In addition, NOX2 has been suggested to be involved in an excessive and unlikely number of disease models [5Casas A.I. et al.Reactive oxygen-related diseases: therapeutic targets and emerging clinical indications.Antioxid. Redox Signal. 2015; 23: 1171-1185Crossref PubMed Scopus (0) Google Scholar], which indicates a possible positive publication bias, as shown by a meta-analysis of NOX2 studies in stroke [88Kleikers P.W. et al.A combined pre-clinical meta-analysis and randomized confirmatory trial approach to improve data validity for therapeutic target validation.Sci. Rep. 2015; 513428Crossref PubMed Google Scholar], or an epiphenomena without therapeutic relevance. In contrast, ROS are part of signaling networks that include ROS targets, such as nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC), and ROS-metabolizing enzymes, in particular those genetically regulated by nuclear factor (erythroid-derived 2)-like 2 (NRF2) [3Dao V.T. et al.Pharmacology and clinical drug candidates in redox medicine.Antioxid. Redox Signal. 2015; 23: 1113-1129Crossref PubMed Scopus (3) Google Scholar, 4David J.A. et al.The Nrf2/Keap1/ARE pathway and oxidative stress as a therapeutic target in type II diabetes mellitus.J. Diabetes Res. 2017; 20174826724Crossref PubMed Scopus (25) Google Scholar]. The differential expression of these players in different subcellular compartments [5Casas A.I. et al.Reactive oxygen-related diseases: therapeutic targets and emerging clinical indications.Antioxid. Redox Signal. 2015; 23: 1171-1185Crossref PubMed Scopus (0) Google Scholar] suggests that a homogenous cell-wide redox level or balance does not exist, but in fact points to there being differential asynchronous hot spots of ROS signaling within the cell. This then makes ROS quite similar to other classical signaling mechanisms [6Cai Z. Yan L.J. Protein oxidative modifications: beneficial roles in disease and health.J. Biochem. Pharmacol. Res. 2013; 1: 15-26PubMed Google Scholar], including those inducing post-translational protein modifications such as phosphorylation. ROS signaling contributes to physiological functions and processes such as the oxidative burst of the innate immune response, cell proliferation and angiogenesis, vasodilation, hearing, hormone synthesis, insulin secretion, and insulin sensitivity [3Dao V.T. et al.Pharmacology and clinical drug candidates in redox medicine.Antioxid. Redox Signal. 2015; 23: 1113-1129Crossref PubMed Scopus (3) Google Scholar, 7Pi J. et al.Reactive oxygen species as a signal in glucose-stimulated insulin secretion.Diabetes. 2007; 56: 1783-1791Crossref PubMed Scopus (300) Google Scholar, 8Ray R. et al.Endothelial Nox4 NADPH oxidase enhances vasodilatation and reduces blood pressure in vivo.Arterioscler. Thromb. Vasc. Biol. 2011; 31: 1368-1376Crossref PubMed Scopus (0) Google Scholar]. Dysfunction in ROS signaling includes formation of excessive amounts of ROS, appearance of ROS at nonphysiological subcellular sites or in cell types that normally do not form relevant amounts of ROS, or shifting from a physiological to a nonphysiological type of ROS [e.g., from hydrogen peroxide (H2O2) to superoxide]. These mechanistic insights are now leading to new therapeutic concepts for which DM is one of best understood and most suitable pathologies. Moving forward, ROS-related drug development will have to focus on the delicate task of identifying the main disease-relevant sources of deleterious ROS while at the same time leaving essential physiological sources of ROS and their signaling pathways intact. One example is the selective inhibition of specific NOX isoforms or the use of NRF2 agonists that enhance the expression of endogenous antioxidant enzymes at their physiological sites, which is qualitatively different to exogenous scavenging antioxidants acting broadly, in all cells and all cellular compartments, and thus in a nonphysiological manner. As a complicating factor, during the course of disease, the enzymatic sources of ROS may change. One example of this is the triggering of vascular dysfunction by NOX1, leading to uncoupling of NOS3, which may become a secondary, yet quantitatively more relevant source of ROS [9Youn J.Y. et al.The p47phox- and NADPH oxidase organiser 1 (NOXO1)-dependent activation of NADPH oxidase 1 (NOX1) mediates endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction in a streptozotocin-induced murine model of diabetes.Diabetologia. 2012; 55: 2069-2079Crossref PubMed Scopus (0) Google Scholar]; another example is ROS-induced ROS release in mitochondria [10Zorov D.B. et al.Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release.Physiol. Rev. 2014; 94: 909-950Crossref PubMed Scopus (601) Google Scholar]. In addition to preventing ROS synthesis and metabolism, ROS-induced damage can also be repaired at both the molecular and functional level. This is exemplified by recoupling of uncoupled NOS, allosteric sensitization of the nitric oxide (NO) receptor, sGC, for lowered NO levels, and the activation of oxidatively damaged, heme-free apo-sGC by heme-mimetics, respectively [3Dao V.T. et al.Pharmacology and clinical drug candidates in redox medicine.Antioxid. Redox Signal. 2015; 23: 1113-1129Crossref PubMed Scopus (3) Google Scholar, 5Casas A.I. et al.Reactive oxygen-related diseases: therapeutic targets and emerging clinical indications.Antioxid. Redox Signal. 2015; 23: 1171-1185Crossref PubMed Scopus (0) Google Scholar]. Collectively, these discoveries have revolutionized the field of ROS in general and, as indicated by one of first clinical applications, DM. We here review, first, the physiological roles of ROS (e.g., NO, H2O2, superoxide, and peroxynitrite) in insulin secretion and signaling (Box 2), as well as in physiology and pathophysiology of organs affected by functional and structural damage due to type 2 DM (T2DM). Importantly, the sources and mechanisms of ROS differ in health, early and late stages of disease, and with respect to different cells and organs (Figure 1, Key Figure), sometimes even qualitatively. These differences need to be carefully dissected and defined for T2DM and its characteristic complications as well as other conditions in order to allow future precision antidiabetic interventions. With respect to ROS, the relevant therapeutic targets (see above) form a causal signaling network and in T2DM will be best targeted by mechanism-based network pharmacology, where multiple drugs are combined to synergistically correct a pathological into a near-physiological state [11Langhauser F. et al.A diseasome cluster-based drug repurposing of soluble guanylate cyclase activators from smooth muscle relaxation to direct neuroprotection.NPJ Syst. Biol. Appl. 2018; 4: 8Crossref PubMed Scopus (0) Google Scholar]. These drugs include some new compound classes that act directly on ROS sources and targets but also some registered drugs, which act, at least in part, through indirectly modulating the ROS network. Finally, mechanism-based diagnostics may enrich these new therapeutic approaches with respect to ROS and DM in order to stratify patients for personalized and precision therapy.Box 2Insulin and ROSPancreatic β CellsThe secretion of insulin from pancreatic β cells is mainly regulated by plasma glucose levels. Glucose is taken up via the glucose transporter 2 (GLUT2) and thereafter oxidized to produce ATP. This leads to the closure of KATP channels, depolarization of the plasma membrane, opening of voltage-dependent Ca2+ channels, and a subsequent increase in intracellular Ca2+, which enables exocytotic insulin release [89Llanos P. et al.Correction: glucose-dependent insulin secretion in pancreatic beta-cell islets from male rats requires Ca2+ release via ROS-stimulated ryanodine receptors [corrected].PLoS One. 2015; 10e0140198PubMed Google Scholar]. Two ROS species, NO and H2O2, increase intracellular Ca2+ and thereby facilitate insulin release. Low levels of NO from NOS1 do so by stimulating cGMP formation through sGC, and cGMP in turn activates cGMP-dependent protein kinase (PKG), which inhibits KATP channels [90Schmidt H.H. et al.Insulin secretion from pancreatic B cells caused by l-arginine-derived nitrogen oxides.Science. 1992; 255: 721-723Crossref PubMed Google Scholar]; mitochondrial glucose metabolism leaks small amounts of superoxide, which, upon dismutation to H2O2, stimulates ryanodine receptors to also increase intracellular Ca2+ [7Pi J. et al.Reactive oxygen species as a signal in glucose-stimulated insulin secretion.Diabetes. 2007; 56: 1783-1791Crossref PubMed Scopus (300) Google Scholar, 89Llanos P. et al.Correction: glucose-dependent insulin secretion in pancreatic beta-cell islets from male rats requires Ca2+ release via ROS-stimulated ryanodine receptors [corrected].PLoS One. 2015; 10e0140198PubMed Google Scholar]. Importantly, the use of ROS by pancreatic β cells comes at a risk. Compared with most other cells, pancreatic β cells have some of the lowest expression and activity levels of ROS-metabolizing (antioxidant) enzymes [7Pi J. et al.Reactive oxygen species as a signal in glucose-stimulated insulin secretion.Diabetes. 2007; 56: 1783-1791Crossref PubMed Scopus (300) Google Scholar], making them more vulnerable than other cells to the potential cytotoxic effects of ROS, such as DNA and protein damage.In early stages of T2DM, production of NO and ROS begins to exceed the antioxidant resistance of pancreatic β cells. Peripheral insulin resistance (see below) and high glucose and fatty acid levels trigger pancreatic β cells to compensate by releasing more and more insulin in a mitochondrial ROS-dependent manner [91Prentki M. Nolan C.J. Islet beta cell failure in type 2 diabetes.J. Clin. Invest. 2006; 116: 1802-1812Crossref PubMed Scopus (981) Google Scholar]. In later stages, this mechanism is exhausted as superoxide anion induces a leak of protons across the mitochondrial inner membrane, decreases the mitochondrial membrane potential and ATP production, and eventually leads to a lower insulin secretion [92Krauss S. et al.The mitochondrial uncoupling-protein homologues.Nat. Rev. Mol. Cell Biol. 2005; 6: 248-261Crossref PubMed Scopus (447) Google Scholar]. Moreover, upregulation of the renin-angiotensin system (RAS) and inflammatory cytokines further increase superoxide overproduction from additional sources (Figure 1) (i.e., NOX1 and NOX2) [87Weaver J.R. et al.Role of NADPH oxidase in beta cell dysfunction.in: Islam M.S. Islets of Langerhans. 2nd edn. Springer, 2014Crossref Google Scholar]. The additional induction of NOS2, which produces NO, toxifies superoxide to yield peroxynitrite which not only further reduces insulin secretion but also induces pancreatic β cell death [93Suarez-Pinzon W.L. et al.An inhibitor of inducible nitric oxide synthase and scavenger of peroxynitrite prevents diabetes development in NOD mice.J. Autoimmun. 2001; 16: 449-455Crossref PubMed Scopus (0) Google Scholar].Insulin-Sensitive TissuesIn peripheral insulin-sensitive tissues such as skeletal muscle, fat cells, and liver, insulin controls the switch from lipolysis/fatty oxidation during fasting to lipid storage/glucose oxidation following feeding [94Taniguchi C.M. et al.Critical nodes in signalling pathways: insights into insulin action.Nat. Rev. Mol. Cell Biol. 2006; 7: 85-96Crossref PubMed Scopus (1574) Google Scholar]. Binding of insulin to the insulin receptor (IR) phosphorylates substrate proteins, IRS1 and IRS2, activating phosphatidylinositol 3-kinase (PI3K)-Akt (protein kinase B) signaling, which leads to the translocation and activation of glucose transporters (mainly GLUT4 in muscles and fat cells) and subsequent glucose uptake [94Taniguchi C.M. et al.Critical nodes in signalling pathways: insights into insulin action.Nat. Rev. Mol. Cell Biol. 2006; 7: 85-96Crossref PubMed Scopus (1574) Google Scholar, 95Mahadev K. et al.The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction.Mol. Cell. Biol. 2004; 24: 1844-1854Crossref PubMed Scopus (377) Google Scholar].ROS come into play in insulin signaling through activation of PI3K and alternative protein kinase C (PKC) activation to increase NOX4 activity, forming H2O2 [95Mahadev K. et al.The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction.Mol. Cell. Biol. 2004; 24: 1844-1854Crossref PubMed Scopus (377) Google Scholar]. H2O2 augments insulin-IR-PI3K signaling twofold, by inhibiting protein tyrosine phosphatase 1B (PTP1B) and the phosphatase and tensin homologue, PTEN, which dephosphorylates IR and downregulates PI3K signaling [94Taniguchi C.M. et al.Critical nodes in signalling pathways: insights into insulin action.Nat. Rev. Mol. Cell Biol. 2006; 7: 85-96Crossref PubMed Scopus (1574) Google Scholar, 95Mahadev K. et al.The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction.Mol. Cell. Biol. 2004; 24: 1844-1854Crossref PubMed Scopus (377) Google Scholar], and by activating MAP kinase phosphatase-1, which dephosphorylates IRS1 [96Schroder K. et al.Nox4 acts as a switch between differentiation and proliferation in preadipocytes.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 239-245Crossref PubMed Scopus (0) Google Scholar].Further increased ROS production is associated with peripheral insulin resistance, a main feature of T2DM [97Den Hartigh L.J. et al.Adipocyte-specific deficiency of NADPH oxidase 4 delays the onset of insulin resistance and attenuates adipose tissue inflammation in obesity.Arterioscler. Thromb. Vasc. Biol. 2017; 37: 466-475Crossref PubMed Scopus (18) Google Scholar]. In early stages of T2DM, NOX4 causes fat and liver cell apoptosis, fibrosis, and inflammation [97Den Hartigh L.J. et al.Adipocyte-specific deficiency of NADPH oxidase 4 delays the onset of insulin resistance and attenuates adipose tissue inflammation in obesity.Arterioscler. Thromb. Vasc. Biol. 2017; 37: 466-475Crossref PubMed Scopus (18) Google Scholar, 98Tiganis T. Reactive oxygen species and insulin resistance: the good, the bad and the ugly.Trends Pharmacol. Sci. 2011; 32: 82-89Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar]; NOX2 decreases skeletal muscle insulin-induced Akt phosphorylation, GLUT4 expression and translocation, and thereby glucose uptake [99Wei Y. et al.Angiotensin II-induced skeletal muscle insulin resistance mediated by NF-kappaB activation via NADPH oxidase.Am. J. Physiol. Endocrinol. Metab. 2008; 294: E345-E351Crossref PubMed Scopus (0) Google Scholar]. In later stages, mitochondrial ROS formation is induced in skeletal muscles, fat cells, and liver, activating serine kinases and further impeding insulin signaling [97Den Hartigh L.J. et al.Adipocyte-specific deficiency of NADPH oxidase 4 delays the onset of insulin resistance and attenuates adipose tissue inflammation in obesity.Arterioscler. Thromb. Vasc. Biol. 2017; 37: 466-475Crossref PubMed Scopus (18) Google Scholar, 98Tiganis T. Reactive oxygen species and insulin resistance: the good, the bad and the ugly.Trends Pharmacol. Sci. 2011; 32: 82-89Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar]. In skeletal muscles, upregulation of the RAS system further activates NOX2 to aggravate insulin resistance [99Wei Y. et al.Angiotensin II-induced skeletal muscle insulin resistance mediated by NF-kappaB activation via NADPH oxidase.Am. J. Physiol. Endocrinol. Metab. 2008; 294: E345-E351Crossref PubMed Scopus (0) Google Scholar]. Pancreatic β Cells The secretion of insulin from pancreatic β cells is mainly regulated by plasma glucose levels. Glucose is taken up via the glucose transporter 2 (GLUT2) and thereafter oxidized to produce ATP. This leads to the closure of KATP channels, depolarization of the plasma membrane, opening of voltage-dependent Ca2+ channels, and a subsequent increase in intracellular Ca2+, which enables exocytotic insulin release [89Llanos P. et al.Correction: glucose-dependent insulin secretion in pancreatic beta-cell islets from male rats requires Ca2+ release via ROS-stimulated ryanodine receptors [corrected].PLoS One. 2015; 10e0140198PubMed Google Scholar]. Two ROS species, NO and H2O2, increase intracellular Ca2+ and thereby facilitate insulin release. Low levels of NO from NOS1 do so by stimulating cGMP formation through sGC, and cGMP in turn activates cGMP-dependent protein kinase (PKG), which inhibits KATP channels [90Schmidt H.H. et al.Insulin secretion from pancreatic B cells caused by l-arginine-derived nitrogen oxides.Science. 1992; 255: 721-723Crossref PubMed Google Scholar]; mitochondrial glucose metabolism leaks small amounts of superoxide, which, upon dismutation to H2O2, stimulates ryanodine receptors to also increase intracellular Ca2+ [7Pi J. et al.Reactive oxygen species as a signal in glucose-stimulated insulin secretion.Diabetes. 2007; 56: 1783-1791Crossref PubMed Scopus (300) Google Scholar, 89Llanos P. et al.Correction: glucose-dependent insulin secretion in pancreatic beta-cell islets from male rats requires Ca2+ release via ROS-stimulated ryanodine receptors [corrected].PLoS One. 2015; 10e0140198PubMed Google Scholar]. Importantly, the use of ROS by pancreatic β cells comes at a risk. Compared with most other cells, pancreatic β cells have some of the lowest expression and activity levels of ROS-metabolizing (antioxidant) enzymes [7Pi J. et al.Reactive oxygen species as a signal in glucose-stimulated insulin secretion.Diabetes. 2007; 56: 1783-1791Crossref PubMed Scopus (300) Google Scholar], making them more vulnerable than other cells to the potential cytotoxic effects of ROS, such as DNA and protein damage. In early stages of T2DM, production of NO and ROS begins to exceed the antioxidant resistance of pancreatic β cells. Peripheral insulin resistance (see below) and high glucose and fatty acid levels trigger pancreatic β cells to compensate by releasing more and more insulin in a mitochondrial ROS-dependent manner [91Prentki M. Nolan C.J. Islet beta cell failure in type 2 diabetes.J. Clin. Invest. 2006; 116: 1802-1812Crossref PubMed Scopus (981) Google Scholar]. In later stages, this mechanism is exhausted as superoxide anion induces a leak of protons across the mitochondrial inner membrane, decreases the mitochondrial membrane potential and ATP production, and eventually leads to a lower insulin secretion [92Krauss S. et al.The mitochondrial uncoupling-protein homologues.Nat. Rev. Mol. Cell Biol. 2005; 6: 248-261Crossref PubMed Scopus (447) Google Scholar]. Moreover, upregulation of the renin-angiotensin system (RAS) and inflammatory cytokines further increase superoxide overproduction from additional sources (Figure 1) (i.e., NOX1 and NOX2) [87Weaver J.R. et al.Role of NADPH oxidase in beta cell dysfunction.in: Islam M.S. Islets of Langerhans. 2nd edn. Springer, 2014Crossref Google Scholar]. The additional induction of NOS2, which produces NO, toxifies superoxide to yield peroxynitrite which not only further reduces insulin secretion but also induces pancreatic β cell death [93Suarez-Pinzon W.L. et al.An inhibitor of inducible nitric oxide synthase and scavenger of peroxynitrite prevents diabetes development in NOD mice.J. Autoimmun. 2001; 16: 449-455Crossref PubMed Scopus (0) Google Scholar]. Insulin-Sensitive Tissues In peripheral insulin-sensitive tissues such as skeletal muscle, fat cells, and liver, insulin controls the switch from lipolysis/fatty oxidation during fasting to lipid storage/glucose oxidation following feeding [94Taniguchi C.M. et al.Critical nodes in signalling pathways: insights into insulin action.Nat. Rev. Mol. Cell Biol. 2006; 7: 85-96Crossref PubMed Scopus (1574) Google Scholar]. Binding of insulin to the insulin receptor (IR) phosphorylates substrate proteins, IRS1 and IRS2, activating phosphatidylinositol 3-kinase (PI3K)-Akt (protein kinase B) signaling, which leads to the translocation and activation of glucose transporters (mainly GLUT