11 REVIEW Reactive oxygen and nitrogen species generation, antioxidant defenses, and b-cell function: a critical role for amino acids P Newsholme, E Rebelato1, F Abdulkader1, M Krause2,3, A Carpinelli1 and R Curi1 School of Biomedical Sciences, Curtin University, PO Box U1987, Perth, Western Australia 6845, Australia 1Department of Physiology and Biophysics, Institute of Biomedical Sciences, University Sao Paulo (USP), Sao Paulo, Brazil 2Biomedical Research Group, Department of Science, Institute of Technology Tallaght, Dublin, Ireland 3UCD Institute for Sport and Health, Dublin, Ireland (Correspondence should be addressed to P Newsholme; Email: [email protected]; M Krause at UCD Institute for Sport and Health; Email: [email protected], [email protected]) Abstract Growing evidence indicates that the regulation of intracellular systems are expressed in b cells at higher levels. Herein, we discuss reactive oxygen species (ROS) and reactive nitrogen species the key mechanisms of ROS/RNS production and their (RNS)levelsisessentialformaintainingnormalb-cell glucose physiological function in pancreatic b cells. We also hypothesize responsiveness. While long-term exposure to high glucose that specific interactions between RNS and ROS may be the induces oxidative stress in b cells, conflicting results have been cause of the vulnerability of pancreatic b cells to oxidative damage. published regarding the impact of ROSon acute glucose exposure In addition, using a hypothetical metabolic model based on the and their role in glucose stimulated insulin secretion (GSIS). data available in the literature, we emphasize the importance of Although b cells are considered to be particularly vulnerable to amino acid availability for GSH synthesis and for the maintenance oxidative damage, as they express relatively low levels of some of b-cell function and viability during periods of metabolic peroxide-metabolizing enzymes such as catalase and glutathione disturbance before the clinical onset of diabetes. (GSH) peroxidase, other less known GSH-based antioxidant Journal of Endocrinology (2012) 214, 11–20 Introduction target proteins. Being a charged species, superoxide cannot freely cross biological membranes but may do so via anion Reactive oxygen species (ROS) such as superoxide anion channels. However, the fate of superoxide in cells and tissues %K (O2 ), hydrogen peroxide (H2O2), and the hydroxyl radical is mostly determined by the activity of various site-specific % K (OH ) plus the related peroxynitrite (ONOO ) molecule are enzymes (extracellular, cytoplasmic, and mitochondrial), the generally thought to cause cell dysfunction and ultimately superoxide dismutase (SOD) family, that convert superoxide death due to alteration of i) metabolic pathway activity into molecular oxygen and hydrogen peroxide (Fig. 1). (Newsholme et al. 2007a, 2009b) and/or ii) the structure of H2O2 is an even less reactive species that is uncharged and cellular membranes, DNA, or proteins (Boveris et al. 1972, can diffuse across membranes through aquaporins. Despite its Turrens 1997, Johnson et al. 1999, Chandra et al. 2000, low reactivity, some proteins contain specific cysteine residues Limon-Pacheco & Gonsebatt 2009). However, the half-life that are prone to oxidation by hydrogen peroxide, which are and reactivity of these various species are very different, which critical to hydrogen peroxide-based signaling systems. H2O2 % is an indication of the different biological functions of these can be converted to the OH , a highly reactive species (see molecules (Droge 2002). Fig. 1). Superoxide can also react with nitric oxide (NO), %K K Formation of the O2 can be considered to be the initial resulting in the formation of peroxynitrite (ONOO ), see step for the subsequent formation of other ROS. It is Fig. 1. Cell-associated oxidative damage may not be simply a generated by the single electron reduction of molecular question of the overall concentration of ROS, but rather the oxygen (O2). Superoxide, compared with to other free type of ROS formed with respect to relative reactivity. radicals, is a poorly reactive species and can exist in solution Hydrogen peroxide is the substrate for the majority of the for a considerable time (and thus diffuse) before reacting with antioxidant systems in the cell. These antioxidant systems other free radicals or with specific clusters of iron–sulfur in (many of which are enzymes) are required to minimize the Journal of Endocrinology (2012) 214, 11–20 DOI: 10.1530/JOE-12-0072 0022–0795/12/0214–011 q 2012 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/25/2021 06:56:41AM via free access 12 P NEWSHOLME and others . Amino acids and b-cell redox regulation Figure 1 ROS/RNS species formation and antioxidant defense. ROS and NO may be generated by appropriate/inappropriate stimuli. These molecules may become toxic unless rapidly removed by the pathways described. GSSG, glutathione disulfide; GSH, glutathione; GSHRED, glutathione reductase; GSHPER, glutathione peroxidase; CAT, catalase; SOD, superoxide dismutase; ETC, electron transport chain; NOX, NADPH oxidase; IKK, I k B kinase; NFkB, nuclear factor kB; iNOS, inducible nitric oxide synthase. % reaction controlling the conversion of H2O2 to HO ,a availability for GSH synthesis and for the maintenance of highly reactive molecule. Superoxide may also chemically b-cell function and viability during periods of metabolic combine with the reactive nitrogen species (RNS), NO in a disturbance before the clinical onset of diabetes. diffusion-controlled reaction, resulting in the formation of K peroxynitrite (ONOO ). When cellular antioxidant defenses are overcome, ROS specificity is lost leading to The impact of ROS in pancreatic b cells reaction with non-readily oxidizable amino acid residues, as well as with unsaturated lipids and DNA, therefore leading to b Cells adequately deal with the physiological challenges of oxidative stress. substrate availability imposed both acutely and chronically Pancreatic b cells are considered to be particularly depending on the nutritional and metabolic states (Schmitt vulnerable to oxidative damage (Lenzen 2008b), as it has et al. 2011). The availability and the type of antioxidant been reported that they express relatively low levels of catalase defenses in these cells are dictated by demand. There is now a and glutathione (GSH) peroxidase, which would contribute consensus that the chronically high circulating levels of to lipotoxicity, glucotoxicity, or a combination termed glucose (glucotoxic concentrations) and/or lipid (glucolipo- glucolipotoxicity in b cells chronically exposed to nutrients, toxic or lipotoxic concentrations) associated with type 2 diabetes favoring apoptosis (Robertson et al. 1992, Newsholme et al. induce oxidative stress in different cell types (Newsholme 2007a,b, Gehrmann et al. 2010). Interestingly, impairment, or et al.2007a, Gehrmann et al.2010). In type 1 diabetes, otherwise, of b-cell function may depend on the antioxidant induced by autoimmune b-cell destruction, death is associated system involved in the scavenging process, as specific with cytokine-mediated oxidative stress (Morgan et al. 2007, manipulation in antioxidant defenses may result in different Lenzen 2008a). outcomes (Lenzen 2008b). Oxidative stress is currently viewed as an imbalance Herein, we critically revise the major mechanisms of between pro- and antioxidants in favor of the former, ROS/RNS production and implications for the physiological which implicates a loss of redox signaling. It can be triggered function of pancreatic b cells. In addition, using a by excessive ROS production as well as by low antioxidant hypothetical metabolic model, based on the data available enzyme activities. A well-known source of electrons in the literature, we emphasize the importance of amino acid for reduction of molecular oxygen is the mitochondrion. Journal of Endocrinology (2012) 214, 11–20 www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/25/2021 06:56:41AM via free access Amino acids and b-cell redox regulation . P NEWSHOLME and others 13 The increase in superoxide formation in the electron Numerous papers have described the negative effects of transport chain is associated with a high (inner) mitochondrial NO generation in the b cell including attenuation of glucose- membrane potential. This causes a decrease in the electron stimulated insulin secretion and stimulation of apoptosis, flow through the respiratory chain, increasing the probability originating from the observation that pro-inflammatory of superoxide formation by the retained electrons at various cytokines induced gene and protein expression of iNOS sites in the mitochondrial respiratory chain. However, an and subsequent NO generation, which was associated with additional specialized enzyme-based system can also generate caspase activation and cell death (Rizzo & Piston 2003). superoxide in a regulated fashion. This enzyme complex, the It is possible that NOX2-derived ROS in the latter NADPH oxidase (NOX), is a member of a family of enzyme conditions may also trigger concomitant nitrosative stress isoforms that are able to transfer electrons from NADPH to (Michalska et al. 2010), thus suggesting that in toxic %K molecular oxygen to generate O2 . NOX activation is more conditions, a key mediator for dysfunction and apoptosis widely associated with efficient killing of pathogens