Regulation of insulin secretion and production of

REVIEW

REVIEW

Islets 3:5, 213-223; September/October 2011; ©2011 Landes Bioscience

Regulation of insulin secretion and production of reactive oxygen species by free fatty acids in pancreatic islets Maria Fernanda Rodrigues Graciano,1,* Maíra M.R. Valle,1 Anjan Kowluru,2,3 Rui Curi1 and Angelo R. Carpinelli1 2

1 Department of Physiology and Biophysics; Institute of Biomedical Sciences; University of São Paulo; São Paulo, SP Brazil; Department of Pharmaceutical Sciences; Eugene Applebaum College of Pharmacy; Wayne State University; Detroit, MI USA; 3 β-Cell Biochemistry Research Laboratory; John D. Dingell VA Medical Center; Detroit, MI USA

Key words: oxidative stress, fatty acids, b-cells, insulin release, NADPH oxidase, hydrogen peroxide, superoxide Abbreviations: ACS, acyl-CoA synthetase; ARBs, angiotensin II receptor blockers; CAT, catalase; CPT-I, carnitine palmitoyltransferase I; DAG, diacylglycerol; FA, fatty acids; GPx, glutathione peroxidase; GCLC, glutamylcysteine ligase; GSIS, glucose-stimulated insulin secretion; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like-peptide 1; GPR, G protein-coupled receptor; HSL, hormone-sensitive lipase; iNOS, induced form of nitric oxide synthase; IP3, inositol triphosphate; K ATP, ATP sensitive-potassium channels; LC-CoA, long-chain acyl-coenzime A; LDH, lactate dehydrogenase; OAA, oxaloacetate; LTCC, L-type calcium channels; PIP2, phosphatidylinositol-4,5-bisphosphate; RAS, renin-angiotensin system; ROS, reactive oxygen species; SNPs, single nucleotide polymorphisms; SOD, superoxide dismutase; TAG, triacylglycerol; TCA cycle, tricarboxylic acid cycle; UCP, uncoupling protein

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

Free fatty acids regulate insulin secretion through metabolic and intracellular signaling mechanisms such as induction of malonyl-CoA/long-chain CoA pathway, production of lipids, GPRs (G protein-coupled receptors) activation and the modulation of calcium currents. Fatty acids (FA) are also important inducers of ROS (reactive oxygen species) production in β-cells. Production of ROS for short periods is associated with an increase in GSIS (glucose-stimulated insulin secretion), but excessive or sustained production of ROS is negatively correlated with the insulin secretory process. Several mechanisms for FA modulation of ROS production by pancreatic β-cells have been proposed, such as the control of mitochondrial complexes and electron transport, induction of uncoupling proteins, NADPH oxidase activation, interaction with the renin-angiotensin system, and modulation of the antioxidant defense system. The major sites of superoxide production within mitochondria derive from complexes I and III. The amphiphilic nature of FA favors their incorporation into mitochondrial membranes, altering the membrane fluidity and facilitating the electron leak. The extra-mitochondrial ROS production induced by FA through the NADPH oxidase complex is also an important source of these species in β-cells.

Reactive oxygen species (ROS) are important components of intracellular redox signaling cascades. They are produced by the metabolism of different substrates such as fatty acids (FA) and glucose. Mitochondrial production of ROS plays an important role *Correspondence to: Maria Fernanda Rodrigues Graciano; Email: [email protected] Submitted: 04/15/11; Revised: 06/15/11; Accepted: 06/17/11 DOI: 10.4161/isl.3.5.15935

in the pancreatic β-cell secretory function.1 However, evidence is accumulating to suggest that pancreatic islets also produce ROS through NADPH oxidase activity, and this process is involved in glucose-stimulated insulin secretion (GSIS).2-4 Production of ROS for short periods is associated with an increase with GSIS, but excessive or sustained production of ROS is negatively correlated with the insulin secretory process.1,2 Chronic exposure to relatively high levels of ROS leads to impairment of pancreatic β-cell function and diabetes.5,6 In this review, the mechanisms involved in fatty acid-mediated insulin secretion and of ROS production in pancreatic islets and b-cells are discussed. Our main focus is the acute effect of the FA on islet functions and on the signaling pathways involved in the amplification of the glucose-induced insulin secretion. These early signals are also important to elucidate the chronic effects of FA in conditions such as insulin resistance, obesity and type 2 diabetes. Mechanisms by which FA Stimulate Insulin Secretion FA play an important role in β-cell function. FA deprivation in islet reduces GSIS,7,8 which is restored by exogenous free FA.9 Short time exposure (1 h) of pancreatic islets to FA augments GSIS;10-13 however, if chronically maintained in excess, saturated FA reduce insulin biosynthesis14 and secretion.15-17 The potency of FA to promote glucose-induced insulin release increases with the chain length and decreases with the degree of unsaturation.18,19 There are pronounced differences in cell effects of saturated and unsaturated FA, with the latter displaying a tendency to promote cell viability under conditions which otherwise would be cytotoxic. The mechanisms involved in this cytoprotective action remain under intense investigation, but

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©201 1L andesBi os c i enc e. Donotdi s t r i but e. Figure 1. Mechanisms by which FA stimulate insulin secretion. Pyruvate can be converted to acetyl-CoA via the pyruvate dehydrogenase complex (PDH) or to oxaloacetate (OAA) via pyruvate carboxylase (PC) in pancreatic islets. The cataplerotic activity of PDH, in association with flux through the anaplerotic enzyme PC, links glycolysis with OAA/citrate synthesis and, in the fed state or in high glucose concentrations, with the synthesis of malonyl-CoA. Malonyl-CoA inhibits mitochondrial long-chain fatty acid uptake at the carnitine palmitoyltransferase I (CPT-I) level, and thereby promotes FA re-esterification. High glucose concentrations raise malonyl-CoA levels and decrease FA oxidation, which leads to LC-CoA accumulation. LC-CoA may additionally be esterified to diacylglycerol (DAG) and triacylglycerol (TAG) in the presence of glycerol-3-phosphate provided by glucose metabolism. DAG formation and PKC activation may synergize with the classic pathways of insulin secretion—K ATP closure and calcium influx—to promote full insulin secretion. GPR40 activation by FA stimulates the Gαq-PLC (phospholipase C) signaling pathway, leading to calcium release from endoplasmic reticulum stores and to DAG production. Endogenous lipolysis by the action of hormone-sensitive lipase (HSL) regulates insulin secretion through the generation of FA and other lipid-signaling molecules. ACS, acyl-CoA synthetase; LDH, lactate dehydrogenase; LTCC, L-type calcium channels; PL, phospholipids.

the rapid inhibition of caspase 3 and the reduction of endoplasmic reticulum stress by the unsaturated FA could be involved.20 Malonyl-CoA/LC-CoA (long-chain acyl-Coenzyme A) metabolic pathway. Circulating levels of FA control GSIS as occurs in fasted rats. Under this condition, b-cells are not able to convert glucose into malonyl-CoA, possibly as a consequence of low pyruvate dehydrogenase activity.21 Malonyl-CoA inhibits mitochondrial long-chain fatty acid uptake by inhibiting carnitine palmitoyltransferase I (CPT-I), thereby promoting FA reesterification. As a result, there is no suppression of CPT-I activity in fasted rats (which avoids the rise of the cytosolic acyl-CoA concentration) and the insulin secretory process is suppressed. This condition renders the b-cell dependent upon a high external FA supply to counterbalance the deficit.22 When the glucose level is low, FA are converted into LC-CoAs by acyl-CoA

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synthetase (ACS) and enter the mitochondria, being oxidized via the β-oxidation pathway for ATP production23 (Fig. 1). Pyruvate is converted into acetyl-CoA via the pyruvate dehydrogenase complex (PDH) or to oxaloacetate (OAA) via pyruvate carboxylase (PC), in general with approximately equal amounts entering each metabolic flux in the mitochondria.24,25 The cytosolic substrate flux via lactate dehydrogenase (LDH) is limited by the low activity of this enzyme in pancreatic islets.26 The cataplerotic activity of PDH, in association with the flux through the anaplerotic PC, links glycolysis with OAA/citrate synthesis and, in the fed state or in high glucose concentrations, with the synthesis of malonyl-CoA. Factors that lead to PDH inhibition, therefore, favor acetyl-CoA production via β-oxidation of long-chain FA. Therefore, intracellular FA homeostasis, in part mediated via the malonyl-CoA nutrient-sensing mechanism, plays a key role

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in pancreatic β-cell function (Fig. 1). These processes are early events in the induction of insulin secretion in pancreatic β-cells.27 High glucose concentrations raise malonyl-CoA levels and decrease FA oxidation, which leads to LC-CoA accumulation. LC-CoA facilitates the fusion of secretory granules with the β-cell plasma membrane, thus promoting insulin secretion.28 The nonmetabolizable analogue 2-bromopalmitate (an inhibitor of CPT-I, which inhibits fatty acid oxidation and is expected to increase cytosolic long chain acyl-CoAs) also stimulates insulin release.29 LC-CoA may additionally be esterified to diacylglycerol (DAG) and triacylglycerol (TAG) in the presence of glycerol3-phosphate provided by glucose metabolism. DAG formation and PKC activation may synergize with the classic pathways of insulin secretion—ATP sensitive-potassium channels (K ATP) closure and calcium influx—to promote full insulin secretion30 (Fig. 1). GPR40 activation and production of lipid-signaling molecules. The GPR (G protein coupled receptor) isoforms 40, 41 and 43 are highly homologous but differ in substrate specificity and tissue distribution. They were formerly known as orphan receptors, however, it was found that FA are agonists of these receptors.31 GPR41 and GPR43 are specifically activated by the short-chain FA acetic, propionic and butyric acids. mRNA expression of both receptors was detected in mouse pancreatic islets as well as in insulin-producing MIN6 cells.32 Long-chain FAs can signal directly via the FA receptor GPR40. This Gαq membrane receptor is highly expressed in pancreatic b-cells and potentially activated by the most prevalent FA in plasma.31,33,34 Taste buds35 and intestinal K and L cells express this receptor,36,37 where they are able to signal fat ingestion, contributing for the secretion of the incretins glucagon-like-peptide (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Therefore, FA can directly stimulate insulin secretion through GPR40 in β-cell surface and, indirectly, potentiate incretin secretion. FA binding to GPR40 activates a heterotrimeric G protein, containing the α subunit of the Gq protein, which is known to stimulate PLC activity. This enzyme converts phosphatidylinositol-4,5-bisphosphate (PIP2) into DAG and inositol triphosphate (IP3). DAG is a well-known PKC activator and IP3 participates in calcium extrusion from the endoplasmic reticulum (Fig. 1). The RNAi-mediated knockdown of GPR40 expression in MIN6 cells abolishes the increase of insulin secretion induced by linoleic and γ-linolenic acids.31 The relevant participation of GPR40 for β-cell function has also been shown in vivo. Latour and collaborators (2007) showed that insulin secretion response to intralipids is reduced by approximately 50% in GPR40 knockout (KO) mice.38 Lan and collaborators (2008) also demonstrated a reduction of insulin secretion by 50% in response to elevated seric fatty acid concentrations in GPR40 KO mice.39 These results show that the signaling induced by GPR40 pathway and the intracellular fatty acid metabolism are complementary for the fatty acid induction of insulin secretion. At least two single nucleotide polymorphisms (SNPs) with functional effects, and located in the coding region of the human GPR40 gene, have been described in references 40 and 41. One of these SNPs leads to an amino acid substitution

(arginine to histidine) at the position 211 in the third intracellular loop of the receptor that was reported to cause enhanced insulin secretion in Japanese men.40 The other SNP also produces an amino acid substitution (Gly180Ser),41 and the less frequent variant (Ser180) has been associated with elevated body-mass index in Europeans and a reduction of insulin secretion following an oral glucose load. Similarly, insulin secretion was also reduced in this group during an oral lipid load. Studies in transfected HeLa cells with the Ser180 variant showed low cytosolic calcium levels compared to those expressing the Gly180 receptor in response to FA load. Isolated rat pancreatic islets export a substantial amount of FA to the incubation medium either in the absence or presence of glucose. After incubation for 1 h, the addition of 5.6 mM glucose raises the medium content of palmitic and stearic acids compared with islets incubated in the absence of glucose. Martins et al concluded that the synthesis and release of saturated and, to a lesser extent, unsaturated FA from glucose-exposed islets represent an amplification pathway for insulin secretion.42 The FA released through triglyceride/FFA cycling and partially secreted from pancreatic islets may activate the GPR40 pathway via autocrine and/or paracrine mechanisms43 (Fig. 1) The FA are supplied to pancreatic b-cells from intracellular triglycerides and phospholipids and from plasma FFA and lipoproteins.43 The FFA originated from plasma triglycerides can access β-cells by the action of lipoprotein lipases.44 In addition, the action of hormone-sensitive lipase (HSL) in b-cells reinforces the concept that endogenous lipolysis participates in the regulation of insulin secretion through generation of FA or other lipidsignaling molecules.45 In fact, the islet triglyceride stores may play an important role in GSIS via HSL-lipolysis (Fig. 1). The HSL KO mice present reduced GSIS both in vivo and in isolated islets. Increases in glucose concentration induce HSL expression, which occurs concomitantly with an augmentation of basal insulin secretion.46,47 These observations indicate that the FA exported from the β-cells may act as signaling molecules in GSIS. Acylation of specific proteins leads to the amplification of insulin secretion. Myristoylation and palmitoylation are involved in directing proteins to appropriate membrane sites, thus stabilizing protein-protein interactions and regulating certain enzyme activities in the mitochondria.48 Various proteins can be acylated: α subunits of G-proteins, tyrosine kinases receptors and proteins involved in the regulation of ion channel activity and exocytosis.29,49,50 FA have also been proposed to regulate ionic channels in pancreatic b-cells.29,51 Cerulenin, an inhibitor of protein acylation, inhibits the augmentation of calcium-induced insulin release promoted by palmitate.49 Calcium currents. In Chinese hamster ovary cells expressing human, mouse and rat GPR40, saturated FA with lengths ranging from C12 to C16 and C18 and C22 unsaturated FA all present calcium influx-inducing activities.31 FA increase calcium influx in β-cells by a mechanism dependent on PLC activation;52,53 and L-type calcium channels (LTCC).52-54 The palmitic acid activation of GPR40 is dependent upon high glucose concentrations. This suggests that GPR40 signaling requires calcium influx through LTCC that are pre-activated by glucose,



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resulting in increased calcium influx and augmented insulin secretion.53 The outward voltage-gated potassium currents are required for the β-cells membrane repolarization after glucose stimulation, limiting calcium influx and insulin secretion.55 Linoleic acid activation of GPR40 reduces the activity of the voltage-gated rectifier potassium channels, leading to reduced potassium conductance and prolonging the opening of the LTCC. This, in fact, prolongs the membrane depolarization period during action potential firing, elevating calcium influx and amplifying the insulin secretion induced by FA.56 Chronic effects of FA. Several mechanisms are involved in FA-induced impairment of insulin secretion. One of these mechanisms involve excessive ROS generation by mitochondrial and extramitochondrial sources such as NADPH oxidase, accompanied by reduction of antioxidant defense via glutathione depletion;57,58 FA-mediated NFκB activation;59 induction of iNOS (the induced form of nitric oxide synthase, encoded by the NOS-2 gene in humans), a process also associated with GPR40 signaling; 60 FA-derived ceramide production involved in β-cell death; 61 endoplasmic reticulum stress; 62 and induction of mitochondrial dysfunction.63 The metabolic effects of FA are also involved in b-cell dysfunction and induce the reduced glucose oxidation. In this process, the increased NADH production via FA β-oxidation inhibits islet pyruvate dehydrogenase activity, leading to a decrease in the conversion of pyruvate into acetyl CoA and promoting a reduction in glucose oxidation.21 As mentioned above, we focused here on the short-time effects of FA. Detailed information on chronic effects can be obtained in other sources.21,57,60,62-64

electrons for entry at complex I, NADH dehydrogenase, which is also known as NADH ubiquinone oxidoreductase; or complex II, succinate dehydrogenase, through acetyl-CoA metabolism in the TCA cycle. In addition, FA metabolism provides electrons through the electron-transport flavoprotein via FADH2, which is a product of β-oxidation, independent of the TCA cycle.72 The major sites of superoxide production within mitochondria derive from complex I and III.73 Complex I superoxide arises from bound flavin reduced FMNH2 by NADH and is released nearly exclusively to the matrix side of the inner membrane. Complex III is the cytochrome bc1 complex and the superoxide is generated during Q cycle, wherein coenzyme Q undergoes redox cycling through a reactive semiquinone species72 and generates superoxide to both the matrix and outward to the intermembrane and extramitochondrial space74 (Figs. 2 and 3). Superoxide production has been demonstrated to participate as a signal to acutely enhance insulin secretion by various stimuli, such as glucose and FA.1,3,4,75 More details of this signaling process induced by FA will be discussed in the section dealing with NADPH oxidase. Modulation of the electron transport. A general condition that favors mitochondrial superoxide generation is the highly reduced state of the electron carriers at specific sites. This enables the leak of electrons out of the enzymatic electron transport route (respiratory chain). Therefore, mitochondrial ROS production depends on the redox state of electron-donating centers of complexes I and III, such as flavin mononucleotide (FMN), Fe-S clusters and Q binding sites76 (Fig. 3). Enhanced cellular concentrations of LC-CoA, as observed in obesity, may increase transmembrane potential and shift the redox state of coenzyme Q toward more reduced values,77 thus promoting mitochondrial superoxide production. FA interact with components of the respiratory chain, thereby inhibiting electron transport.78 β-oxidation of FA supports reverse electron transport.79 In this mechanism, electrons are transferred from FA to the flavoprotein (electron transfer flavoprotein) and enter the respiratory chain at the coenzyme Q level, a reaction mediated by flavoprotein-quinone oxidoreductase, thus increasing the superoxide production associated to the complex I (Fig. 3). FA can interact with the respiratory chain by binding to cytochrome c in complex III,80 which interrupts the electron transport and contributes to enhance ROS production. In this way, FA might induce ROS production due to the depletion of cytochrome c from mitochondria, thereby interrupting the electron flow from complex III to complex IV (Fig. 3). The amphiphilic nature of FA favors their incorporation into phospholipid bilayers or mitochondrial membranes and alters the membrane fluidity. This may facilitate the electron leak from the inner mitochondrial membrane and one-electron reduction of O2.79 Ceramides are amides of long-chain FA and sphingosine, essential building blocks of sphingolipids and components of biological membranes. Ceramides have been recognized as important signaling molecules in cell proliferation, differentiation and, in particular, cell death. They stimulate respiratory chainassociated ROS production by depletion of mitochondrial


Mitochondrial ROS Production Induced by FA Biologically, ROS include the superoxide radical O2•-, hydrogen peroxide H2O2, and the hydroxyl radical, OH*. At the physiological pH, superoxide spontaneously dismutates, which is more efficiently performed by superoxide dismutase (Mn-SOD in the mitochondrial matrix, Cu/Zn-SOD in the cytosol) to form H2O2. H2O2 is converted to oxygen and water by catalase (CAT) and by glutathione peroxidase (GPx).65 ROS are involved in pathological conditions such as diabetes,6 cardiovascular diseases66-68 and neurodegeneration familial amyotrophic lateral sclerosis.69 ROS also play a role in physiological processes such as vascular smooth muscle function,70 insulin signaling pathway71 and insulin secretion,1-3 by acting as second messengers. Mitochondria are generally considered sources of cellular ROS, but are also recognized as organelles with a high capacity of antioxidative defense through Mn-superoxide dismutase (Mn-SOD), matrix glutathione and glutathione peroxidase (Fig. 2). Substrates for the tricarboxylic acid cycle (TCA cycle) enter the mitochondrial matrix through pyruvate dehydrogenase, carrier proteins or one of multiple shuttle mechanisms. The metabolism of the substrates results in electron donation to specific complexes or sites. Fatty acyl-CoAs enter through CPT-I for b-oxidation in pancreatic β-cells. FA oxidation generates

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©201 1L andesBi os c i enc e. Donotdi s t r i but e. Figure 2. ROS generating systems and antioxidants defenses in pancreatic islets. (1) Fatty acid modulation of NADPH oxidase catalyzes the superoxide production in phagocytic cells and pancreatic islets. DAG and calcium increases might be involved in PKC activation, which phosphorylates NADPH oxidase subunits, inducing the translocation of the cytoplasmic subunits to the plasma membrane core for the NADPH oxidase holoenzyme assembly. (2) The extracellular SOD (EC-SOD) dismutates superoxide to H2O2 that can diffuse through aquaporin channels (AQP) in the plasma membrane to elicit an intracellular signaling response. Superoxide can also initiate intracellular signaling by permeation of the plasma membrane through anion channels (Cl-channel-3, ClC-3). (3) Antioxidant defenses involve the cytosolic Cu/Zn-SOD (superoxide dismutase), the mitochondrial Mn-SOD and glutathione peroxidase (GPx) and catalase (CAT) in cytosolic, mitochondrial and peroxisomal compartments. (The size of the letters expresses differential expression of the antioxidant defenses in b-cells). Also, the GSH/GSSG (glutathione reduced/glutathione oxidized) ratio is an important antioxidant defense system in b-cells. Short and medium chain FA are exclusively b-oxidized in the mitochondria (4), whereas long-chain FA are b-oxidized in the mitochondria and the peroxisomes (5) and very long-chain FA are handled preferentially by peroxisomes. In mitochondria, the electrons are transferred to FAD; however, in peroxisomes, O2 is the electron acceptor that leads to formation of hydrogen peroxide. The complexes I and III are the main source of mitochondrial superoxide, and UCP2 acts as a negative regulator of mitochondria-derived ROS production.

cytochrome c and by direct inhibition of electron transport.81 Other mechanisms are associated to the mitochondrial ROS production by FA, such as the interaction with the antioxidant enzymes, as will be later discussed in the section on antioxidant defenses. β-oxidation of FA occurs in mitochondria and peroxisomes in higher eukaryotes. Short and medium chain (C4–C8) FA are exclusively β-oxidized in the mitochondria, whereas C10–C16 FA are β-oxidized in mitochondria and peroxisomes (C14–C16). Long and very long-chain (C17-C24) FA are handled preferentially by peroxisomes.82 In mitochondria, the electrons are transferred to flavin adenine nucleotide (FAD) and nicotinamide adenine dinucleotide (NAD +); however, in peroxisomes, O2 is

the electron acceptor that leads to formation of hydrogen peroxide. This reaction is catalyzed by specific peroxisomal acyl-CoA oxidase isoforms in rat and humans. So, peroxisomal FA metabolism may contribute to the production of hydrogen peroxide also in pancreatic islets.83 Uncoupling proteins. The mitochondrial membrane potential is generated by charge differences across the inner membrane, and that electrical potential is required for ATP production. This potential depends on substrate utilization and is generated by proton pumping at complexes I, III and IV and offset by proton transfer in the opposite direction, a process called proton leak. A prominent amount of proton leak is mediated by the uncoupling proteins (UCPs).84 UCP2 is the most ubiquitous form


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A signaling role of UCP2 could be important during the period of low glucose concentration, such as during sleep, when FA oxidation is enhanced, resulting in superoxide production.94,95 As a consequence, the mild uncoupling by UCP2 guarantees that the β-cell responds properly to the lack of glucose, as it will reduce insulin secretion despite the availability of an adequate substrate for ATP production.96 Moreover, UCP2 KO protects mice from fatty acid-induced impairment in GSIS.97 In physiological conditions with higher glucose and fatty acid concentrations, as after a meal, the superoxide induction of UCP decreases the proton motive force, which reduces potential oxidative damage but impairs insulin secretion.96 The transcription of the UCP2 gene is highly inducible under conditions of oxidative stress, such as those induced by lipopolysaccharide, free FA and high-fat diet.98-100 Superoxide and the lipid peroxidation product 4-hydroxy-2-nonenal both activate UCP2, increasing proton conductance in the mitochondrial inner membrane.79 A high fat ketogenic diet increases UCP2 mRNA and protein levels and reduces ROS production in the brain.101 Conversely, a low fat diet given to immature rats reduces UCP2 levels and increases ROS production and seizure-induced excitotoxicity.102 Thus, the ketogenic diet may be neuroprotective by diminishing ROS production through activation of UCP2 in the brain.101

Figure 3. ROS generation in the mitochondrial electron transport chain and the modulation by FA. The major sites of superoxide production within mitochondria derive from complex I and III of the respiratory chain. FA inhibit the electron transport within complexes I and III, thus facilitating the electron leak and enabling one-electron reduction of oxygen to superoxide. FA might induce ROS production due to the depletion of cytochrome c from mitochondria, thereby interrupting the electron flow from complex III to complex IV, increasing the reduction state of upstream electron carriers. Fum, fumarate; Succ, succinate; R FeS, Rieske iron-sulfur protein; cyt c1, cyt c, cyt a/a3: the respective cytochromes. Interactions of FA in electron transport are indicated by dotted lines. Solid lines show the direction of electron transfer. Adapted from P. Schönfeld, L. Wojtczak (2008).79


and it is also expressed in pancreatic β-cells.85 UCP1 dissipates caloric energy as heat, by uncoupling mitochondrial respiration from ATP production. However, UCP2 is not a physiologically relevant “uncoupling protein” like UCP1 and does not contribute to adaptive thermogenesis.86 UCPs are also able to promote the export of FA from mitochondria to the cytosolic compartment, thus protecting against FA overload. For example, the antioxidative activity of UCP3 decreases the matrix content of FA.87 UCPs transfer fatty acid peroxides from the inner to the outer leaflet of the inner mitochondrial membrane, thus extruding these highly toxic peroxidation products.88 UCPs can be activated by extramitochondrially generated superoxide89 and by superoxide generated in the mitochondrial matrix under nonphysiological90 and physiological conditions.91 UCP2 in β-cells decreases GSIS and the removal of UCP2 results in higher ATP levels and improved insulin secretion in mice islets.85 UCP2 functions as a negative regulator of mitochondriaderived ROS production. UCPs may respond to overproduction of matrix superoxide by catalyzing mild uncoupling, which lowers proton motive force and decreases superoxide production from electron transport chain, attenuating superoxide-mediated damage at the cost of slightly lowered efficiency of oxidative phosphorylation.92 UCP2 KO mice, on three highly congenic strain backgrounds, exhibit increased oxidative stress and decreased GSIS.93 Therefore, chronic absence of UCP2 may disrupt the adaptive response to oxidative stress.

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Modulation of NADPH Oxidase Activity by FA

The NADPH oxidase complex is well characterized in phagocytic cells, where it consists of the cytosolic components p47PHOX, p67PHOX, p40PHOX, a low molecular weight G-protein, Rac 1 or Rac 2, and the membrane-associated cytochrome b558, including gp91PHOX or NOX2 (the catalytic subunit, with the transmembrane redox chain) and p22PHOX. In phagocytic cells, PKC activates NADPH oxidase by a phosphorylation-dependent activation of p47PHOX, p67PHOX, and/or Rac.103 In these cells, activation of the NADPH oxidase complex requires translocation of the cytosolic components to the plasma membrane and their association with gp91PHOX /p22PHOX. p47PHOX phosphorylation leads to a conformational change in this molecule that allows the interaction with p22PHOX. The p47PHOX subunit translocation to the plasma membrane leads the p67PHOX into contact with gp91PHOX, bringing the p40PHOX subunit to the complex. Finally, the GTPase Rac interacts with gp91PHOX. This assembled complex is active and produces superoxide by electron transfer from NADPH to oxygen.103 Expression of the components of the NADPH oxidase complex and its homologues (NOX1, NOX4, NOXO1 and NOXA1) has been shown in pancreatic islets.104,105 We demonstrated the induction of p47PHOX translocation to plasma membrane by a glucose stimulus in pancreatic islets.104 Furthermore, NADPH oxidase has been identified in caveolae and lipid rafts in various cell types, such as coronary endothelial cells and vascular smooth muscle cells.106-108 These structures are cholesterol and sphingolipid-rich plasma membrane microdomains where multiple signaling molecules, including GPRs, tyrosine kinase receptors, PKC and G proteins are localized, promoting the compartmentalization of signaling.109

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In phagocytes, gp91PHOX can also be activated within the granules without the need of fusion with surface membranes. However, all NOX family members are transmembrane proteins that transport electrons across biological membranes to reduce oxygen to superoxide. NOX2 is a transmembrane redox chain that connects the electron donor, NADPH, on the cytosolic side of the membrane with the electron acceptor, oxygen, on the outer side of the membrane. It transfers electrons through a series of steps involving a FAD and two assymetrical hemes found in transmembrane domains III and V. Electrons are initially transferred from NADPH to FAD, a process that is regulated by the activation domain of p67PHOX. A single electron is transferred from the reduced flavin FADH2 to the iron center of the inner heme. The inner heme must donate its electron to the outer heme before the second electron can be accepted from the now partially reduced flavin, FADH. Oxygen must be bound to the outer heme to accept the electron.103 Overall, gp91PHOX transfers electrons from intracellular NADPH to extracellular oxygen, generating superoxide anion. The latter is dismutated to H2O2 by the extracellular SOD (EC-SOD). H2O2 can diffuse through aquaporin channels in the plasma membrane to elicit intracellular signaling responses. Superoxide can also initiate intracellular signaling by penetrating the cell membrane through anion channels (Cl-channel-3, ClC-3) 110 (Fig. 2). Short-time exposure to palmitate in the presence of low glucose concentration and pro-inflammatory cytokines increases superoxide production through NADPH oxidase activation in insulin-secreting cells and pancreatic islets.75 Acute palmitate induction of superoxide production in islets was demonstrated to be dependent on the activation of PKC and NADPH oxidase, causing p47 PHOX translocation to plasma membrane and upregulation of the p47PHOX protein content and of the p22PHOX, gp91PHOX and p47PHOX mRNA levels. Palmitic acid oxidation contributes to its induction of superoxide production in the presence of 5.6 mM glucose, as observed in experiments performed with a CPT-I irreversible inhibitor, etomoxir.4 This is probably a consequence of the electron transfer from FA to the flavoprotein that enters the respiratory chain at the coenzyme Q level and, through the reverse electron transport, increases the superoxide production associated to the complex I, as previously discussed. On the other hand, in pancreatic islets of Wistar rats fed with a high fat diet during 3 months, glucose metabolism is increased, insulin secretion elevated in the presence of high glucose levels, protein expression of NADPH oxidase subunits is reduced and both ROS production and apoptosis are diminished.111 The downregulation of the NADPH oxidase complex has a role in the compensation of the deleterious effect of lard, promoting the equilibrium of the cellular redox state (as demonstrated by the absence of differences in oxidative stress markers)111 to maintain insulin secretion and to avoid the initial event of insulin resistance in obese rats. FA stimulation of insulin secretion in the presence of high glucose concentration is reduced by inhibition of NADPH oxidase activity.4 In fact, NADPH oxidase inhibition has been associated with reduced glucose oxidation, calcium influx and

insulin secretion in high glucose concentrations.2,3 In cardiac myocytes, endotelin-1 increases ROS production via NADPH oxidase, which plays a key role in calcium influx through L-type calcium channels. This probably occurs due to the redox modification of cysteine residues on the cardiac L-type calcium channel.112 Chronic effects of palmitic and oleic acids inducing ROS production are restored by suppression of gp91PHOX,113 and the chronic effect of palmitate-induced reduction of GSIS is prevented by incubation of mouse islets with the NADPH oxidase inhibitor apocynin.114 On the other hand, the concomitant incubation of the unsaturated FA arachidonic acid with palmitic acid dose-dependently reduces the saturated FA induction of ROS and NO production in BRIN-BD11 cells. Arachidonic acid reduced the expression of iNOS, NFκB and p47PHOX and improved GSH/ GSSG ratio in palmitic acid-treated cells.115 As mentioned, fatty acid-induced ROS production for short time stimulates insulin secretion and, in chronic stimulation, reduces GSIS. There is possibly an optimal range of ROS content to maintain β-cells function, and a variation of the redox state due to excessive ROS formation or marked ROS reduction results in reduced insulin secretion. The renin-angiotensin system and NADPH oxidase. The pancreatic islets are exposed not only to systemic but also to locally produced components of the renin-angiotensin system (RAS). RAS, in conditions such as obesity and diabetes mellitus type 2, is inappropriately upregulated. Angiotensinogen, the angiotensin converting enzyme (ACE), and the angiotensin receptors 1 and 2 (AT-1 and AT-2) have all been found in rodent pancreatic islets.116-118 In the human pancreas, renin precursors and AT-1 have been found in β-cells, as well as in endothelial cells of the pancreatic vasculature.119 Increasing concentrations of angiotensin II impairs GSIS in a dose-dependent manner in mouse islets and this effect is abolished by losartan, an AT-1 receptor antagonist.116,120 Angiotensin II induces a dose-dependent superoxide generation via NADPH oxidase activation and increases protein and mRNA levels of NADPH oxidase subunits (p47PHOX and gp91PHOX).121 Hyperglycemia per se can activate RAS in human islets, and under high glucose concentrations, angiotensin converting enzyme inhibitors exert beneficial effects on β-cell, improving insulin secretion and reducing nitrotyrosine and p22PHOX mRNA levels.122 Thus, RAS triggers the production of ROS via NADPH oxidase complex in pancreatic islets, regulating insulin secretion. Obesity-induced diabetes mellitus type 2 in db/db mice has been related to b-cell dysfunction, likely via activation of pancreatic RAS and upregulation of AT-1 receptors in the pancreas. Angiotensin II receptor blockers (ARBs) increase both insulin production and secretion. Furthermore, hyperglycemia, glucose intolerance, and the onset of diabetes are delayed by ARBs, without affecting insulin resistance.123 ARBs have received a great deal of attention as therapeutic tools for obesity-related metabolic disorders. The increased expression of p22PHOX and gp91PHOX is correlated with increased oxidative stress in islets of the type 2 diabetes models, OLEFT



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rats and db/db mice. The inhibitory effect of AT1 ARB valsartan on the expression of these components in islets of db/db mice occurs concomitantly with a reduction of oxidative stress and preservation of insulin content.124 Telmisartan, another ARB, improved insulin sensitivity and reduced the incidence of type 2 diabetes in patients with hypertension.125 In rats fed a high-fat and high-carbohydrate diet, telmisartan was shown to reduce weight gain and significantly reduce the levels of plasma glucose, insulin and triglycerides.126 The ARB irbesartan attenuates oxidative stress in islets of Zucker diabetic fat rats and this process may be a consequence of RAS blockade, inhibiting NADPH oxidase activation.127 The ARBs candesartan and telmisartan both decrease palmitate-induced ROS accumulation in MIN6 cells and in mouse islets, an effect dependent on PKC and NADPH oxidase activation.128,129 Also, the treatment with candesartan recovered palmitate-induced decrease of intracellular insulin content.129 FA and RAS activation modulate common intracellular signals, such as NADPH oxidase activation, that control b-cell function. This could explain the positive effects of ARBs in high-fat diet and diabetic conditions. Free FA modulation of Rac1 activation by Tiam1. The activation of small Rho-GTPase proteins, including Rac1, depends on the change of the bound GDP to GTP. This process is regulated by several factors, such as Rho GDIs (GDP dissociation inhibitor), GEFs (guanine nucleotide exchange factors) and GAPs (GTPase-activating proteins).69 Several proteins play these three roles in different situations and tissues. In pancreatic islets, the main GEF responsible to activate rac1 is Tiam1.130 Tiam1 (T-lymphoma invasion and metastasis) is a GEF involved in many cellular process, but in β-cell it is associated with GSIS and activation of NADPH oxidase complex.130,131 As NOX2 is a ROS producer, the activity of Tiam1, and consequently of Rac1, can be related to β-cells dysfunction caused by oxidative stress. The incubation of the β-cell line INS-1 832/13 with palmitate causes activation of Tiam1/Rac1 and ROS production by NOX2. This palmitate action is mediated by ceramide. The use of a pharmacological inhibitor of Tiam1 (NSC23766) reduces this response, and the dysfunction generated by palmitate in pancreatic b-cells possibly occurs via a Tiam1-Rac1 signaling pathway.132

not only for rodent pancreatic islets but also for the rat β-cell lineage RINm5F.135 In 2004, the level of expression of glutamylcysteine ligase (GCLC), the enzyme that regulates the de novo synthesis of glutathione, was shown in pancreatic islets to be similar to other tissues.136 The reasons for this islet low antioxidant defense are still being debated. Why such important cells are so vulnerable to reactive oxygen species? A co-evolution of β-cells, brain cells and corticosteroids and cortisol receptors may be at the origin of this peculiarity of the endocrine pancreas.137 The initial clues were obtained by the fact that both human and rodent females present low antioxidant capacity in pancreatic islets.138,139 The fact that during pregnancy females present insulin resistance provides an indication as to why evolution maintained low levels of antioxidant enzymes in β-cells.137 The insulin resistance progresses slowly during pregnancy, supplying extra circulating glucose according to the fetus needs.140 The molecular mechanism that prevents the excess of insulin secretion in response to peripheral insulin resistance is augmented ROS content.137 The brain needs a constant supply of glucose and cannot store it.141 In the same line of reasoning, cortisol and corticosteroid induce insulin resistance to guarantee the fuel supply necessary to the central nervous system during stress.137 However, ROS are involved in insulin secretion, especially H2O2 at low concentrations.1,3,142 In this regard, there must be other physiological reasons for the low antioxidant capacity of pancreatic β-cells. Besides the fact that insulin-producing cells have poor antioxidant defenses and that this fact must have an adaptive meaning, the environmental conditions offered by the current style of life causes chronic stress, glucolipotoxicity, oxidative stress and consequent β-cell failure. Chronic high glucose levels (30 mM) augment the expression of MnSOD in β-cells.135 Chronic treatment with the most common free FA in our diet, palmitate and oleate, modulates the levels of antioxidant enzymes in pancreatic islets. Vandewalle and coworkers found that the incubation of human pancreatic islets with palmitate 0.33 mM for 48 h diminishes the mRNA levels of GPx and SOD. The treatment with 1 μM of rosiglitazone abolished this effect; however, it was not dependent on PPAR-γ.143 In human pancreatic islets, Bikopoulos and co-workers found that chronic incubation (48 h) with 0.4 mM oleate augments mRNA levels of catalase, metallothionein 1F and sequestosome 1, molecules involved in the redox balance.144 The differences between treatments may be due to the different actions of the two free FA. In opposition to palmitate, which presents deleterious effects, oleate is considered less damaging and even protective for islet cells.5


Modulation of the Antioxidant Enzyme Activities in Pancreatic Islets by Free FA The pancreatic islets are particularly susceptible to oxidative stress. In the beginning of the 1980 decade, the activity of antioxidant enzymes GPx, catalase, mitochondrial Mn-SOD and cytosolic Cu/Zn-SOD was shown to be low in the endocrine pancreas when compared to other tissues.133 In 1996, the Lenzen’s group reported that pancreatic islets have considerably low mRNA levels of antioxidant enzymes in comparison with the liver, kidney and brain. The islets present, in comparison to the liver, about 15% of GPx, 38% of Cu/Zn-SOD, 30% of Mn-SOD and very low expression of CAT 134 (Fig. 2). In the following year, this research group showed that the same was true

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Concluding Remarks Several mechanisms for FA induction of ROS production by pancreatic β-cells (mitochondrial complexes and electron transport, NADPH oxidase, antioxidant defenses) were discussed herein. The balance between ROS production and consumption defines an optimal range of ROS concentration to maintain b-cell function. The high susceptibility of pancreatic islets to

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oxidative stress is another factor that contributes to tissue dysfunction caused by prolonged exposure to FA. In this review, some therapeutic strategies to prevent β-cell dysfunction, such as the use of antioxidant agents, modulation of UCP2 and regulation of pro-oxidative signaling factors, such as NADPH oxidase complex and RAS (both of which are common intracellular signaling pathways modulated by FA), were indicated. Nevertheless, more studies are undoubtedly necessary to clarify the molecular mechanisms triggered by FA and involved in the redox balance in pancreatic β-cells. References 1.

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Acknowledgments

Thanks are due to Dr. Luiz R.G. Britto (University of São Paulo) and Dr. Mauro Leonelli (University of São Paulo) for critically reading the manuscript. Financial Support

Our studies have been supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), the Instituto Nacional de Obesidade e Diabetes (INCT) and the US Department of Veterans Affairs.

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Islets

Volume 3 Issue 5

104. Oliveira HR, Verlengia R, Carvalho CR, Britto LR, Curi R, Carpinelli AR. Pancreatic beta-cells express phagocyte-like NAD(P)H oxidase. Diabetes 2003; 52:1457-63. 105. Uchizono Y, Takeya R, Iwase M, Sasaki N, Oku M, Imoto H, et al. Expression of isoforms of NADPH oxidase components in rat pancreatic islets. Life Sci 2006; 80:133-9. 106. Vilhardt F, van Deurs B. The phagocyte NADPH oxidase depends on cholesterol-enriched membrane microdomains for assembly. EMBO J 2004; 23:739-48. 107. Zhang AY, Yi F, Zhang G, Gulbins E, Li PL. Lipid raft clustering and redox signaling platform formation in coronary arterial endothelial cells. Hypertension 2006; 47:74-80. 108. Hilenski LL, Clempus RE, Quinn MT, Lambeth JD, Griendling KK. Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2004; 24:677-83. 109. Cohen AW, Razani B, Schubert W, Williams TM, Wang XB, Iyengar P, et al. Role of caveolin-1 in the modulation of lipolysis and lipid droplet formation. Diabetes 2004; 53:1261-70. 110. Fisher AB. Redox signaling across cell membranes. Antioxid Redox Signal 2009; 11:1349-56. 111. Valle MM, Graciano MF, Lopes de Oliveira ER, Camporez JP, Akamine EH, Carvalho CR, et al. Alterations of NADPH oxidase activity in rat pancreatic islets induced by a high-fat diet. Pancreas 2011; 40:390-5. 112. Mikami A, Imoto K, Tanabe T, Niidome T, Mori Y, Takeshima H, et al. Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel. Nature 1989; 340:230-3. 113. Yuan H, Zhang X, Huang X, Lu Y, Tang W, Man Y, et al. NADPH oxidase 2-derived reactive oxygen species mediate FFAs-induced dysfunction and apoptosis of beta-cells via JNK, p38 MAPK and p53 pathways. PLoS One 5:15726. 114. Michalska M, Wolf G, Walther R, Newsholme P. Effects of pharmacological inhibition of NADPH oxidase or iNOS on pro-inflammatory cytokine, palmitic acid or H2O2-induced mouse islet or clonal pancreatic beta-cell dysfunction. Biosci Rep 2010; 30:445-53. 115. Keane DC, Takahashi HK, Dhayal S, Morgan NG, Curi R, Newsholme P. Arachidonic acid actions on functional integrity and attenuation of the negative effects of palmitic acid in a clonal pancreatic beta-cell line. Clin Sci (Lond) 2011; 120:195-206. 116. Lau T, Carlsson PO, Leung PS. Evidence for a local angiotensin-generating system and dose-dependent inhibition of glucose-stimulated insulin release by angiotensin II in isolated pancreatic islets. Diabetologia 2004; 47:240-8. 117. Leung PS, Chappell MC. A local pancreatic reninangiotensin system: endocrine and exocrine roles. Int J Biochem Cell Biol 2003; 35:838-46. 118. Leung PS, Carlsson PO. Tissue renin-angiotensin system: its expression, localization, regulation and potential role in the pancreas. J Mol Endocrinol 2001; 26:155-64.

119. Tahmasebi M, Puddefoot JR, Inwang ER, Vinson GP. The tissue renin-angiotensin system in human pancreas. J Endocrinol 1999; 161:317-22. 120. Carlsson PO, Berne C, Jansson L. Angiotensin II and the endocrine pancreas: effects on islet blood flow and insulin secretion in rats. Diabetologia 1998; 41:127-33. 121. Hirata AE, Morgan D, Oliveira-Emilio HR, Rocha MS, Carvalho CR, Curi R, et al. Angiotensin II induces superoxide generation via NAD(P)H oxidase activation in isolated rat pancreatic islets. Regul Pept 2009; 153:1-6. 122. Lupi R, Del Guerra S, Bugliani M, Boggi U, Mosca F, Torri S, et al. The direct effects of the angiotensin-converting enzyme inhibitors, zofenoprilat and enalaprilat, on isolated human pancreatic islets. Eur J Endocrinol 2006; 154:355-61. 123. Chu KY, Lau T, Carlsson PO, Leung PS. Angiotensin II type 1 receptor blockade improves beta-cell function and glucose tolerance in a mouse model of type 2 diabetes. Diabetes 2006; 55:367-74. 124. Nakayama M, Inoguchi T, Sonta T, Maeda Y, Sasaki S, Sawada F, et al. Increased expression of NAD(P)H oxidase in islets of animal models of Type 2 diabetes and its improvement by an AT1 receptor antagonist. Biochem Biophys Res Commun 2005; 332:927-33. 125. Vitale C, Mercuro G, Castiglioni C, Cornoldi A, Tulli A, Fini M, et al. Metabolic effect of telmisartan and losartan in hypertensive patients with metabolic syndrome. Cardiovasc Diabetol 2005; 4:1-8. 126. Benson SC, Pershadsingh HA, Ho CI, Chittiboyina A, Desai P, Pravenec M, et al. Identification of telmisartan as a unique angiotensin II receptor antagonist with selective PPARgamma-modulating activity. Hypertension 2004; 43:993-1002. 127. Tikellis C, Wookey PJ, Candido R, Andrikopoulos S, Thomas MC, Cooper ME. Improved islet morphology after blockade of the rennin-angiotensin system in the ZDF rat. Diabetes 2004; 53:989-97. 128. Saitoh Y, Hongwei W, Ueno H, Mizuta M, Nakazato M. Telmisartan attenuates fatty-acid-induced oxidative stress and NAD(P)H oxidase activity in pancreatic beta-cells. Diabetes Metab 2009; 35:392-7. 129. Saitoh Y, Hongwei W, Ueno H, Mizuta M, Nakazato M. Candesartan attenuates fatty acid-induced oxidative stress and NAD(P)H oxidase activity in pancreatic beta-cells. Diabetes Res Clin Pract 2010; 90:54-9. 130. Veluthakal R, Madathilparambil SV, McDonald P, Olson LK, Kowluru A. Regulatory roles for Tiam1, a guanine nucleotide exchange factor for Rac1, in glucose-stimulated insulin secretion in pancreatic betacells. Biochem Pharmacol 2009; 77:101-13. 131. Mertens AE, Roovers RC, Collard JG. Regulation of Tiam1-Rac signalling. FEBS Lett 2003; 546:11-6. 132. Syed I, Jayaram B, Subasinghe W, Kowluru A. Tiam1/ Rac1 signaling pathway mediates palmitate-induced, ceramide-sensitive generation of superoxides and lipid peroxides and the loss of mitochondrial membrane potential in pancreatic beta-cells. Biochem Pharmacol 2010; 80:874-83.

133. Grankvist K, Marklund SL, Taljedal IB. CuZnsuperoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase in pancreatic islets and other tissues in the mouse. Biochem J 1981; 199:393-8. 134. Lenzen S, Drinkgern J, Tiedge M. Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues. Free Radic Biol Med 1996; 20:463-6. 135. Tiedge M, Lortz S, Drinkgern J, Lenzen S. Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells. Diabetes 1997; 46:1733-42. 136. Tran PO, Parker SM, LeRoy E, Franklin CC, Kavanagh TJ, Zhang T, et al. Adenoviral overexpression of the glutamylcysteine ligase catalytic subunit protects pancreatic islets against oxidative stress. J Biol Chem 2004; 279:53988-93. 137. Rashidi A, Kirkwood TB, Shanley DP. Metabolic evolution suggests an explanation for the weakness of antioxidant defences in beta-cells. Mech Ageing Dev 2009; 130:216-21. 138. Tonooka N, Oseid E, Zhou H, Harmon JS, Robertson RP. Glutathione peroxidase protein expression and activity in human islets isolated for transplantation. Clin Transplant 2007; 21:767-72. 139. Cornelius JG, Luttge BG, Peck AB. Antioxidant enzyme activities in IDD-prone and IDD-resistant mice: a comparative study. Free Radic Biol Med 1993; 14:409-20. 140. Ladyman SR, Augustine RA, Grattan DR. Hormone interactions regulating energy balance during pregnancy. J Neuroendocrinol 2010; 22:805-17. 141. Fehm HL, Kern W, Peters A. The selfish brain: competition for energy resources. Prog Brain Res 2006; 153:129-40. 142. Rebelato E, Abdulkader F, Curi R, Carpinelli AR. Low doses of hydrogen peroxide impair glucose-stimulated insulin secretion via inhibition of glucose metabolism and intracellular calcium oscillations. Metabolism 2010; 59:409-13. 143. Vandewalle B, Moerman E, Lefebvre B, Defrance F, Gmyr V, Lukowiak B, et al. PPARgamma-dependent and -independent effects of rosiglitazone on lipotoxic human pancreatic islets. Biochem Biophys Res Commun 2008; 366:1096-101. 144. Bikopoulos G, da Silva Pimenta A, Lee SC, Lakey JR, Der SD, Chan CB, et al. Ex vivo transcriptional profiling of human pancreatic islets following chronic exposure to monounsaturated fatty acids. J Endocrinol 2008; 196:455-64.



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