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www.landesbioscience.com Islets 213
Islets 3:5, 213-223; September/October 2011; ©2011 Landes Bioscience
REVIEW REVIEW
Reactive oxygen species (ROS) are important components of
intracellular redox signaling cascades. They are produced by the
metabolism of different substrates such as fatt y acids (FA) and glu-
cose. Mitochondrial production of ROS plays an important role
*Correspondence to: Maria Fernanda Rodrigues Graciano;
Email: mafe@icb.usp.br
Submi tted: 04/15/11; Revised: 06/15/11; Accepted: 06/17/11
DOI: 10.4161/isl.3.5.15935
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 uidity
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.
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
1Departm ent of Physiology and Biop hysics; Institute of Biom edical Sciences; Unive rsity of São Paulo; São Pau lo, SP Brazil;
2Depart ment of Pharmaceutical S ciences; Eugene Appleb aum College of Pharmac y; Wayne State University ; Detroit, MI USA;
3β-Cell Bioch emistry Research L aboratory; John D. D ingell VA Medical Center; De troit, 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; R AS, renin-angiotensin system; ROS,
reactive oxygen species; SNPs, single nucleotide polymorphisms; SOD, superoxide dismutase; TAG, triacylglycerol; TCA cycle,
tricarboxylic acid cycle; UCP, uncoupling protein
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 cor-
related 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-medi-
ated 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 aug-
ments 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 display-
ing 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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214 Islets Volume 3 Issue 5
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 dehy-
drogenase 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 cyto-
solic substrate flux via lactate dehydrogenase (LDH) is limited by
the low activity of this enzyme in pancreatic islets.26 The cataple-
rotic 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 synthe-
sis of malonyl-CoA. Factors that lead to PDH inhibition, there-
fore, 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
the rapid inhibition of caspase 3 and the reduction of endoplas-
mic 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 carni-
tine palmitoyltransferase I (CPT-I), thereby promoting FA re-
esterification. As a result, there is no suppression of CPT-I activ-
ity 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 exter-
nal FA supply to counterbalance the deficit.22 When the glu-
cose level is low, FA are converted into LC-CoAs by acyl-CoA
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 carbox ylase (PC) in pancreatic islets. The cataplerotic activity of PDH, in association with ux 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-esterication. High glucose concentrations raise malonyl- CoA levels and decrease FA oxidation, which leads to LC-CoA accumulation.
LC-CoA may additionally be esteried 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—KATP closure and calcium inux—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 chan-
nels; PL, phospholipids.
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www.landesbioscience.com Islets 215
(arginine to histidine) at the position 211 in the third intracellu-
lar loop of the receptor that was reported to cause enhanced insu-
lin secretion in Japanese men.40 The other SNP also produces an
amino acid substitution (Gly180Ser),41 and the less frequent vari-
ant (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 con-
cluded 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 lipo-
proteins.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 regula-
tion of insulin secretion through generation of FA or other lipid-
signaling 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 stabiliz-
ing 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 exocy-
tosis.29,4 9,50 FA have also been proposed to regulate ionic chan-
nels 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 express-
ing 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 activa-
tion;52,53 and L-type calcium channels (LTCC).52-54 The palmitic
acid activation of GPR40 is dependent upon high glucose con-
centrations. This suggests that GPR40 signaling requires cal-
cium influx through LTCC that are pre-activated by glucose,
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 diacylglyc-
erol (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—ATP sensitive-potassium channels (KATP )
closure and calcium influx—to promote full insulin secretion30
(Fig. 1).
GPR40 activation and production of lipid-signaling mol-
ecules. The GPR (G protein coupled receptor) isoforms 40, 41
and 43 are highly homologous but differ in substrate specific-
ity 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,3 6,37 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 phosphatidylinosi-
tol-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 knock-
out (KO) mice.38 Lan and collaborators (2008) also demon-
strated 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
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216 Islets Volume 3 Issue 5
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 mitochon-
dria 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 pro-
cess 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 pro-
duction 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 flavo-
protein) 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 phospho-
lipid bilayers or mitochondrial membranes and alters the mem-
brane 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 bio-
logical membranes. Ceramides have been recognized as impor-
tant signaling molecules in cell proliferation, differentiation
and, in particular, cell death. They stimulate respiratory chain-
associated ROS production by depletion of mitochondrial
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 acti-
vation 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 fir-
ing, 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 mitochon-
drial and extramitochondrial sources such as NADPH oxidase,
accompanied by reduction of antioxidant defense via glutathi-
one 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 mito-
chondrial 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 men-
tioned 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
Mitochondrial ROS Production Induced by FA
Biologically, ROS include the superoxide radical O2
•-, hydrogen
peroxide H2O2, and the hydroxyl radical, OH*. At the physio-
logical 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 diabe-
tes,6 cardiovascular diseases66-68 and neurodegeneration familial
amyotrophic lateral sclerosis.69 ROS also play a role in physiologi-
cal 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 dis-
mutase (Mn-SOD), matrix glutathione and glutathione peroxi-
dase (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 spe-
cific complexes or sites. Fatty acyl-CoAs enter through CPT-I
for b-oxidation in pancreatic β-cells. FA oxidation generates
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www.landesbioscience.com Islets 217
the electron acceptor that leads to formation of hydrogen perox-
ide. This reaction is catalyzed by specific peroxisomal acyl-CoA
oxidase isoforms in rat and humans. So, peroxisomal FA metabo-
lism may contribute to the production of hydrogen peroxide also
in pancreatic islets.83
Uncoupling proteins. The mitochondrial membrane poten-
tial 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 uncou-
pling proteins (UCPs).84 UCP2 is the most ubiquitous form
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 preferen-
tially by peroxisomes.82 In mitochondria, the electrons are trans-
ferred to flavin adenine nucleotide (FAD) and nicotinamide
adenine dinucleotide (NAD+); however, in peroxisomes, O2 is
Figure 2. ROS generating systems and antioxidants defenses in pancreatic islets. (1) Fatty acid modulation of NADPH oxidase catalyzes the superoxide
production in phagocy tic 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 diuse 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 cy tosolic, mitochondrial and peroxisomal compar tments. (The size of the letters expresses dierential expres-
sion 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 mito-
chondria 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.
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218 Islets Volume 3 Issue 5
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 physi-
ological conditions with higher glucose and fatty acid concentra-
tions, as after a meal, the superoxide induction of UCP decreases
the proton motive force, which reduces potential oxidative dam-
age but impairs insulin secretion.96
The transcription of the UCP2 gene is highly inducible under
conditions of oxidative stress, such as those induced by lipopoly-
saccharide, free FA and high-fat diet.98-10 0 Superoxide and the lipid
peroxidation product 4-hydroxy-2-nonenal both activate UCP2,
increasing proton conductance in the mitochondrial inner mem-
brane.79 A high fat ketogenic diet increases UCP2 mRNA and pro-
tein 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
Modulation of NADPH Oxidase Activity by FA
The NADPH oxidase complex is well characterized in phago-
cytic 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, includ-
ing g p91PHOX or NOX2 (the catalytic subunit, with the trans-
membrane redox chain) and p22PHOX. In phagocytic cells, PKC
activates NADPH oxidase by a phosphorylation-dependent acti-
vation of p47PHOX, p67PHOX, and/or Rac.103 In these cells, activa-
tion of the NADPH oxidase complex requires translocation of the
cytosolic components to the plasma membrane and their associa-
tion 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, bring-
ing the p40P HOX subunit to the complex. Finally, the GTPase Rac
interacts with gp91PHOX. This assembled complex is active and pro-
duces superoxide by electron transfer from NADPH to oxygen.103
Expression of the components of the NADPH oxidase com-
plex 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 sphingo-
lipid-rich plasma membrane microdomains where multiple sig-
naling molecules, including GPRs, tyrosine kinase receptors,
PKC and G proteins are localized, promoting the compartmen-
talization of signaling.109
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 mito-
chondria 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 mito-
chondrial membrane, thus extruding these highly toxic peroxida-
tion 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 mitochondria-
derived ROS production. UCPs may respond to overproduction
of matrix superoxide by catalyzing mild uncoupling, which low-
ers proton motive force and decreases superoxide production from
electron transport chain, attenuating superoxide-mediated dam-
age at the cost of slightly lowered efficiency of oxidative phos-
phorylation.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.
Figure 3. ROS generation in the mitochondrial elec tron 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 elec tron transport within complexes I and III, thus
facilitating the electron leak and enabling one-electron reduction of
oxygen to superoxide. FA might induce ROS produc tion due to the
depletion of c ytochrome c from mitochondria, thereby interrupting the
electron ow from complex III to complex IV, increasing the reduction
state of upstream electron carriers. Fum, fumarate; Succ, succinate;
R FeS, Rieske iron-sulfur protein; cy t 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
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www.landesbioscience.com Islets 219
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 modi-
fication 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 pre-
vented by incubation of mouse islets with the NADPH oxidase
inhibitor apocynin.114 On the other hand, the concomitant incu-
bation 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). R AS, in conditions such as obesity and diabetes mel-
litus 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 abol-
ished by losartan, an AT-1 receptor antagonist.116 ,12 0 Angiotensin
II induces a dose-dependent superoxide generation via NADPH
oxidase activation and increases protein and mRNA lev-
els of NADPH oxidase subunits (p47PH OX and g p91PHOX).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, improv-
ing 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 pan-
creatic 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, with-
out affecting insulin resistance.123
ARBs have received a great deal of attention as therapeu-
tic tools for obesity-related metabolic disorders. The increased
expression of p22PHOX a nd gp91P HOX is correlated with increased
oxidative stress in islets of the type 2 diabetes models, OLEFT
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 ini-
tially transferred from NADPH to FAD, a process that is regu-
lated 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, g p91PHOX trans-
fers 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 intracellu-
lar signaling responses. Superoxide can also initiate intracellular
signaling by penetrating the cell membrane through anion chan-
nels (Cl-channel-3, ClC-3) 110 (Fig. 2).
Short-time exposure to palmitate in the presence of low glu-
cose 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 oxi-
dase, causing p47PHOX translocation to plasma membrane and
upregulation of the p47PHOX protein content and of the p22PHOX,
gp91PHOX and p47P HOX mRNA levels. Palmitic acid oxidation
contributes to its induction of superoxide production in the
presence of 5.6 mM glucose, as observed in experiments per-
formed 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 lev-
els, 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 resis-
tance in obese rats.
FA stimulation of insulin secretion in the presence of high
glucose concentration is reduced by inhibition of NADPH oxi-
dase activity.4 In fact, NADPH oxidase inhibition has been
associated with reduced glucose oxidation, calcium influx and
©2011 Landes Bioscience.
Do not distribute.
220 Islets Volume 3 Issue 5
not only for rodent pancreatic islets but also for the rat β-cell
li nea ge RINm5F.135 In 2004, the level of expression of glutamyl-
cysteine 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 pres-
ent 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 anti-
oxidant 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 periph-
eral 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 anti-
oxidant defenses and that this fact must have an adaptive mean-
ing, 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 treat-
ments 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
Concluding Remarks
Several mechanisms for FA induction of ROS production by
pancreatic β-cells (mitochondrial complexes and electron trans-
port, NADPH oxidase, antioxidant defenses) were discussed
herein. The balance between ROS production and consump-
tion defines an optimal range of ROS concentration to maintain
b-cell function. The high susceptibility of pancreatic islets to
rats and db /db mice. The inhibitory effect of AT1 ARB val-
sartan on the expression of these components in islets of
db/db mice occurs concomitantly with a reduction of oxidative
stress a nd preservation of insul in content.124 Telmi sarta n, 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 candes-
artan 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 activa-
tion 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 dia-
betic 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 pro-
cess 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 tis-
sues. 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 com-
plex.130,131 As NOX2 is a ROS producer, the activity of Tiam1,
and consequently of Rac1, can be related to β-cells dysfunc-
tion 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 medi-
ated 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
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 endo-
crine 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 fol-
lowing year, this research group showed that the same was true
©2011 Landes Bioscience.
Do not distribute.
www.landesbioscience.com Islets 221
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.
oxidative stress is another factor that contributes to tissue dys-
function 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 intra-
cellular 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.
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