Am J Physiol Cell Physiol 317: C420–C433, 2019.

First published June 19, 2019; doi:10.1152/ajpcell.00141.2019.

REVIEW Metabolism, Oxidative Stress and Cell Signaling

Oxidative stress pathways in pancreatic ␤-cells and insulin-sensitive cells and

tissues: importance to cell metabolism, function, and dysfunction
Philip Newsholme,1 Kevin N. Keane,1 Rodrigo Carlessi,1 and Vinicius Cruzat2
1
School of Pharmacy and Biomedical Sciences, and Curtin Health Innovation Research Institute, Curtin University, Perth,
Western Australia, Australia; and 2Faculty of Health, Torrens University Australia, Melbourne, Victoria, Australia
Submitted 24 April 2019; accepted in final form 12 June 2019

Newsholme P, Keane KN, Carlessi R, Cruzat V. Oxidative stress pathways in

pancreatic ␤-cells and insulin-sensitive cells and tissues: importance to cell metab-
olism, function, and dysfunction. Am J Physiol Cell Physiol 317: C420 –C433,
2019. First published June 19, 2019; doi:10.1152/ajpcell.00141.2019.—It is now
accepted that nutrient abundance in the blood, especially glucose, leads to the
generation of reactive oxygen species (ROS), ultimately leading to increased
oxidative stress in a variety of tissues. In the absence of an appropriate compen-
satory response from antioxidant mechanisms, the cell, or indeed the tissue,
becomes overwhelmed by oxidative stress, leading to the activation of intracellular
stress-associated pathways. Activation of the same or similar pathways also appears
to play a role in mediating insulin resistance, impaired insulin secretion, and late
diabetic complications. The ability of antioxidants to protect against the oxidative
stress induced by hyperglycemia and elevated free fatty acid (FFA) levels in vitro
suggests a causative role of oxidative stress in mediating the latter clinical
conditions. In this review, we describe common biochemical processes associated
with oxidative stress driven by hyperglycemia and/or elevated FFA and the
resulting clinical outcomes: ␤-cell dysfunction and peripheral tissue insulin resis-
tance.
manganese superoxide dismutase; oxygen species; superoxide dismutase-2; uncou-
pling protein

INTRODUCTION tive stress due to enhanced glucose metabolism and associated

Overview of the Development of Type 1 and Type 2 Diabetes
Type 1 diabetes (T1DM) is characterized by inflammation of
the pancreatic islets, ␤-cell dysfunction, and death, dependent
on proinflammatory cytokine and other mediators released
from infiltrating immune cells, including powerful free radical
species such as superoxide and nitric oxide (NO) (120). The
immune mediated attack on the ␤-cells is a very specific type
of autoimmunity. In contrast, type 2 diabetes (T2DM) is
characterized by insulin resistance, excessive hepatic glucose
production, and decreased insulin secretion. Insulin resistance
most often precedes the onset of type 2 diabetes, is present in
many of the general population, and is multifactorial (56, 93).
Reactive oxygen species (ROS) generation is critical to the
dysfunction of the pancreatic ␤-cell and the development of
insulin resistance (137) and is discussed in detail in this review.
Early insulin resistance is initially overcome by hyperinsulin-
emia, so normalizing glucose levels in the blood. However,
persistently elevated glucose may result in intracellular oxida-
Address for reprint requests and other correspondence: P. Newsholme,
School of Pharmacy and Biomedical Sciences, Curtin University, Perth, WA,
Australia, 6102 (e-mail: philip.newsholme@curtin.edu.au).
C420 0363-6143/19 Copyright © 2019 the American Physiological Society
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mitochondrial ROS production, especially in the pancreatic
␤-cell but also in many insulin-sensitive tissues (120), nonen-
zymatic glycation of proteins, and glucose autoxidation (120).
Oxidative stress may overwhelm antioxidant responses, acti-
vating stress-sensitive cellular signaling pathways, as de-
scribed below.
Oxidative Stress and Diabetes: An Overview
Although oxygen is responsible for the expansion of life on
Earth, there are two sides to this molecule: it is essential to
sustain energy generation in the mitochondria of cells, but it
can be damaging if not fully reduced to water. Oxygen in the
air is a relatively nonreactive chemical, but when exposed to
high-energy electron-transferring chemical reactions where re-
duction is incomplete, it can be converted to various highly
reactive chemical forms collectively designated ROS. In mam-
malian cells, a delicate balance exists between the generation
of reactive oxygen and reactive nitrogen species (ROS and
RNS, respectively), and the removal of such molecules through
the action of antioxidants, which may be either chemical [e.g.,
glutathione (GSH)] or enzymatic in structure [e.g., catalase
(CAT)]. ROS and RNS are generated as products of normal
cell metabolism and respiration and are absolutely required for
appropriate cellular function. For example, hydrogen peroxide
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 OXIDATIVE STRESS, CELL METABOLISM AND FUNCTION
(H2O2) is a central redox signaling molecule that can regulate
cell proliferation, migration, and differentiation via an influ-
ence on signal pathway transduction and posttranslational
modifications such as sumoylation (166). Depending on the
location of generation of the ROS and RNS, once the endog-
enous ROS/RNS levels exceed the subcellular buffering anti-
oxidant capacity of the cell, the localized excess reactive
species alter the function of various biological molecules such
as proteins, lipids, and DNA through chemical modification
including nitrosylation, peroxidation, and carbonylation, thus
promoting dysfunction. This is discussed further below in the
sections on Superoxide-Generating Systems in Pancreatic Is-
lets and Other ROS Species and Their Sources. Ultimately, this
disturbance in redox homeostasis, resulting in oxidative stress,
promotes impaired function of tissues and organs, which leads
to chronic disorders at the whole body level. While the level of
ROS/RNS can be increased by exogenous sources such as
excessive dietary nutrients or tobacco smoking, low endoge-
nous levels can rapidly accumulate via a reduction in or
impairment of the antioxidant arsenal of the cell. Therefore, the
relative levels of ROS, RNS, and antagonistic antioxidants are
critical for maintaining cellular redox homeostasis, preserving
appropriate cell, tissue, and organ function, and therefore
preventing various chronic disorders such as diabetes. An
imbalance in redox homeostasis is particularly damaging to the
pancreatic ␤-cell, as discussed in detail below.
ROS AND ␤-CELL FUNCTION
Superoxide-Generating Systems in Pancreatic Islets
Mitochondrial respiration consumes molecular oxygen,
which is the ultimate acceptor of electrons generated by glu-
cose and fatty acid catabolism. During the course of normal
oxidative phosphorylation, up to 4% of all oxygen consumed is
converted into the free radical superoxide (O2·⫺) (16). Super-
oxide may be converted into other ROS and may react with
RNS to generate the highly destructive peroxynitrite molecule
(120). Superoxide molecules are quickly converted to H2O2 by
specific isoforms of the enzyme superoxide dismutase (SOD).
The H2O2 is then either detoxified to H2O and O2 by glutathi-
one peroxidase (GPx, in the mitochondria) or is detoxified by
catalase in peroxisomes. However, in the presence of higher
concentrations of the transition metals such as Cu2⫹ or Fe2⫹,
H2O2 can be converted to the highly reactive hydroxyl radical
and OH⫺ (Fenton reaction).
The electron transport chain (ETC) is a major source of
cellular ROS, and O2·⫺ is one of the most abundant, though
short-lived, ROS species in cells (7, 63). Electron “leak”
mainly occurs at ETC complex I and III, when elections
derived from NADH or FADH2 that have escaped normal
processing by the ETC react with molecular oxygen to form
O2·⫺, that is rapidly converted to H2O2 and subsequently
detoxified by CAT or GPx (see above), which essentially
nullifies the initial toxicity from electron leak. However, this
flux may be disrupted in the ␤-cells, as they express relatively
low levels of CAT and Gpx (103), around 5% of that reported
for liver cells (161). In addition, ␤-cells may also be chroni-
cally exposed to glucose overload in the diabetic milieu,
leading to elevated glycolytic flux and glucose oxidation,
elevated tricarboxylic acid (TCA) cycle activity, and increased
Ca2⫹ oscillations. Together, these metabolic reactions increase
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C421
the probability of further electron leak, O2·⫺generation, and the
potential pathological consequences that may affect ␤-cell
function in terms of reduced insulin secretion or promote ␤-cell
dysfunction and death, reducing overall pancreatic insulin
output (53), as described further in the following section.
The role of other nutrients, such as circulating free fatty
acids (FFA), which are elevated in obesity and T2DM, on
mitochondrial ROS and O2·⫺ are less well defined. It is
reasonable to consider that increased circulating FFA levels
will lead to increased uptake by ␤-cells and subsequently used
as a metabolic fuel by the mitochondria via ␤-oxidation. The
resulting flux to acetyl-CoA will prime the TCA cycle to
produce more NADH and FADH2, which may again lead to
raised O2·⫺ production. It has been reported that palmitate
exposure can increase mitochondrial O2·⫺ levels in the INS-1E
␤-cell line (101), and oleate promoted mitochondrial H2O2
formation in MIN6 ␤-cells, which was likely to be mediated by
the reaction of O2·⫺ produced by complex I and SOD (90). It
was also reported that exposure to oleate and palmitate induced
ETC uncoupling, increased mitochondrial membrane depolar-
ization, decreased ATP production, and consequently reduced
insulin secretion in several ␤-cell lines and isolated islets (19,
90). However, other studies have detected oleate-induced O2·⫺
in the cytosol of murine islets (91, 92), which was derived from
NADPH oxidase activity (NOX). Palmitate can also activate
NOX in ␤-cells and islets, leading to increased O2·⫺ produc-
tion, and in acute palmitate exposure conditions NOX can
potentiate glucose-stimulated insulin secretion (GSIS) (62,
114, 148). Consequently, NOX is a critical source of intracel-
lular O2·⫺, as it can be stimulated by nutrients such as glucose
or saturated fatty acids and key stimulus-secretion coupling
factors [e.g., Ca2⫹ influx and protein kinase C activation
(121)].
In phagocytic immune cells such as macrophages, mono-
cytes, and neutrophils, NOX functions to transfer electrons
from NADPH to molecular oxygen to generate O2·⫺ to kill
invading pathogens (122). The generalized NOX complex
consists of five major components, including membrane-em-
bedded gp91phox and gp22phox, and cytosolic p40phox, p47phox,
and p67phox (121). In some cases, activity is also regulated by
the small G protein Rac (121). Plasma membrane recruitment
of the cytosolic components leads to NOX activation, NADPH
utilization, and O2·⫺ production. It has been reported that pancre-
atic ␤-cells express several functional isoforms of NADPH oxi-
dase, and impairment of p47phox translocation to the plasma
membrane reduced glucose oxidation, Ca2⫹ flux, and GSIS in
␤-cells (115). Due to the activity of NOX in immune cell
function, and the recently identified positive and negative roles
of NOX isoforms in ␤-cell function (129), NOX dysregulation
in T1DM and T2DM under conditions of proinflammatory
cytokines (such as IL-1␤, TNF-␣, and INF-␥) and glucose
excess can result in cellular dysfunction and impairment of
insulin secretion (120).
The availability of the NOX substrate NADPH, which is
generally involved in biosynthetic pathways, for example fatty
acid synthesis, and in the regeneration of reduced glutathione,
is central to NOX activity. NADPH can be produced in the
mitochondria by specific enzymes, but the vast majority of
cellular production comes from the pentose phosphate pathway
(PPP). In essence, generation of NADPH in high-glucose
conditions can be considered as a protective function, as the
C422 OXIDATIVE STRESS, CELL METABOLISM AND FUNCTION
PPP shuttles excess glucose away from energy- and ROS-
generating processes (i.e., glycolysis and oxidative phosphor-
ylation) into biosynthetic pathways, while NADPH is also
required for converting oxidized glutathione into the reduced
state so that it can nullify additional ROS species. However,
excessive NADPH generated via nutrient overload may also
lead to elevated O2·⫺ production by promoting NOX activity.
Furthermore, it is understood that, following exposure of
␤-cells to glucose, there is a larger increase in the NADPH/
NADP ratio compared with the NADH/NAD ratio (63), and
consequently NOX is activated in response to glucose stimu-
lation (114). NOX also functions as a useful mechanism to
regenerate NADP from NADPH, thus allowing further metab-
olism of glucose via the PPP, albeit producing O2·⫺ in the
process. This is particularly important as ␤-cells possess a
relatively low level of lactate dehydrogenase (LDH) and con-
sequently are limited in their ability to regenerate NAD⫹ from
NADH through lactate production to promote glycolytic flux.
Key experiments have shown that inhibition of NOX (77, 115)
and other extramitochondrial oxidoreductase enzymes (63)
impaired insulin release, as NOX is required for optimal GSIS
via O2·⫺ production (115). The regulation of NOX in ␤-cell
function is complex and is possibly dependent on the genera-
tion of other intracellular ROS species, such as H2O2 (115), as
well as the level of glucose. Priming of the antioxidant defense
system by low/initial ROS levels may be important for subse-
quent elevations of ROS generated by the enhancement of
metabolic activity. Interestingly, using fasting and fed-state
animals, it was reported that increased NOX activity and ROS
generation prevented hypoglycemia in fasting conditions via a
reduction in insulin output (117). Conversely, in high-glucose
or leucine-stimulated conditions, the increased production of
NADPH was diverted from NOX activity and into antioxidant
defense, which mitigated excess ROS production and was
important for continued insulin secretion (117). Therefore, it
would appear that NOX activity, NADPH, O2·⫺, and their
related fluxes based on nutrient or proinflammatory cytokine
stimulation are intimately involved in ␤-cell function and fate.
Finally, ␤-cell O2·⫺ can also be produced in other cell
compartments by additional oxidase enzymes, including in the
cytosol by xanthine oxidase (XOD) (179), and in the smooth
endoplasmic reticulum (SER) by cytochrome P450 enzymes
(23, 126). A recent in vitro study demonstrated that intermittent
high glucose (11.1 to 25 mM rotating every 24 h), significantly
increased XOD activity and O2·⫺ in the INS-1 ␤-cell line
(179). This also led to decreased cell viability and an accumu-
lation of cells in the G0/G1 cell cycle stage. It was indicated
that large fluctuations in glucose concentration were more
detrimental to ␤-cell function than chronically elevated levels.
This may have implications for those with uncontrolled blood
glucose, although the functionality of the GSIS response in
these treated cells was not explored. Cytochrome P450 en-
zymes play a key role in detoxifying compounds in the SER
and are ubiquitously expressed in the liver but are also present
in human islets (48, 96). Some SER enzymes, such as
CYP2E1, have been demonstrated to be a risk factor for
T2DM, and increased hepatic expression has been shown in
obesity (104, 171). The activity of these enzymes can directly
promote O2·⫺ and other ROS, but they can also be involved in
bioactivation of toxic compounds such as nitrosamines, that are
usually derived from poor nutritional sources such as processed
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or smoked meats (96). With the ability to generate O2·⫺, other
ROS, and nitrosamines, cytochrome P450 enzymes have fur-
ther potential to generate highly reactive RNS (71, 75) and
have been reported to increase DNA damage in BRIN-BD11
␤-cells in vitro (96). Nitric oxide can regulate insulin secretion
by enhancing insulin granule exocytosis through S-nitrosyla-
tion of syntaxin 4, a protein regulating vesicle-plasma mem-
brane docking (173). However, these regulatory aspects are
generally mediated by low to medium ROS/RNS levels, and it
has been reported in the INS-1 ␤-cell line that H2O2 in the
range of 1– 4 ␮M can promote insulin release, while levels
above or below this range impede insulin exocytosis (63, 135).
␤-Cell signaling pathways are exquisitely designed to link
nutrient-sensing capabilities to insulin secretion, the primary
function of the cell. This is achieved by coupling rapid uptake
and oxidative degradation of glucose to intracellular ATP
production, which, via interaction with ATP sensitive K⫹
channels, leads to plasma membrane depolarization, Ca2⫹
influx, and appropriate insulin exocytosis (119). Elevated glu-
cose entering ␤-cells is mainly oxidized to CO2 through gly-
colysis and TCA activity (53, 150), but it can also enhance
anaplerosis and thus biosynthetic pathways, through formation
of ␣-ketoglutarate, oxaloacetate, and citrate required for pro-
tein (150) and lipid synthesis (116), but, as described above, it
increases the probability of electron leak and ROS generation.
Other ROS Species and Their Sources
Given that O2·⫺ is the main source of cellular ROS species,
it is important to also consider that it is a central regulator in
the generation of other ROS/RNS molecules. As illustrated
above, O2·⫺ can be converted to H2O2 through the action of
SOD, and H2O2 is then detoxified by catalase. However, in
circumstances where O2·⫺-to-H2O2 fluxes are elevated but the
subsequent H2O2 detoxifying activity of catalase is low [which
is the case for ␤-cells (103)], then both species can react
together to form the highly reactive hydroxyl radical (HO·) and
hydroxide (52, 122, 149). Importantly, this Fenton chemistry
reaction will only occur in the presence of metal ions, partic-
ularly iron (Fe2⫹ and Fe3⫹), and the probability of it occurring
rises with increased intracellular metal ion concentrations (7,
163). This has implications for disorders including hemochro-
matosis and diabetes, where iron overload is or can be present,
respectively (157), and the risk is intensified within glucotoxic,
lipotoxic, or glucolipotoxic environments due to high meta-
bolic activity. In addition, O2·⫺ can react with ·NO to form a
dangerous RNS species, peroxynitrite (ONOO⫺) (67, 122).
Both HO· and ONOO⫺ are highly reactive species that can
rapidly promote significant cellular damage in rat and human
islets in vitro (29, 37, 67), and the latter was reported to be
elevated in the islets of NOD mice (139). However, while HO·
and ONOO⫺ may play a key role in ␤-cell oxidative stress (29,
37, 67), HO· production is more relevant in response to cyto-
kine insult, inflammation, and cellular dysfunction, as it was
reported that ONOO⫺ was not generated by ␤-cells under
proinflammatory conditions (12, 67).
␤-Cell production of HO· can also occur via other mecha-
nisms, including the reaction of ·NO or nitrite with H2O2
(generated by high levels of SOD and reduced catalase, as
outlined above) (67, 122). In this context, exposure of RINm5f
␤-cell lines to IL-1␤ alone induced ·NO production via the
 OXIDATIVE STRESS, CELL METABOLISM AND FUNCTION
expression of inducible nitric oxide synthase (iNOS), while the
addition of TNF␣ and INF␥ increased the mitochondrial accu-
mulation of H2O2. This proinflammatory combination led to
the production of HO·, which was highly toxic to the ␤-cells.
However, this cytokine-mediated toxicity was abrogated by the
supply of catalase (67), demonstrating the importance of raised
H2O2 accumulation and reduced catalase activity in ␤-cell
physiology. Additional work also revealed that ␤-cells respond
to cytokine challenge by changing the expression status of
RNS-generating enzymes such as the various isoforms of NOS.
The constitutive form of NOS in ␤-cells, neuronal NOS
(nNOS), is mainly expressed within insulin vesicles (66, 94)
and catalyzes the conversion of L-arginine to ·NO and L-citrul-
line, using molecular oxygen and NADPH as substrates (120).
However, when challenged with cytokines, nNOS expression
decreased in ␤-cells as a compensatory mechanism to offset a
cytokine-mediated rise in iNOS activity (66). So in this sce-
nario, a RNS-generating enzyme played a protective role in the
cell by reducing one source of RNS in response to stress.
Conversely, while some studies have demonstrated that induc-
ible production of ·NO is detrimental to insulin secretion (27)
and ␤-cell proliferation (138), it is apparent that endogenous
·
NO production is a physiological regulator of insulin secretion
in ␤-cells at lower concentrations (153). The production of ·NO
by nNOS is important for liberation of glucokinase from
insulin-secretory granules to the cytosol via nitrosylation
(144). Gucokinase is a key rate-determining enzyme and as
such is required for appropriate glycolytic flux and for gener-
ating a high ATP/ADP ratio, needed for insulin release. Inter-
estingly, ·NO donors such as hydroxylamine and 3-morpholi-
nosydnonimine also promoted insulin release in INS-1 cells. It
was observed that intracellular ·NO levels were increased in
response to glucose and Ca2⫹ influx in INS-1 ␤-cells and rat
islets, while scavenging of ·NO blocked insulin secretion (153).
Thus, low levels are involved in physiological signaling, while
higher levels are detrimental to ␤-cell function. This is due to
the high reactivity of ·NO and its promotion of nitrosative
stress, which can target heme-containing proteins such as those
in the ETC. In fact, ·NO can compete with O2 for binding to
ETC complex IV, changing the ⌬␺, ⌬pH, and Ca2⫹ retention
(54), while it can also induce S-nitrosylation of ETC com-
plexes I, III, and IV and the Fo/F1 ATPase (38, 54). These
covalent modifications alter protein structure and thus function,
leading to impaired ATP generation and optimum insulin
release.
Finally, H2O2 is a nonradical ROS and is chemically more
stable in comparison to other ROS species (134). H2O2 is
cytotoxic and elicits toxic effects through protein modification
via its interaction with protein thiol groups (121). While H2O2
does not promote changes in DNA structure directly, the
H2O2-induced production of HO·, as indicated above (134), can
lead to DNA damage and cell death. The formation of H2O2
can occur in various subcellular compartments mainly due to
the distribution of different SOD isoforms and the subcellular
generation of O2·⫺ (mitochondria and cytosol). However, it can
also be produced by other biochemical reactions including the
degradation of FFA by ␤-oxidation in peroxisomes (134).
Various peroxisomal enzymes have the capacity to generate
H2O2, including acyl CoA-oxidase, D-aspartate oxidase, L-a-
hydroxy oxidase, and XOD (134), but it is possibly those
involved in FFA metabolism that are more pertinent in the
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C423
context of H2O2-induced impaired ␤-cell function. Utilizing
INS-1E and RINm5f ␤-cell lines along with rat islets, Elsner et
al. (42) demonstrated that the peroxisome is a major site of
H2O2 production in ␤-cells and that only saturated FFA with
carbon lengths greater than 14 were toxic to ␤-cells. Interest-
ingly, peroxisomal and cytosolic catalase overexpression pro-
tected these cells from palmitate-induced toxicity by reducing
the intracellular formation of H2O2. However, overexpression
of catalase in the mitochondria did not alter H2O2 levels and
did not protect ␤-cells (42). Conversely, other research indi-
cated that the production of H2O2 may be required for appro-
priate intracellular signaling to allow insulin release. It can
positively modulate mitochondrial Ca2⫹ flux (97), which is a
major regulator of insulin release in ␤-cells (79). Furthermore,
Morgan et al. (115) also reported that suppression of NOX in
conditions of reduced H2O2 production led to a decrease in
GSIS. Recent investigations have demonstrated that insulin can
potentiate the toxicity of H2O2 insult in ␤-cells. Exposure of
MIN-6 and RINm5f to insulin (100 nM) alone increased cell
death over 24, 48, and 72 h (147), while exposure to a
combination of insulin and H2O2 (70 ␮M) resulted in further
cell death over and above exposure to the same concentration
of H2O2 alone (147). Additional studies in rodent and human
islets confirmed that the cytotoxic response occurred concom-
itantly with markers of ER stress and alterations in BCL
proteins (15). These findings have stimulated interest with
regard to whether or not the early overproduction and secretion
of insulin in obese and insulin-resistant humans may harm
␤-cell function (4). Taken together, there appears to be a
delicate balance between ROS/RNS production and detoxifi-
cation that is critical for ␤-cell physiology. Further descriptions
of ROS and their relevant chemistry can be found in a recent
review published in the Biochemical Journal (120).
SOD, CAT, and GPx
Antioxidants are required to attenuate and/or neutralize the
deleterious effects of reactive substances, and can be grouped
as enzymatic and nonenzymatic factors. Superoxide dismutase
(SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), and gluta-
thione-peroxidases (GPx, EC 1.11.1.9) are the main enzymatic
antioxidants and have been investigated for the treatment of
diseases and catabolic circumstances resulting from oxidative
damage and/or oxidative stress (1).
As previously described, high concentrations and/or inade-
quate removal of reactive products, especially O2·⫺ results in
oxidative stress, which has been a cause and/or a consequence
of most pathological conditions, such as cardiovascular dis-
eases, cancer, atherosclerosis, hypertension, and type 1 and 2
diabetes (40, 46). For instance, some important RNS signal
transduction molecules, such as ·NO, can be rapidly removed
by reacting with O2·⫺, resulting in the production of ONOO⫺,
a compound with powerful oxidant properties (see above). This
reaction highlights the importance of all compartmentalized
SOD isoforms as the first line of defense against oxidative
stress.
Pharmacological treatments with SOD (39) and/or SOD
mimetics [e.g., Mn(II)(pyane)Cl2] (155) was reported to reduce
liver and hippocampus oxidative stress, respectively, in dia-
betic animals. Studies also report antioxidant effects using
chemically modified SOD (e.g., carboxymethylcellulose-SOD,
C424 OXIDATIVE STRESS, CELL METABOLISM AND FUNCTION
and polymethyl vinyl ether-co-maleic anhydride-SOD) in
streptozotocin (STZ)-induced diabetic rats (108). The defi-
ciency of some trace minerals such as zinc has also been
implicated in the effectiveness of Cu-Zn-SOD and therefore
associated with reduced insulin secretion and action (100). For
instance, long-term low zinc intake from drinking groundwater
was reported to be associated with an increased risk of devel-
oping type 1 diabetes (6, 156). In obese animal models, such as
mutant db/db mice and mice subjected to a high-fat diet (HFD),
however, zinc supplementation improved glucose homeostasis
by restoration of ␤-cell function (25, 151).
CAT is an antioxidant enzyme present in almost all aerobic
organisms and is considered the detoxification-completing step
initiated by SOD. It is primarily located in the peroxisomes and
absent in the mitochondria of almost all tissues (except in the
heart) (21, 140). The main function of CAT is to catalyze the
degradation/reduction of H2O2 to H2O and molecular oxygen.
In addition, CAT reacts with H⫹ donors, such as methanol,
ethanol, formic acid, or phenols, with peroxidase activity.
Specific mutations/polymorphisms in CAT gene may result in
catalase deficiency (also known as acatalasemia), as reported in
Japanese, Swiss, and Hungarian individuals (59, 60, 158). The
deficiency or mutation of CAT has been associated with low
H2O2 removal and, therefore, oxidative stress leading to vari-
ous diseases and abnormalities, such atherosclerosis, hyperlip-
idemia, gangrene, hypertension, neurodegeneration, and diabe-
tes (60). At the ␤-cell level, low CAT level results in the
inability to protect the pancreatic ␤-cells from toxic quantities
of H2O2; however, this is more likely to be observed when
␤-cells are stimulated with glucose. Furthermore, acatalasemic
mice treated with alloxan are more prone to exhibit low insulin
level due to the decline in the number of viable ␤-cells
(compared with normal-CAT animals) (159). Although con-
ventional antioxidant treatments for low CAT activity are
available, there is a lack of scientific evidence evaluating their
effect. In addition, conventional antioxidant treatments for
acatalasemic-induced type 1 or 2 diabetes may increase ROS
synthesis through endomembrane signaling cascades, commu-
nication networks, and metabolic regulatory complexes. A
recent alternative has been studied by using a cell-penetrating
derivative of the peroxisomal enzyme catalase (also called
CAT-SKL). This highly specific supplementation has shown a
significant reduction in oxidative stress associated with dia-
betic retinopathy in in vitro and in vivo animal models (55).
H2O2 conversion to H2O and molecular oxygen can also
occur through GPx (selenium-dependent glutathione peroxi-
dase). There are at least eight types of GPx, namely GPx1– 8,
however, the most well described and abundant in cells is
GPx1. This antioxidant enzyme is mainly located in mamma-
lian cell mitochondria, and to a lesser extent in the cytosol.
GPx also detoxifies the cell from organic and inorganic per-
oxides and therefore provides protection against a variety of
oxidative stressors (76). In Gpx1⫺/⫺ mice, Wang et al. (170)
observed a significant reduction in ␤-cell mass and GSIS,
allied with hyperinsulinemia, mild hyperglycemia, and im-
paired ATP production. In another study, Gpx1⫺/⫺ mice sub-
jected to HFD for 12 wk exhibited chronic and systemic
oxidative stress associated with hyperglycemia. Although no
alteration in insulin sensitivity was observed, chronic oxidative
stress induced by ROS dramatically reduced ␤-cell mass and
insulin secretion associated with Pdx1 gene expression (112).
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The phenotype exhibited by Gx1⫺/⫺ mice is considered closely
associated with cell dysfunction described for type 1 diabetes
(102, 170).
The activity of GPx is dependent on the availability of
micronutrients such as selenium (Se). Se concentration in the
bloodstream has been used to assess whole body Se status and
as an indirect antioxidant indicator of GPx activity. Many in
vivo and in vitro studies demonstrated the potential of seleno-
proteins and GPx-related antioxidant effects induced by sele-
nium and therefore led to the idea that an increased Se intake
through the diet or supplementation would attenuate the oxi-
dative stress observed in diabetes (143). However, later stud-
ies, including epidemiological studies, observed that both ex-
cessive or low selenium intake is associated with increased risk
of diabetes, following a U-shaped curve (76, 128, 142, 143).
This is particularly important for well-nourished populations,
where Se deficiency is less likely to occur (76, 143), and
therefore a nutritional assessment associated with the determi-
nation of Se concentration in plasma of diabetic patients is
recommended before considering increments in Se intake.
Gene Expression Mechanisms Responsible for ROS Effects
on Insulin Secretion
It is clear that ROS/RNS act as signaling molecules and that
elevated levels negatively affect ␤-cell function including in-
sulin secretion (Fig. 1). However, the precise mechanisms by
which ROS elicit these effects are complex. Due to their highly
reactive nature, ROS can alter the native conformation of
proteins by chemical modification like nitrosylation, carbony-
lation, and peroxidation among others, and this leads to im-
paired function. Some of the most common targets include
proteins associated with membrane ion transport, metabolic
enzymes, and proteins regulating gene expression and signal
transduction (53, 122). For example, H2O2 modulates proin-
flammatory NF-␬B signaling and regulates its translocation
into the nucleus. However, the effects are dependent on the
concentration of H2O2 and the inflammatory conditions. For
instance, H2O2 alone does not promote nuclear translocation of
NF-␬B but enhances this process in the presence of TNF (36).
Conversely, when concentrations were increased, translocation
was inhibited (36). Other studies have reported that oxidized
NF-␬B had a significantly reduced ability to bind DNA and
mediate gene transcription, although this was restored with
antioxidant enzymes (83). More recent work has demonstrated
that NOX-generated ROS was critical for redox balance and
reduced the LPS-induced interaction of NF-␬B with DNA,
rather than altering the nuclear translocation (68). The ability
of NF-␬B to interact with DNA is influenced by the redox
status of cysteine moieties located within various subunits,
including Cys62 of p50 and Cys38 of p65 (56). Thiol groups
and cysteine residues are sensitive to redox homeostasis and
are key targets for ROS (73), which alters subsequent activity.
One of the classical examples of ROS-mediated negative
effects on ␤-cell function and insulin secretion was a report of
ROS impacting insulin gene expression via major redox-sen-
sitive transcription factors, pancreatic and duodenal homeobox
1 (PDX1), and V-Maf avian musculoaponeurotic fibrosarcoma
oncogene homolog A (MafA) (24). Both are targets for ROS
(85, 121), and exposure of rat islets to H2O2 reduced the
binding interaction of PDX-1 with DNA, leading to decreased
 OXIDATIVE STRESS, CELL METABOLISM AND FUNCTION C425

Fig. 1. Interplay between nutrient metabolism and reactive oxygen species (ROS) generation in regulating ␤-cell function. Exposure to high nutrient load
including glucose (Gluc) and free fatty acids (FFA) leads to enhanced mitochondrial metabolism and possibly increased ROS production in ␤-cells. If not
neutralized by the antioxidant arsenal, elevated ROS levels may alter electron transport efficiency, decreasing ATP production and raising the mitochondrial
membrane potential (MMP), which leads to electron slippage from normal transport processes. The reduced ability of ␤-cells to increase or maintain ATP levels
in response to nutrient stimulation may lead to impaired insulin secretion through decreased action on ATP-sensitive K⫹ channels. Elevated ROS generation
through other mechanisms, such as the proinflammatory NADPH oxidase (NOX) activity, may also impair insulin gene transcription and biosynthesis,
contributing to reduced insulin output. However, increased ROS may activate the Keap1-Nrf2 antioxidant pathway by enhancing the cytoplasmic to nuclear
translocation of Nrf2. Gln, glutamine; Arg, arginine, Glu, glutamine; iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase; GCK,
glucokinase; Cat, catalase; Gpx, glutathione peroxidase; ARE, antioxidant response elements; SOD, superoxide dismutase.

insulin gene expression (85). For PDX-1, it appears that the
negative effects of ROS are mediated through the c-Jun NH2-
terminal kinase (JNK) signaling and the forkhead box protein
O1 (FOXO1) transcription factor (53, 84). Inhibition of the
JNK pathway protected ␤-cells from ROS, while activation
reduced insulin gene expression (84, 85). Exposure of HIT-T15
␤-cells to H2O2 for 48 h led the cytoplasmic-nuclear translo-
cation of FOXO1, while PDX-1 was expressed mainly in the
cytoplasm. In addition, JNK activation also induced nuclear
translocation of FOXO1, whereas inhibition of FOXO1 expres-
sion with siRNA maintained nuclear expression of PDX-1 and
importantly insulin secretion (86). A similar observation was
reported with respect to the transcription factor MafA, which
regulates insulin gene transcription along with ␤-cell development
and proliferation (125, 169). H2O2 also promoted cytoplasmic
localization of MafA and prevented its positive influence on
insulin gene expression (169). Work in animal models demon-
strated that overexpression of antioxidants such as GPx main-
tained nuclear expression of MafA and ␤-cell function (65). It was
proposed that in hyperglycemic conditions the cytoplasmic trans-
location of MafA is an early indicator of ␤-cell demise (65, 169).
Therefore, it appears that redox status can affect the opposing
subcellular localization of key insulin gene transcription factors
and their potential regulatory proteins.
Novel ␤-Cell Cell-Antioxidant Defenses in Diabetes: the
Nrf2-Keap1 Pathway
Redox homeostasis/balance is essential for cell homeostasis
(30, 78, 98, 113, 137), and in the case of pancreatic ␤-cells it

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C426 OXIDATIVE STRESS, CELL METABOLISM AND FUNCTION
is very important to minimize the deleterious effects of excess
nutrients or inflammatory factors on ␤-cell function, i.e., insu-
lin secretion and cell death (see above). In normal or elevated
nutrient/immune stress circumstances, redox component levels
are regulated at the transcriptional level of the cell. The
Nrf2/Keap1 pathway regulates the expression of over 100
genes and functions related to oxidative stress and cell sur-
vival, including the above mentioned enzymatic and non-
enzymatic antioxidants, growth factors and transcription fac-
tors related to inflammation (Fig. 1) (34). Indeed the role of the
Nrf2/Keap1 pathway in mediating redox balance in ␤-cells has
been further explored, with dysregulation contributing to dia-
betic pathologies (34). In basal conditions, Keap1 marks the
Nrf2 transcription factor for ubiquitination in the cytoplasm.
After oxidative insult, redox-sensitive changes in Keap1 cys-
teine moieties lead to the dissociation of the Keap1-Nrf2
dimer, allowing the entry of Nrf2 into the nucleus, where
various genes are upregulated, including those involved in
antioxidant defenses such as ␥-glutamylcysteine synthetase
(GCLC), glutathione S-transferase-A2 (GSTA2), NADPH qui-
none oxidoreductase (NQO-1), superoxide dismutase (SOD1),
and heme oxygenase-1 (HO-1)(137). Murine studies have
reported that knockout and suppression of Nrf2 led to increased
intracellular ROS and aggravated ␤-cell damage in islets and
␤-cells lines (174). Conversely, overexpression of Nrf2 re-
duced DNA adduct formation and reestablished insulin secre-
tion (174). This pathway also reduced inflammation associated
with NF-␬B activation (154), which indicated effects beyond
redox balance. However, the precise effects of this pathway on
insulin-secretory machinery and insulin release and homeosta-
sis are not fully understood, as in Nrf2 knockout mice, de-
creased blood glucose, lower body fat and body weight along
with enhanced insulin signaling was observed (136, 178).
Several studies have reported changes in Nrf2 expression in
diabetic individuals, and the direction of these changes appears
to be related to the presence of diabetic complications and
response to oxidative stress conditions, including upregulation
in diabetic nephropathy and retinopathy (80, 180) and down-
regulation in the diabetic myocardium (160). Although the role
played by Nrf2/Keap1 in mediating oxidative stress in diabetes
remains to be fully understood, the pathway has been consid-
ered a target for pharmacological and nutritional therapeutics.
For example, the drug dimethyl fumarate (BG-12) is an acti-
vator of Nrf2 downstream pathways, leading to cytoprotective,
anti-inflammatory, and antioxidant effects and is already a
target for the treatment of multiple sclerosis (57). The nutri-
tional approach also shed light in some specific nutrient acti-
vators of Nrf2, including vitamin D, zinc, and bioactive com-
pounds found in foods and herbs (e.g., curcumin, sulforaphane,
cinnamic aldehyde, resveratrol, magnesium lithospermate B,
catechins, pterostilbene) (5, 34, 81). Furthermore, some amino
acids such as glutamine and arginine exhibit Nrf2-activating
properties in proliferating cells of the intestinal crypt of healthy
animals (167) and in left ventricular heart tissue of alloxan-
induced hyperglycemic rats (141), respectively. Interestingly,
arginine and glutamine are also sources of glutamate, the
supply of which can be rate limiting for the synthesis of
glutathione (GSH) through GCLC in oxidative stress circum-
stances (30, 31, 146). The Nrf2/Keap1/ARE pathway is also a
target in cancer/tumor cells and autoimmune diseases, includ-
ing T1DM. The activation of Nrf2 downstream cascades may
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contribute to the resistance and survival of supercells and
therefore the evolution of cancer and chemoresistance (69).
Furthermore, the balance of T helper cell (Th)1 and Th2
immune responses are critical for immunity, and studies have
shown that both responses can be regulated by the Nrf2
downstream pathway in different ways. For instance, Rockwell
et al. (145) and Kikuchi et al. (87) reported that Nrf2 promotes
Th2 cytokine production via Th1 response suppression in mice
exposed to food preservatives (i.e., allergy animal model), and
pulmonary fibrosis, respectively. However, in diabetic condi-
tions (1, 2), the demise of pancreatic ␤-cells appears to occur
through the Th1 cytokine cascade and results in cytotoxic
quantities of ROS/RNS (93, 131). Sireesh et al. (152) observed
a positive correlation between plasma Nrf2 and Th2 cytokine
(IL-4, IL-13) levels, and a negative correlation with Th1
cytokine (IFN-␥, TNF) levels in early diagnosed type 2 diabe-
tes patients. These findings support other type 1 (72, 93, 127,
131) and type 2 (34, 105) diabetes studies, where reduced
levels of Nrf2 favors Th1 responses and oxidative stress,
potentially contributing to the progression of the disease.
INSULIN RESISTANCE
ROS in the Periphery and Their Role in Insulin Resistance
Insulin resistance is the pathological condition in which periph-
eral tissues (e.g., liver, skeletal muscle, and adipose tissue) fail to
respond to normal levels of insulin. As a result, pancreatic ␤-cells
have to produce and secrete higher amounts of insulin to com-
pensate. This poses a significant biosynthetic and energy burden
on the ␤-cells. When ␤-cells fail to maintain high insulin produc-
tion in response to greater metabolic demand, T2DM results
(120). There is a large body of evidence connecting hyperglyce-
mia and many of the major complications of diabetes including
nephropathy, retinopathy, neuropathy, and macro- and microvas-
cular damage (13, 120). In uncontrolled diabetes, decreased levels
of the antioxidants vitamin E and ␣-lipoic acid (LA) have been
reported (130). There is also evidence of a deficiency in erythro-
cyte catalase, an enzyme responsible for the removal of H2O2, that
can occur in diabetes (60, 61). In vivo studies demonstrate that
oxidative stress due to hyperglycemia occurs before late compli-
cations are clinically evident (123). The stress-activated signaling
pathways indicated above, including NF-␬B, p38 MAPK, JNK/
stress-activated protein kinases (SAPK), advanced glycosylation
end-products (AGE)/receptor for AGE (RAGE), and PKC may all
be activated by oxidative stress. ROS generation is critical for
mediating hyperglycemia-induced cellular damage (124). In bo-
vine endothelial cells, exposure to hyperglycemia initially in-
creased the production of intracellular ROS and activated NF-␬B.
Subsequently, PKC activity, AGE, and sorbitol levels increased. It
is likely that oxidative stress occurs early in the sequence of events
induced by high glucose, followed by activation of other pathways
that lead to cellular dysfunction and damage (120, 123).
Studies in human populations have described a strong link
between insulin resistance and elevated oxidative stress (43,
58, 132, 133, 165). Using a euglycemic-hyperinsulinemia
clamp approach, (considered to be the best method to estimate
even small variations in insulin sensitivity (88), in a population
of obese and nonobese men, Urakawa et al. (165), observed a
positive association between insulin resistance and circulating
levels of 8-epi-PGF2␣, a marker of lipid peroxidation. Differ-
ent models to assess insulin resistance, such as the homeostasis
 OXIDATIVE STRESS, CELL METABOLISM AND FUNCTION
model assessment of insulin resistance (HOMA-IR) have also
been reported to positively associate with oxidative stress
levels in various populations (89, 110, 132, 133). Additionally,
genes involved in providing cellular protection against oxida-
tive stress are often found to be dysregulated in both insulin-
resistant and T2DM patients (2, 14, 22). For instance, the
expression levels of Mn-SOD in skeletal muscle tissue have
been reported to be significantly decreased in obese patients
and also in pregnant women that were overweight or were
clinically diagnosed with gestational diabetes (11, 44). In
support of this view, overexpression of Mn-SOD ameliorated
insulin sensitivity in a rat model of HFD-induced skeletal
muscle insulin resistance (8). Conversely, downregulation of
redox control enzymes or antioxidant protein-associated tran-
scriptional factors such as Nrf2, has been reported to contribute
to oxidative stress-induced insulin resistance (160).
In this scenario, ectopic lipid accumulation and mitochon-
drial dysfunction, particularly in the skeletal muscle tissue, are
important physiological hallmarks of insulin-resistant subjects.
They have been reported to be paralleled and also preceded by
increased ROS production in the latter sites (9, 109) (Fig. 2).
C427
Thus, oxidative stress may indeed play a causal role in the
development of insulin resistance. Houstis et al. (74) used two
models to induce insulin resistance in adipocytes, TNF and
dexamethasone treatment. Importantly, in both models, ele-
vated ROS production was detected before any insulin insen-
sitivity could be observed, suggesting that exacerbated oxida-
tive stress is upstream of insulin signaling defects. In the same
study, insulin resistance was successfully ameliorated to vary-
ing degrees using various distinct approaches designed to
reduce cellular ROS levels, highlighting that insulin resistance
can be attenuated by suppression of redox stress. Not surpris-
ing, perhaps, is the ever-increasing list of publications provid-
ing direct and indirect evidence that a reduction in intracellular
ROS levels, either through genetic manipulations or through
the use of antioxidants, can alleviate insulin resistance and
glucose intolerance (3, 20, 70, 95, 106).
Mitochondrial electron transport overload due to excess
dietary nutrient metabolism appears to be central to the mech-
anism leading to elevated ROS generation, as described above
(1, 162). Incomplete fatty acid metabolism is associated with
accumulation of ROS that can directly impair insulin receptor

↑↑ Pro-inflammatory ↑ Impaired glucose disposal

Insulin
↑ Hyperinsulinemia
cytokines Gluc
e.g.TNF-α 7 Gluc
IL-6 Insulin Gluc 1 ↑ Ectopic lipid
G LU accumulation
IL-1β Receptor T4
Adipokines
1

ine
y tok tors Tyrosine
C ep
c phosphorylation
Re
IRS ↓ ↓ GLUT 4 translocation
2
↑ Fatty acid
↓ Glucose
↑ IRS-1 serine phosphorylation transport into
uptake
PI3K ↓ mitochondria
↑ JKK, p38, IKKβ, NFκB, PKC
5 T
PDK1 ↓ G
3
↑↑ Glut 4 vesicle
↑ Beta-oxidation
↑↑ ETC ↑↑ ETC
GSK3 AKT ↓
4
6 Mitochondrial
↑↑ dysfunction
↓ Glycogen synthesis and storage M
Metabolic
by
by-products

↓ Represent processes reduced in insulin resistance

↑ Represent processes exacerbated in insulin resistance
Skeletal muscles
Fig. 2. Role of reactive oxygen species (ROS) in lipid-induced skeletal muscle insulin resistance. Ectopic lipid accumulation leads to increased fatty acid transport
into the sarcoplasm and mitochondria of skeletal muscle cells. Excess fatty acids are metabolized through ␤-oxidation, resulting in mitochondrial electron
transport overload and increased formation of metabolic by-products and ROS. Stress response kinases (JNK, p38, PKC) are activated by ROS and can directly
impair insulin receptor signaling through serine phosphorylation of insulin receptor substrate 1 (IRS-1). This, in turn, inhibits tyrosine phosphorylation of IRS-1
in response to insulin binding, subsequently blocking downstream insulin signaling through PI3K/PDK1/Akt and, consequently, GLUT-4 translocation as well
as glycogen synthase activation. As a result, there is a decrease in insulin-stimulated glucose transport and glycogen synthesis, all together leading to impaired
glucose disposal and insulin hypersecretion as a compensatory mechanism. Red arrows pointing down represent processes reduced in insulin resistance; blue
arrows pointing up represent processes exacerbated in insulin resistance. Akt, protein kinase B; ETC, electron transport chain; Gluc, glucose; GLUT-4, glucose
transporter type 4; GSK-3, glycogen synthase kinase-3; IKK-␤, inhibitor of NF-␬B kinase-␤; IRS-1, insulin receptor substrate-1; JNK, c-Jun NH2-terminal
kinase; NF-␬B, nuclear factor-␬B; PDK1, phosphoinositide-dependent kinase-1; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; RNS, reactive nitrogen
species; ROS, reactive oxygen species.

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C428 OXIDATIVE STRESS, CELL METABOLISM AND FUNCTION
signaling through activation of several of the protein kinases
and transcription factors that control insulin sensitivity, such as
JNK, p38 MAPK, I␬B kinase (IKK-␤), NF-␬B, and PKC
(177). In particular, JNK and p38 MAPK respectively can
directly and indirectly induce serine phosphorylation of insulin
receptor substrate (IRS)-1/2 (35, 49, 50, 82, 118), an important
negative feedback regulatory mechanism of insulin signaling
(64), and widely reported to participate in cellular insulin
resistance (41). Mechanistically, serine phosphorylation of
IRS-1/2 induces their dissociation from the insulin receptor,
ubiquitination, and proteasome degradation (26), ultimately
promoting insulin resistance through reduced insulin signaling.
Antioxidant supplementation has been frequently tested in
human populations in various trials designed to evaluate their
efficacy to improve insulin sensitivity. Vitamins C, E, and
␤-carotene have been tested either alone or in combination and
have been generally reported to improve insulin sensitivity
outcomes. A short-term 8-wk placebo-controlled trial reported
that a combination of vitamins C, E, and ␤-carotene supple-
mentation resulted in a significant improvement in HOMA and
a concomitant reduction in fasting insulin levels in young
obese subjects (168). When tested alone, low doses (200 IU) of
vitamin E did not produce detectable improvements in fasting
insulin or glucose after 27 wk of supplementation in T2D
patients (10). However, high doses of 800 IU resulted in
notable improvements in fasting plasma glucose and insulin
levels in overweight individuals at 3 mo of treatments, which
was not sustained at 6 mo after the beginning of the supple-
mentation regimen (107). Vitamin C when supplemented in
combination with metformin has been demonstrated to consti-
tute a superior therapy in terms of glycemic control improve-
ments compared with metformin alone (33). ␤-Carotene when
supplied in the form of a fruit and vegetable juice concentrate
to overweight children attenuated insulin resistance, which was
also associated with a detectable increase in plasma levels of
␤-carotene (17). Omega 3 fatty acids also resulted in positive
outcomes when tested for their potential to ameliorate insulin
resistance. A 90-day placebo-controlled trial demonstrated that
1,800 mg of a combination of ecosapentaenoic acid and doco-
sahexaenoic acid daily improved several glycemic control
markers in a heterogeneous population of T2D patients with
5–10 yr of disease diagnostic (164). Indeed, a recent meta-
analysis pooling data from 17 studies with a total number of
672 participants revealed that short-term fish oil supplementa-
tion is associated with improved insulin sensitivity in people
with various metabolic conditions (51). The proposed mecha-
nisms for the action of omega 3 fatty acids on insulin sensi-
tivity likely involve their effects on mitochondrial bioenerget-
ics and endoplasmic reticulum and have recently been expertly
reviewed (99). Another antioxidant molecule, lycopene, a ca-
rotenoid pigment found in tomatoes, has recently been reported
in animal models to improve insulin sensitivity, although
clinical evidence is still lacking to support its use in humans for
the specific purpose of controlling insulin resistance (175,
176). Resveratrol, traditionally associated with improvements
in cardiovascular outcomes, was recently assessed in two system-
atic review and meta-analysis studies evaluating its effects on
glucose homeostasis parameters in T2D patients. Insulin resis-
tance as assessed by HOMA was significantly ameliorated in T2D
patients receiving resveratrol; data were pooled from nine ran-
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domized controlled trials involving 283 participants and supple-
mentation ranged from 4 wk to 12 mo (181).
GSH and Mechanisms to Therapeutically Protect the Redox
State
Nonenzymatic antioxidants are a group of compounds that
can react directly or in conjunction with enzymatic antioxi-
dants against ROS and RNS. A variety of substances can be
classified as a nonenzymatic antioxidant, such as polyphenols,
ascorbic acid, vitamin A, ␣-lipoic acid, thioredoxin, melatonin,
coenzyme Q, ␤-carotenoids, and ␣-tocopherols (45). However,
the most important and most concentrated nonenzymatic anti-
oxidant ubiquitously distributed in eukaryotes is glutathione
(␥-L-glutamyl-L-cysteinylglycine, GSH). GSH is composed of
three amino acid residues cysteine, glutamic acid, and glycine
and reacts with ROS in nonenzymatic reaction or as an electron
donor in the peroxide reduction catalyzed by GPx (32, 47).
Around 85–90% of GSH is found in the cytosol, and ~10 –15%
is located in organelles such as the mitochondria, nuclear
matrix, and peroxisomes. Since GSH is a critical antioxidant,
therapeutic approaches to increase systemic and/or tissue-
specific glutathione concentrations are of considerable interest,
especially through supplementation studies involving glu-
tamine, GSH or N-acetylcysteine (NAC) (3, 18, 21, 33–35, 37,
117, 136). For instance, in vitro and in vivo studies have
demonstrated a reciprocal relationship between low glutamine
availability and GSH in ␤-cells, skeletal muscle, and liver
tissues, with a concomitant rise in oxidative stress (28, 111,
172). Importantly, a recent in vitro study by the Newsholme
laboratory has shown that glutamine deprivation leads to rapid
induction of oxidative and endoplasmic reticulum stress, ulti-
mately resulting in marked ␤-cell dysfunction (18). However,
few studies in humans have demonstrated improvement in the
whole body and ␤-cell oxidative stress or glucose homeostasis.
CONCLUDING REMARKS
This review has evaluated several metabolic cell signaling
mechanisms emphasizing the control of the pathways of mito-
chondrial ROS generation, NADPH-oxidase-dependent ROS
generation, and the acute and chronic roles of antioxidants in
reducing oxidative stress. The modulation of various steps of
ROS generation and subsequent signaling that are induced by
hyperglycemia could be potential therapeutical targets in dia-
betes, e.g., attenuating ROS generation, cytokine production,
and cellular dysfunction and death. It is known that, besides its
osmotic effects on cells, hyperglycemia activates mitochon-
drial and NADPH-oxidase ROS production, which leads to
oxidative stress in diabetes. Inflammatory reactions can be
promoted by hyperglycemia ROS production and they may be
aggravated by the action of AGEs, due to the interaction with
the responsible receptor (RAGE). ROS and AGE generation
can lead to chronic low-grade inflammation that may contrib-
ute to diabetic complications. Any attempt to control these
processes could modulate some of the consequences of hyper-
glycemia. NADPH-oxidase-specific and antioxidant levels
have been suggested as being the best targets to reduce hyper-
glycemia-induced oxidative stress, but more targeted therapies
need to be found. In addition, future translational studies in
humans should reveal whether some nutrient-based antioxi-
dants can attenuate oxidative stress occurring in diabetes.
 OXIDATIVE STRESS, CELL METABOLISM AND FUNCTION
ACKNOWLEDGMENTS
The authors thank Curtin University School of Pharmacy and Biomedical
Sciences, Curtin Health Innovation Research Institute, and Faculty of Health,
Torrens University Australia, for provision of excellent research facilities and
support. Part of this review was written while P. Newsholme was on Academic
Program Study Leave at the Rowett Institute, University of Aberdeen, Scot-
land.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
K.N.K., R.C., and V.C. prepared figures; P.N., K.N.K., R.C., and V.C.
drafted manuscript; P.N., K.N.K., R.C., and V.C. edited and revised manu-
script; P.N., K.N.K., R.C., and V.C. approved final version of manuscript.
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