Physiol Rev 89: 27–71, 2009;
doi:10.1152/physrev.00014.2008.
Positive and Negative Regulation of Insulin Signaling
by Reactive Oxygen and Nitrogen Species
NAVA BASHAN, JULIA KOVSAN, ILANA KACHKO, HILLA OVADIA, AND ASSAF RUDICH
Department of Clinical Biochemistry and the Nutrition Center, Faculty of Health Sciences,
Ben-Gurion University of the Negev, Beer-Sheva, Israel
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I. Introduction
A. What are ROS and RNS and how do they function in biological systems?
B. ROS and RNS in normal physiology and in pathophysiology
C. Increased oxidative stress in insulin resistance states: obesity and diabetes
D. Key message from section I
II. The Oxidant-Antioxidant Balance in Health and in Insulin Resistance States
A. Systems for oxidants generation
B. The antioxidant systems
C. Key message from section II
III. Physiological Roles of ROS and RNS in Insulin Action
A. Oxidants have insulin-mimicking effects
B. Insulin induces ROS and RNS production
C. RNS and ROS are second messengers in insulin signal transduction
D. Insulin-like effects of ROS and RNS through alternative pathways
E. Key message from section III
IV. Cellular and Molecular Mechanisms for Impaired Insulin Action Induced by Oxidants
A. IR and IRS1 serine phosphorylation and enhanced degradation
B. Impaired signal transmission from PI 3-kinase to PKB
C. Disruption of spatial organization of the insulin signal: potential role for the actin cytoskeleton
D. Direct modification of insulin signaling components by oxidants
E. Alterations in gene regulation
F. Key message from section IV
V. Manipulation of the Oxidant/Antioxidant Balance to Enhance Insulin Action
A. Cellular systems
B. Experimental animal studies
C. Human studies
D. Key message from section V
VI. Summary and Conclusions
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Bashan N, Kovsan J, Kachko I, Ovadia H, Rudich A. Positive and Negative Regulation of Insulin Signaling
by Reactive Oxygen and Nitrogen Species. Physiol Rev 89: 27–71, 2009; doi:10.1152/physrev.00014.2008.—
Regulated production of reactive oxygen species (ROS)/reactive nitrogen species (RNS) adequately balanced by
antioxidant systems is a prerequisite for the participation of these active substances in physiological processes,
including insulin action. Yet, increasing evidence implicates ROS and RNS as negative regulators of insulin
signaling, rendering them putative mediators in the development of insulin resistance, a common endocrine
abnormality that accompanies obesity and is a risk factor of type 2 diabetes. This review deals with this dual,
seemingly contradictory, function of ROS and RNS in regulating insulin action: the major processes for ROS and
RNS generation and detoxification are presented, and a critical review of the evidence that they participate in
the positive and negative regulation of insulin action is provided. The cellular and molecular mechanisms by
which ROS and RNS are thought to participate in normal insulin action and in the induction of insulin resistance
are then described. Finally, we explore the potential usefulness and the challenges in modulating the oxidantantioxidant balance as a potentially promising, but currently disappointing, means of improving insulin action
in insulin resistance-associated conditions, leading causes of human morbidity and mortality of our era.
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0031-9333/09 $18.00 Copyright © 2009 the American Physiological Society
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I. INTRODUCTION
A. What are ROS and RNS and How Do They
Function in Biological Systems?
Physiol Rev • VOL
B. ROS and RNS in Normal Physiology
and in Pathophysiology
As outlined above, life in an aerobic environment is
constantly accompanied by generation of ROS and RNS.
In fact, multiple functions vital for normal physiology
make use of regulated ROS/RNS generation and subsequent alterations in redox state, one example of which is
the insulin signaling cascade. Yet, when normal regulation
of ROS/RNS generating processes and/or mechanisms to
shut-off ROS/RNS-initiated signals are disrupted, RS play
an active role in pathophysiological processes, including
in obesity and diabetes in which insulin signaling is impaired. A discussion of ROS/RNS generation and antioxidant mechanisms and the evidence for their dysregulation
in obesity and diabetes are presented in sections IC and II.
Below is an overview of ROS/RNS participation in normal
physiology and pathophysiology (Figs. 1 and 2).
1. Triggers for ROS/RNS generation
Production of ROS and RNS can occur in response to
diverse stimuli (Fig. 1A). These include extracellular factors signaling through plasma membrane receptors, like
hormones and growth factors, including platelet-derived
growth factor (216, 413), epithelial growth factor (12),
insulin (as detailed in sect. III), proinflammatory cytokines
like tumor necrosis factor (TNF)-␣, (69), and physicalenvironmental factors (like ultraviolet irradiation) (167,
371). In addition, intracellular factors or processes that
trigger ROS generation include nutrient metabolism (detailed in sect. IIA1B), endoplasmic reticulum stress (143,
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All biological systems functioning in aerobic conditions are exposed to oxidants, either generated intentionally or as byproducts. Multiple different species of oxidants are being generated, and as the first letter of the
terms reactive oxygen species (ROS) and reactive nitrogen species (RNS) implies, they are generally reactive
substances. In some ways, one can compare the function of oxidants in biological systems to that of acids,
which we are more used to think about in quantitative
terms. Reactive species (RS) are characterized by the
following.
They are a very diverse group of substances: there
are multiple types of ROS and RNS. The most widely
known ROS are superoxide ion (O2•⫺), hydrogen peroxide (H2O2), and peroxyl radical (OH•), and the common
RNS are nitric oxide (NO) and peroxynitrite (ONOO⫺).
ROS and RNS chemically interact, as do acids: a very
intricate chemistry of ROS and RNS generates multiple
species from those originally generated, occasionally resulting in a “hybrid RS” (like the generation of peroxynitrite from the rapid chemical interaction between NO and
O2•⫺). Furthermore, such interaction may convert a
non-RS to a RS, generating chain reactions. In that regard,
an “antioxidant” (or reductant) can be converted into an
oxidant, reminiscent of an acid’s conversion into its cognate base. Whereas pKa defines the thermodynamic balance of this conversion for a specific acid, the redox
potential of a specific RS is more difficult to measure.
Additionally, although acids react spontaneously, biological systems also include enzymes to accelerate some
reactions (like carbonic anhydrase for carbonic acid).
Similarly, enzymes are also involved in some reactions of
RS, like the conversion of O2•⫺ to H2O2 by superoxide
dismutase (SOD). A detailed description of ROS and RNS
chemistry and biochemistry is well beyond the scope of
this review, and the interested reader is referred to other
reviews on the topic (27, 155, 341, 345).
Different ROS and RNS vary in their chemical reactivity, i.e., their tendency to react chemically with
neighboring molecules: in principle, the more reactive a
RS is, the shorter its half-life, since it more rapidly
interacts with other molecules. In a biological system,
this of course results in different distances a particular
RS could travel from its site of generation: a less reactive ROS like H2O2 has a half-life of seconds and can
therefore theoretically travel relatively large distances,
even crossing cells and tissues, whereas the diffusion
distance of OH• is in the range of a small protein due to
its high reactivity.
Chemical characteristics determine the biological
milieu of activity: RS can be electrically charged or
electroneutral, hydrophobic or hydrophilic, governing
their ability to cross membranes and/or to partition
between aqueous and lipophilic (membranes) environments.
Oxidants are balanced by reductants (antioxidants): for
normal function (physiological conditions), homeostasis needs to be maintained, reminiscent of the balance
between acids and bases in biological systems. In the case
of acid-base balance, it is measured easily by the pH. For
ROS and RNS, redox balance needs to be maintained, but
is poorly defined and has no agreed-upon measures.
ROS and RNS have critical biological functions essential for normal physiology, just as acids do. Yet, overproduction, deficiency in the balancing forces [bases (in
the case of acid-base), reductants (in the case of redox
balance)], and/or generation of abnormal species (certain
organic acids; specific ROS and RNS) result in impaired
homeostasis and is associated with pathology. This point
related to RS is further discussed in the following section.
ROS, RNS, AND INSULIN ACTION
29
.
FIG. 1. Major triggers, generating mechanisms, types, and targets of reactive oxygen species (ROS) and reactive nitrogen species (RNS)
in biological systems.
.
166; detailed in sect. IIA1C), and the detoxification of
various xenobiotics (135, 399).
2. Generation mechanisms
Generation mechanisms (Fig. 1B, and further detailed in sect. IIA) include designated systems for intentional ROS/RNS generation like the NADPH oxidase (22)
and nitric oxide synthase (4, 316), mitochondrial electron
transfer (474), as well as enzymatic systems in which ROS
are generated as a byproduct. These include various oxiPhysiol Rev • VOL
dases (lipoxygenase, xanthine oxidase) and detoxification
mechanisms like the P-450 system (471).
The biological outcome of ROS/RNS generation depends on multiple factors partly presented in section IA.
Particularly important is which are the specific reactive
oxygen and/or nitrogen species that are being generated,
what is their cellular location and duration of production
(temporal-spatial coordinates), how much is generated, and
whether it overwhelms antioxidant defense systems. Together, the biological consequence of ROS and RNS gener-
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BASHAN ET AL.
ation is very much dependent on the “biological context,”
representing a highly combinatorial signal with certain specificity (302).
3. “Cellular/molecular targets” of ROS/RNS generation
4. Functional outcome of RS generation
Functional outcome of RS generation (Fig. 2) ranges
from the physiological to the pathophysiological range.
Vascular tone (149), cell adhesion (59, 416), immune responses (145), and growth factors and hormone action
(96, 179) are examples of RS participation in normal
physiology. Conversely, a causative role of RS has been
TABLE
1.
Different approaches to assess “oxidative stress” in biological systems
Approach
Examples
Markers of increased pro-oxidant activity
Markers of decrease in antioxidant activity
Altered cellular redox state
Oxidative damage parameters
Increase in oxidant-generating systems (NADPH oxidase,
mitochondrial ROS, NOS)
Direct measurements of ROS/RNS generation (reduction of NBT,
oxidant-sensitive dyes, direct radicals measurement by ESR)
Low-molecular-weight antioxidants (vitamins C and E, GSH)
Antioxidant enzymes (SOD, GR, catalase)
Total antioxidant capacity
Resistance to an external oxidant
Overall reducing activity (cyclic voltametry)
GSH/GSSG ratio
Lipid oxidation (TBARS, isoprostanes, 4-HNE)
Protein oxidation (protein carbonylation, S-nitrosylation,
nitrotyrosine, glutathionylation, 4-HNE conjugation)
DNA oxidation (8-hydroxydeoxyguanosine, dihydropropidium iodide)
ROS, reactive oxygen species; NOS, nitric oxide synthase; RNS, reactive nitrogen species; NBT, nitroblue tetrazolium; ESR, electron spin
resonance; SOD, superoxide dismutase; GR, glutathione reductase; 4-HNE, 4-hydroxynonenal; TBARS, thiobarbituric acid reactive substances.
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The “cellular/molecular targets” of ROS/RNS generation (Fig. 1C) in response to a specific stimulus are
the consequence of the biological context in which they
are generated and their specificity. Oxidants can react
with multiple cellular components (proteins, lipids, nucleic acids), generating reversible or irreversible oxidative modifications. They also activate various signaling
cascades, some of which are designated for sensing and
responding to “stress,” like the “stress-activated protein
kinases” of the mitogen-activated protein (MAP) kinase
family, JNK, and p38 MAP kinase (109, 353). Such signaling pathways alter proteins’ functions, half-life,
and/or gene regulation. As a principle, RS involvement in
normal physiological processes involves carefully regulated production in a tight spatial-temporal manner, leading to reversible oxidative modifications. Pathophysiological processes mediated by RS are more likely to involve
irreversible modifications of cellular components, be it
proteins, lipids, or DNA. Accumulation of irreversibly
modified biomolecules is a reasonable definition of the
ill-defined term oxidative stress (discussed further below,
and presented in Table 1).
implicated in ageing-related diseases (251), malignant
transformation (405), atherosclerosis (268, 403), neurodegenerative diseases (236), obesity, and diabetes (detailed
below).
The diversity of RS-mediated processes and their
broad range of involvement in physiology and pathophysiology make the definition of “pathological RS-mediated
processes” particularly challenging. To date, there is no
agreed-upon direct measurement of oxidative stress in
biological systems. Defining oxidative stress as the imbalance between pro- and anti-oxidant mechanisms has led
to the use of indicators of each side of the balance (Table
1). However, this view is somewhat limited. First, an
increase in antioxidants might be interpreted as “lower
oxidative stress.” Yet, it may in fact indicate a biological
adaptive response to an increase in pro-oxidant processes. This would imply that an elevation, rather than
decrease, in antioxidant defense, indicates increased oxidative stress. Second, if an increase in pro-oxidant processes is fully balanced by an upregulation of antioxidant
mechanism, a new steady-state is reached without adverse effects to the biological system. So, should oxidative stress be defined as an increase in the amount of
oxidation products of biological macromolecules (lipids,
proteins, and/or DNA, Table 1)? Finally, is an increase in
“oxidative stress” the equivalent of “oxidative damage,” or
does the latter also indicate a functional consequence to
the oxidative insult? These different ways of defining
oxidative stress, along with the different, mostly indirect
ways of measuring it (237), provide the fundamental basis
for confusion, controversies, and contradictory conclusions in the field (11). Keeping these limitations in mind,
the following section summarizes current evidence that
insulin resistance-related conditions, obesity and type 2
diabetes, are associated with increased oxidative stress.
ROS, RNS, AND INSULIN ACTION
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FIG. 2. Examples for ROS and RNS participation in
physiological and pathophysiological processes.
A wealth of evidence largely from the last 15 years
suggests increased oxidative stress in obesity (10, 13, 72,
122, 228, 410) and diabetes (20, 109, 122, 132, 137, 343,
462). Decreased antioxidant capacity, increased production of ROS, and elevated oxidation products of lipids,
DNA, and proteins have been reported in plasma, urine,
and various tissues, suggestive of systemic and organspecific oxidative stress. The more recent evidence for
systemic oxidative stress includes detection of increased
circulating and urinary levels of the lipid peroxidation
product F2-isoprostane (8-epi-prostaglandin F2␣) in both
type I and type II diabetic patients (81, 82, 289), and in
obesity (35, 83). Remarkably, this marker correlated with
blood glucose levels and responded to antidiabetic intervention (82). In nearly 3,000 obese persons from the Framingham Heart Study, 8-isoprostane level strongly correlated with body mass index (228). Increase in plasma
protein carbonylation, a group of different oxidative protein modifications characterized by various carbonyl moieties, was also reported in both type 1 and 2 diabetes (47,
426; reviewed in Ref. 78).
The evidence for oxidative stress in key target tissues
for insulin action has been somewhat less consistent.
Although this may largely reflect the inconsistencies (and
limitations) of the various approaches to measure oxidative stress, it may point to true physiological differences
between these tissues.
1. Adipose tissue
Supportive evidence for increased oxidative stress in
adipose tissue in obesity (and diabetes) includes the docPhysiol Rev • VOL
umentation of elevated oxidation products of both lipids
and proteins. Obese mice before developing diabetes exhibited increased H2O2 generation by adipose tissue, accompanied by decreased mRNA levels of SOD, catalase,
and glutathione peroxidase (122). The apparent perturbation of the pro-oxidant/antioxidant balance was manifested by elevated levels of thiobarbituric acid reactive
substances (TBARS)(122) or malondialdehyde (MDA)(126),
crude measures of lipid peroxidation. Developing diabetes in these mice mildly exaggerated these alterations,
which remained unobservable in liver, muscle, or aorta
(122). In addition, total protein carbonylation was reported to be two- to threefold higher in adipose tissue of
high-fat fed (HFF) mice (144). Concomitantly, mRNA levels of glutathione-S-transferase 4, a key enzyme responsible
for reversing lipid peroxidation adducts (4-hydroxynonenal,
4-HNE) on proteins, was decreased three- to fourfold, providing a potential mechanism for increased protein carbonylation in obesity.
2. Liver
Increase in markers of lipid peroxidation has been
reported in the livers of animal models of diabetes and
obesity (91, 113, 284, 415). However, these were not
matched by a decrease in the levels of dietary antioxidants like vitamin E, nor by a protective effect of antioxidant supplementation (113, 415). Obesity and the related
insulin resistance are frequently associated with increased accumulation of lipids (triglycerides) in the liver.
Hence, it remains unclear if the elevation in lipid peroxidation in liver really reflects increased oxidative stress, or
simply the increase in available substrates for lipid peroxidation (415). Furthermore, a decrease in the ratio of
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C. Increased Oxidative Stress in Insulin
Resistance States: Obesity and Diabetes
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BASHAN ET AL.
3. Muscle
Evidence for oxidative stress in muscle in human
obesity and diabetes is also somewhat limited. A recent
study reported no increase in 4-HNE in muscle of obese
diabetics compared with obese nondiabetic persons
(287). In experimental type 1 diabetes, decreased high- or
low-molecular-weight thiols have been reported in skeletal or cardiac muscles, as well as decrease in antioxidant
enzymes (91, 148, 231). Furthermore, increased S-nitrosylation of total muscle proteins as well as of key proteins
in the insulin signaling cascade has been reported in
nutritional and genetic obesity in rodents (49, 470). Despite these, in the KKAy mouse model of obesity and
diabetes skeletal muscle H2O2 production, TBARS and the
expression of antioxidant enzymes were unaltered, although they were affected in adipose tissue (122).
D. Key Message From Section I
RS, a diverse group of oxidant molecules, are involved in a broad range of biological processes ranging
from physiology to pathophysiology. In the latter, an imbalance between pro- and anti-oxidative mechanisms occurs, resulting in “oxidative stress,” an ill-defined term
likely representing an array of different conditions, underlying mechanisms, and accumulation of different oxidatively modified biomolecules. Consequently, there is currently no unifying measure to assess oxidative stress.
Moreover, gaps exist in our understanding of organ-specific oxidative stress in conditions associated with impaired insulin action, like obesity and diabetes. These
may reflect differences in models and parameters utilized
to measure oxidative stress. Conversely, they may represent true organ-dependent differences, and/or the existence of different “types” of oxidative stress in each tissue. Regardless, the current common notion is that oxidative stress is an accompanying feature of the obese/
diabetic state (109). The next section provides further
detail on major oxidant-generating and antioxidant sysPhysiol Rev • VOL
tems and their status in conditions of impaired insulin
action.
II. THE OXIDANT-ANTIOXIDANT BALANCE
IN HEALTH AND IN INSULIN
RESISTANCE STATES
A. Systems for Oxidants Generation
In the context of insulin resistance, excessive ROS/
RNS generation occurs as part of several, partially interrelated, pathophysiological mechanisms, which include
metabolic overload–the overabundance of glucose and
free fatty acids (FFA), inflammation, endoplasmic reticulum (ER) stress and the unfolded protein response (UPR),
and dysregulated hormonal and growth factors regulation
(Fig. 3). These rely on the main cellular sources for ROS
or RNS generation in physiological conditions, which include the NOX family of NADPH oxidases, mitochondrial
respiration, the ER, and nitric oxide synthases (Fig. 4),
each of which is discussed below.
1. ROS generation
A) NADPH OXIDASE. NADPH oxidases are membranebound enzymatic complexes comprising an electron
transport chain that transfers electrons from the donor,
cytosolic NADPH, to the acceptor, O2. Consequently, O2•⫺
(and H2O2) are generated, initially extracellularly or
within organelles (recently reviewed in Ref. 22). This
highly specialized system for intentional ROS generation
was thought for many years to be uniquely utilized by
phagocytic cells for oxidative burst, a process required
for pathogen killing. Yet, in recent years, homologs of the
cytochrome subunit of phagocytic NADPH oxidase were
discovered (NOX1-5 and DUOX1-2) (22). Phagocytes’ capacity to generate ROS is indeed the highest due to the
high level of expression and the specific kinetic properties
of the phagocytic NADPH oxidase NOX2 versus other
NOXs. Nevertheless, the discovery of multiple members
of the NOX family made it clear that most cells have the
capacity to generate ROS through an NADPH oxidase
system. Such ROS generation is increasingly recognized
to be required for diverse physiological functions, including the response to various hormones and growth factors.
Insulin-stimulated ROS production relies on NOX4 in adipocytes (272) and possibly also on NOX2 expressed in
skeletal and cardiac muscle (173, 209). NOX4 shares only
a mild (39%) sequence identity to NOX2 and is distinct
from it in several aspects worth mentioning here. NOX4
was proposed to be localized also within internal membranes (like the ER) and, hence, to generate ROS into the
lumen of such organelles (279, 440). Superoxide may
require a transporter to get into the cytosol, but H2O2,
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reduced to oxidized GSH (GSH/GSSG) in the liver was
reported in a model of type 1 (91), but not in type 2/obesity model (123). Despite this seemingly inconsistent evidence for oxidative stress in obesity and diabetes (possibly attributed to the different models and parameters
used), oxidative stress is accepted as a major driving
force in the pathophysiology of nonalcoholic steato-hepatitis (NASH, “fatty liver”) (reviewed in Refs. 112, 161).
Fatty liver is closely associated with insulin resistance,
yet oxidative stress may be particularly involved in progressive stages of NASH, like the conversion from steatosis to cirrhosis. It is therefore difficult to conclude if
NASH-associated liver oxidative stress is representative
of the liver in obesity and diabetes.
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ROS, RNS, AND INSULIN ACTION
FIG. 3. Major sources of ROS and RNS in pahtophysiology related to insulin resistance and obesity. The main proposed cellular sources for
increased ROS/RNS generation in obesity and diabetes are shown. The processes responsible include the following: i) High metabolic load:
dysregulated carbohydrate and lipid metabolism in obesity and diabetes exposes cells to an overload of nutrients. Excessive mitochondrial oxidation
may result in enhanced ROS generation. In addition, high levels of free fatty acids (FFA) can activate toll-like receptors, constituting an overlap
between ROS generation in response to metabolic and inflammatory cues. Finally, ROS can affect mitochondrial function, potentially resulting in
further ROS generation. ii) Inflammation: obesity has been demonstrated to be associated with a chronic inflammatory state characterized by both
increased circulating levels of proinflammatory cytokines (TNF, IL-6) and macrophage infiltration of adipose tissue. Macrophages can be a direct
source of ROS generation through their high-efficiency NADPH oxidase system (NOX2). Pro-inflammatory cytokines act via membrane receptors
to activate NADPH oxidases (NOX4), to accelerate mitochondrial ROS generation, and to induce endoplasmic reticulum (ER) stress. iNOS is an
NOS-generating system activated by inflammatory cues. iii) ER stress: has been initially proposed to occur in beta cells, but recently proposed to
occur in adipose tissue in obesity. Like mitochondria, dysfunctional ER can be both a source and a consequence of increased ROS generation.
iv) Endocrine dysregulation: impaired endocrine regulation can result in altered circulating levels of hormones (in type 2 diabetes, elevated
circulating insulin levels are a common compensatory response to peripheral insulin resistance). Many hormones activate NADPH oxidases and rely
on ROS generation as part of their signaling cascades. AGE, advanced glycation end products; G6PD, glucose-6-phosphate dehydrogenase; PKC,
protein kinase C; TNF-R, TNF receptor; TLR, toll-like receptor; UPR, unfolded protein response.
likely the consequence of O2•⫺ dismutation within the
lumen by SOD, can cross membranes directly. Second,
different from NOX2 that depends on the phosphorylation
and assembly of several cytosolic components, functional
NOX4 is independent of those for activity (130, 279, 389).
Nevertheless, a requirement for the membranal p22Phox
has been proposed (227, 279), and for the small GTPase
Rac demonstrated in cells endogenously expressing
NOX4 (138, 195). Further discussion of the requirement of
ROS production by NOX4 in normal insulin signaling is
presented in section III.
Physiol Rev • VOL
What is the evidence that NADPH oxidases contribute to enhanced ROS production in conditions related to
insulin resistance? In adipose tissue of obese mice, NOX4
was increased, as were p22Phox, NOX2, and other components of its multiprotein complex (p67Phox, p47Phox, and
p40Phox) (122). NOX2 in adipose tissue of obese mice may
arise either from obesity-associated upregulation of
NOX2 in the adipose cells, or from phagocytic cells infiltrating this tissue in obesity (454, 466). Regardless of the
exact NOX, increased ROS production in 3T3-L1 adipocytes incubated with FFA (122) or in hyperglycemic con-
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(iNOS)
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BASHAN ET AL.
ditions (463) was inhibited by chemical inhibitors thought
to inhibit mainly NADPH oxidases. In HFF mice, increased ROS production by adipocytes was inhibited by
both inhibitors of NADPH oxidases, as well as by inhibiting protein kinase C (PKC)-␦ (420), indicating PKC-␦ as
an upstream regulator of NADPH-mediated adipocyte
ROS production in vivo. Similarly, impaired cardiac and
vascular function (including insulin-induced vasodilation)
in diabetes, HFF, and in response to fatty acids or to TNF
were all reported to be mediated by NADPH oxidasedependent ROS overproduction in the endothelium (60,
224, 257, 335, 395). In humans, expression of p47phox in
endothelial cells correlated with body mass index and
was significantly elevated in obese and overweight compared with lean controls (390). Studies on human leukocyte function in diabetes are somewhat contradictory.
Some studies report higher levels of nonstimulated or
stimulated superoxide generation and/or p47phox in neutrophils’ membranes from diabetics (92, 176, 221, 312),
consistent with a primed state of the NADPH oxidase
complex. Other reports suggest decreased neutrophil
ROS generation particularly in the stimulated state (86,
197, 369, 425). This was proposed to underlie, along with
Physiol Rev • VOL
deficiencies in other neutrophil functions [like phagocytic
capacity (6)], the elevated risk for bacterial infections in
some diabetic patients. These discrepancies either reflect
different patient populations and disease severity, methodologies, and/or the possibility that in vivo basal (unstimulated) ROS generation is elevated, but activation of
the NADPH oxidases in response to infectious stimuli is
impaired.
The exact mechanism for increased NOX-mediated
ROS production in adipocytes in obesity is unclear. With
the use of other cell systems, it was demonstrated that
hypoxia (412, 439), inflammatory cytokines like TNF
(286) and angiotensin II (175, 461), and ER stress (327) all
induce upregulation of NOX4 mRNA and protein levels.
All of these conditions or factors have been implicated as
causative factors in insulin resistance of adipose tissue
and/or muscle (143, 184, 185, 193). Furthermore, peroxisome proliferation activator receptor (PPAR)-␥ ligands
used as insulin sensitizing agents were shown to downregulate protein and mRNA expression of NOX4 (and
NOX2) in endothelial cells (192). In adipose tissue, obesity was shown to increase the expression of glucose-6phosphate dehydrogenase (G6PD) (324). This rate-limit-
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FIG. 4. Major ROS and RNS generation systems. Generation mechanisms of ROS or RNS are depicted in blue boxes, while reactive species are
shown in stars. AGE’s, advanced glycation end products; ER, endoplasmic reticulum; Mito, mitochondria; NO, nitric oxide; ONOO⫺, peroxynitrite;
NOS, nitric oxide synthase; O2•⫺, superoxide; OH•, hydroxyl radical; H2O2, hydrogen peroxide; XaOX, xanthine oxidase.
ROS, RNS, AND INSULIN ACTION
Physiol Rev • VOL
reaction far faster than the dismutation to H2O2, generating peroxynitrite. Likewise, H2O2, through interaction
with transition metals, may form OH• (the Haber-Weiss or
Fenton reactions, Fig. 4). Both OH• and peroxynitrite are
powerful oxidants that propagate oxidative chain of reactions. Hence, the biological outcome of mitochondrial
ROS production and its potential involvement in physiological versus pathological processes depends on the extent and site of superoxide generation, other RS formed
simultaneously, and the balance with detoxifying mechanisms. The reader is referred to several excellent reviews
on ROS and RNS (bio)chemistry in the mitochondria (404,
436, 474).
Increased mitochondrial ROS generation has been
strongly implicated as a mediator between hyperglycemia
and its pathological consequences in the vasculature
(306), kidney (233), neurons (445, 446), retina (260), and
pancreatic beta cells (364). As presented above, superoxide is generated in the mitochondria from complexes of
the respiratory chain in their reduced (electron-bound)
state. Respiratory chain complexes remain electronbound when they cannot transfer electrons downstream.
Most frequently, such conditions arise when the proton
gradient between the mitochondrial matrix and the intermembrane space is high (high membrane potential, ⌬␺),
preventing further outward pumping of H⫹. In such state,
chemical uncouplers or uncoupling proteins (UCPs)
could relieve ROS production, as they allow proton reentry into the mitochondrial matrix and electron transfer to
oxygen. Indeed, this scenario has been suggested in response to high glucose. The increased glucose flux
through glycolysis and the tricarboxylic acid (TCA) cycle
generates excess electron donors (NADH), initially resulting in high ⌬␺ and increased mitochondrial ROS production. In principle, such a mechanism should also operate
in response to fatty acids (an additional source of NADH),
as was shown in endothelial cells (306). Increasing antioxidant capacity or decreasing mitochondrial superoxide
production with uncouplers prevented high-glucose-induced activation of PKCs, NF␬B, generation of advanced
glycation end products (AGEs) and sorbitol accumulation
in endothelial cells (42, 306), all pathogenic mechanisms
for diabetic vascular complications (reviewed in Ref.
348).
In adipose tissue and in skeletal muscle, the primary
targets for the metabolic actions of insulin, evidence supporting these mechanisms is scarce. In 3T3-L1 adipocytes,
high glucose was shown to induce mitochondrial ROS,
which mediated insulin resistance and activation of inflammatory pathways (261). This scenario is essentially
the one proposed for endothelial cells presented above
(306). In contrast, in response to FFA, ROS production in
3T3-L1 adipocytes (122), or in isolated adipocytes from
HFF mice (420), was proposed to be mediated through an
NADPH oxidase, not mitochondria, based on chemical
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ing enzyme of the hexose monophosphate shunt is a
major source of NADPH. Thus both elevated NOX4 expression and increased production of its substrate
NADPH potentially contribute to enhanced O2•⫺ production in adipose tissue in obesity. Interestingly, vascular
cells were shown to have decreased activity of G6PD in
response to hyperglycemia (475), suggesting cell-type specific adaptations to metabolic stress.
Collectively, the NADPH oxidases are a major source
of ROS production in normal physiology and of overproduction in pathophysiological settings related to insulin
action.
B) MITOCHONDRIAL ROS GENERATION. The mitochondrial
electron transport chain is a major source of ROS production. Its primary function is in respiration, delivering electrons from NADH and FADH2 to molecular oxygen while
generating an electrochemical gradient across the inner
mitochondrial membrane. Yet, even under normal physiological conditions, it is estimated that incomplete electron transfer to oxygen resulting in ROS production
(mainly O2•⫺) occurs with at least 0.2–2% of O2 molecules,
generating a large oxidative burden (51, 398). In this
respect, mitochondrial ROS production is a byproduct of
aerobic ATP production. As such, it has been implicated
in various pathophysiological conditions, including degenerative disease, ischemia-reperfusion injury, and aging
(8, 267, 386, 404). Yet, it is increasingly recognized that
mitochondrial ROS generation is regulated and may be
important for various cellular functions, suggesting that
its view exclusively as a toxic byproduct is an oversimplification.
Electrons can leak from the electron transfer chain
generating O2•⫺ mainly from complexes I and III, as a
result of partial reduction of molecular oxygen. Superoxide ion is released into the mitochondrial matrix (by
complexes I and III-Qi), or outwards to the intermembrane space (by complex III-Qo) (404, 436, 474) (Fig. 5).
Given their negative charge, O2•⫺ formed in the inner
matrix cannot readily diffuse out of the mitochondrial
matrix, though a passage through the voltage-dependent
mitochondrial anion channel of the mitochondrial permeability transition pore has been described (156). More
commonly, O2•⫺ in the matrix is rapidly dismutated to the
readily permeable H2O2 by Mn-SOD (SOD2), a process
that can also occur in the intermembrane space or the
cytosol by CuZn-SOD (SOD1) (404). Final detoxification
of mitochondrial superoxides can occur by converting
H2O2 to H2O by catalase (at high H2O2 concentrations) or
glutathione peroxidase (at lower concentrations), as per
the relative Km of the two enzymes (see sect. IIB). These
systems are essential for limiting oxidative damage of
mitochondrial structures, particularly in the inner mitochondrial matrix, as exemplified by the lethal phenotype
of SOD2 knockout mice (441). Furthermore, even in the
presence of SOD, O2•⫺ may react with NO in a chemical
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BASHAN ET AL.
inhibitors. However, what exactly is the mechanism for
altered mitochondrial function in adipocytes and skeletal
muscle?
In both tissues, evidence exists for decreased respiratory capacity associated with insulin resistance and/or
diabetes (287, 334, 460). Theoretically, a decrease in mitochondrial respiration could represent decreased number (mass) of mitochondria, impaired function of the
respiratory chain, and/or a decrease in mitochondrial uncoupling (decrease in UCPs). The latter two can be associated with elevated ROS production due to higher likelihood of respiratory complexes remaining in an electronPhysiol Rev • VOL
bound state. A dysfunctional downstream complex creates a
“bottle neck” for electron transfer, thus retaining upstream
complexes in a reduced state. Such dysfunction of a respiratory complex could be genetically determined, and/or
could represent a consequence of oxidative damage to the
mitochondria, suggesting a pathological futile cycle. Decreased UCPs expression/function is associated with high
⌬␺, which as explained above, is associated with enhanced ROS generation. These different mechanistic possibilities are difficult to differentiate in vivo. In adipocytes, decreased mitochondrial gene expression attributed to reduced mitochondrial mass was documented
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FIG. 5. ROS generation by the ER and its interrelations to mitochondrial ROS generation. ROS can be generated in the ER as part of oxidative
protein folding. To generate correctly folded protein with disulfide bonds formed between the correct cysteines (in green), electrons are donated
to oxidoreductases (PDI, Ero1) and ultimately to molecular oxygen, yielding H2O or ROS. When incorrect disulfide bonds form (in orange), they need
to be reduced by GSH, resulting in a further decrease of GSH/GSSG ratio, altering the redox state within the ER. Alternatively, misfolded proteins
can be directed to degradation through ER-associated degradation machinery (ERAD). Accumulation of misfolded proteins in the ER initiates the
unfolded protein response (UPR), part of which is programmed to enhance antioxidant defense. Calcium ions released from the ER can augment
mitochondrial ROS generation. This occurs through the elevation in electron donors to the electron transport (respiratory) chain through the
stimulation of the tricarboxylic acid cycle. In addition, calcium ions increase cytochrome c release, impairing electron transfer and increasing ROS
generation. This in turn can impair respiratory complexes, further augmenting ROS generation. ATF6, activating transcription factor 6; ATF4,
activating transcription factor 4; eIF2␣, eukaryotic translational initiation factor 2␣; ERAD, ER-associated degradation; IRE, inositol-requiring
enzyme-1; Nrf2, nuclear respiratory factor 2; ox/red-Ero1, ER oxidoreductin 1; ox/red-PDI, oxidized/reduced protein disulfide isomerase; PERK,
PKR-like eukaryotic initiation factor 2␣ kinase; XBP1, X-box protein 1.
ROS, RNS, AND INSULIN ACTION
Physiol Rev • VOL
accumulation of misfolded proteins that initiate the unfolded protein response (UPR), ER-derived ROS generation may increase (275). Furthermore, interorganellar
communication between mitochondria and ER may underlie increased mitochondrial ROS generation originating from ER-derived signals. Intriguingly, excessive activation of the UPR, or “ER stress,” has been implicated in
the pathogenesis of insulin resistance associated with
obesity (and with pancreatic beta cell failure related to
type 2 diabetes). Gaps still exist in the basic understanding of the intricate interrelations between oxidative and
ER stresses, but the two conditions seem to share common downstream signaling pathways that can impinge on
insulin signaling. Thus, as several excellent recent reviews are available describing ER stress and UPR in molecular details (73, 143, 249, 275, 372), this section highlights the proposed mechanisms for ER-based ROS generation, and the possibility that within the context of ER
stress, ROS generation contributes to the pathogenesis of
insulin resistance.
Oxidative protein folding in the ER requires an electron transfer chain, which is less characterized than that
in mitochondrial respiration, but shares some similarities
with it (Fig. 5). Electrons freed from the two cysteines
that form a disulfide bond are donated to ER oxidoreductases like protein disulfide isomerase (PDI), then to ER
oxidoreductin 1 (Ero1), a flavin adenine dinucleotide
(FAD)-containing protein, and finally, to molecular oxygen (433). As in mitochondrial ROS generation, which is
augmented when metabolic load is increased, the ER
electron transfer to oxygen is a likely source of ROS
generation particularly when the demand for it is increased. In such conditions, a more severe redox imbalance may result from the excessive utilization of GSH to
reduce inappropriate disulfide bonds formed between
mispaired cysteine residues. Although the mechanisms
regulating the redox balance within the ER are not well
characterized, it is highly conceivable that the combination of increased ROS generation and GSH consumption
by the ER would on its own induce oxidative stress. Yet,
ER-derived signals can also augment mitochondrial ROS
generation (Fig. 5). As a major Ca2⫹ storage site, perturbation of the ER, like in response to oxidative or ER
stress, can readily result in elevated cytosolic and mitochondrial Ca2⫹ levels (139). This can stimulate mitochondrial ROS production by enhancing generation of reducing equivalents (mitochondrial Ca2⫹ would enhance the
TCA cycle) while interfering with mitochondrial electron
transfer (due to Ca2⫹-induced release of cytochrome c
through the permeability transition pore, inhibition of
complex IV secondary to Ca2⫹-stimulated generation of
NO by nitric oxide synthase) (28, 204). Moreover, Ca2⫹induced opening of the mitochondrial permeability transition pore could also result in depletion of intramitochondrial reducing equivalents. Thus the ER, particularly
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with the initiation of obesity in the leptin-deficient ob/ob
mouse (460). Remarkably, this could be prevented by a
thiazolidinedione (TZD) PPAR-␥ agonists, potentially by
activating transcription factors involved in mitochondrial
biogenesis, like PGC1 (460). Intriguingly, PGC1 can enhance mitochondrial antioxidant defense (upregulating
Mn-SOD), possibly contributing to the capacity of TZDs to
prevent ROS production and protein carbonylation in adipocytes exposed to dexamethasone or TNF (188). Counterintuitively, UCP2 expression was shown to be markedly increased in white adipose tissue of mouse obese
models compared with lean controls (346, 414). Thus, in
adipose tissue, only minimal evidence exists for enhanced
mitochondrial ROS production and is associated with
decreased mitochondrial mass.
In skeletal muscle, evidence for mitochondrial ROS
overproduction in both human and rodent models is
scarce, and in some studies was not documented even
when such evidence was documented in adipose tissue
(122, 287). Yet, in skeletal muscle of insulin-resistant or
diabetic people, it appears that decreased respiratory
function is a consequence of reduced mitochondrial mass
and/or increased type II (glycolytic) muscle fibers (287,
370), and could underlie the development of insulin resistance without enhanced ROS production (334). Whether
skeletal muscle UCP expression is altered in diabetes also
remains controversial (17, 379). A recent study proposed
that in muscle of diet-induced insulin resistant mice, mitochondrial dysfunction is in fact a consequence, not the
cause, of increased ROS production (38).
Thus clear evidence linking mitochondrial ROS production and impaired insulin action in muscle and fat is
limited. Nevertheless, a mitochondrial source of ROS/
RNS production in these tissues, possibly from endothelial cells, is possible. Its relative contribution to overall
oxidative burden and its physiological/ pathophysiological significance in vivo awaits further studies.
C) ER ROS GENERATION. The ER is an additional organelle
that recently attracted attention as a source of ROS generation (73, 275). The ER is where an estimated one third
of translated proteins undergo full assembly and posttranslational protein modifications, which are required
for their normal subcellular targeting and biological functions. Among other ER-based posttranslational modifications like glycosylation, the local environment within the
ER supports the generation of disulfide bonds, which are
critical for proteins’ correct tertiary and quaternary structure stabilization. This is achieved by the presence of
oxidoreductases and a relatively oxidizing environment
(lower GSH-to-GSSG ratio) compared with the cytoplasm.
It is estimated that oxidative protein folding may account
for as much as 25% of total cellular ROS generation (433).
When the requirement for ER-supported protein folding
increases, whether as a consequence of increased demand for newly synthesized proteins or secondary to
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Physiol Rev • VOL
the requirement of normal UPR response for beta-cell
survival and function. More recently, ER stress was proposed as a putative mechanism also for insulin resistance,
particularly in adipose tissue and the liver (143, 300, 314,
315). Adipose tissue from both nutritional and genetic
models of obesity exhibit markers of increased ER stress
(314), including phosphorylation of eIF2␣, PERK, and
elevated expression of the ER chaperone GRP78. Mice
with heterozygous deletion of XBP1, a genetic means of
exaggerating ER stress, faired worse metabolically compared with wild-type controls when placed on HFF (314).
Similarly, genetic loss of a protective protein induced by
the UPR, ORP150, caused impaired glucose tolerance,
even when this loss was restricted to the liver (300, 313).
Furthermore, mice with a mutant eIF2␣ also have more
severe metabolic response to HFF (373). Although this
may be attributed also to beta-cell dysfunction, the possibility that defective eIF2␣ signaling impaired the antioxidant response to ER stress makes it particularly noteworthy herein. The cause-effect relationship between ER
stress and insulin resistance was further offered by the
demonstration that impaired insulin response can be relieved in mice by chemical chaperones that reduce ER
stress (315), or by overexpression of ORP150.
The mechanism for the contribution of ER stress to
insulin resistance, and particularly, the role ROS/RNS
exert in this process, is still largely unknown. Strongest
evidence exists for IRE-mediated phosphorylation of the
stress-activated MAP kinase JNK as a molecular mechanism connecting ER stress and impaired insulin signaling
(314). This kinase has been implicated in phosphorylating
insulin receptor substrate protein 1 (IRS1) on serine residues that are inhibitory for insulin-initiated signal transduction (see also sect. IVA). Indeed, cells deficient in IRE
exhibit lower activation of JNK and lower Ser phosphorylation of IRS1 in response to tunicamycin (314), a commonly used inducer of experimental ER stress. Yet, JNK
can be activated by inflammatory cytokines and directly
by oxidants, both of which can either be or not be related
to ER stress, as described above for ER-related ROS
generation. Therefore, it would seem fair to conclude that
current understanding of the role of ROS generation in the
context of ER stress as a mechanism for insulin resistance
still requires further studies. These may be particularly
challenging given the complex interrelations between oxidative and ER stresses and would still require proof for
playing a significant role in the pathogenesis of human
insulin resistance.
2. RNS generation: NO synthases
NO synthases (NOS) are the major source of NO
production, an RNS that perhaps best reflects the complex and ambivalent roles of RS in physiology and pathophysiology. Its major physiological action is in mediating
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when the UPR is activated, may be a significant source of
ROS generation and of oxidative stress.
Although the section above describes how ER stress/
UPR is a likely source of oxidative stress, the two phenomena are more intricately interrelated. Oxidative stress
can also be a cause, rather than a consequence, of ER
stress, as it can be the primary reason for protein misfolding. In addition, the UPR constitutes a complex response (reviewed in Ref. 249) that can also induce antioxidative capacity as one of its arms (Fig. 5). Specifically,
one of the primary effectors of the UPR is the activation
of PKR-like ER kinase (PERK). The best characterized
functional consequence of PERK activation is the inhibition of protein translation through Ser51 phosphorylation
of the translation initiation factor eIF2␣. This arm of the
UPR serves to decrease the load of “client proteins” on
the ER machinery. However, another substrate of PERK is
Nrf2 (74), a member of ubiquitously expressed transcription factors that are best known to be activated in response to oxidative stress, and to upregulate detoxifying
and antioxidant enzymes. This function is mediated by
Nrf2 binding to the antioxidant response element (ARE)
in the promoters of the genes encoding for these enzymes
(361). Nrf2 target genes include heme oxygenase 1, glutathione-S-transferase, and the rate-limiting enzyme in
glutathione biosynthesis ␥-glutamylcysteine synthetase
(457). Hence, in the context of the UPR, PERK-mediated
Nrf2 activation serves to enhance the increased demand
for reduced glutathione (GSH), which as mentioned
above, frequently accompanies ER stress. An additional
“antioxidant” mode of the UPR is the upregulation of the
ER-associated degradation (ERAD) machinery through
which misfolded proteins are targeted to degradation
(208). This response, mediated by IRE through activation
of Xbp1, decreases the load of proteins that consume GSH
for reducing inappropriate disulfide bonds, hence preventing deterioration of the GSH/GSSG ratio and oxidative stress (166). Collectively, ER stress and oxidative
stress are intimately interrelated. Each can cause the
other, they share common signaling pathways (the activation of stress-responsive kinases like JNK and other
effectors like CHOP), and also, they share the attempt to
enhance antioxidant defense through Nrf2.
In the context of obesity and type 2 diabetes, ER
stress has been mostly implicated as a potential mechanism for pancreatic beta-cell failure (recently reviewed in
Ref. 372). The response to obesity-related insulin resistance is an attempt to increase circulating insulin levels to
maintain glucose homeostasis, thereby increasing the synthetic load on the beta cells. This challenge on the ER
folding machinery predisposes the beta cells to ER stress,
and renders them particularly sensitive to defective UPR
response. Indeed, PERK knockout mice and humans with
mutant PERK (Wolcott-Rallison syndrome) develop diabetes due to beta-cell apoptosis (87, 160), demonstrating
ROS, RNS, AND INSULIN ACTION
Physiol Rev • VOL
NF␬B, and via various stress and inflammation-responsive
kinases like p38 MAPK and ERK (244). Differently from
eNOS and nNOS, its dependence on calmodulin is unaffected by Ca2⫹ (464), suggesting that iNOS is regulated
differently from the two other NOSs by both gene expression and allosteric regulation. Furthermore, under inflammatory states, O2•⫺ generation may also be enhanced.
This likely reflects enhanced production through NADPH
oxidases (NOXs; see sect. IIA1A), but may also be the
consequence of increased mitochondrial ROS generation
(see sect. IIA1B). Alternatively, O2•⫺ generation may be
the results of “uncoupled” NOS activity itself due to reduced availability of tetrahydrobiopterin (BH4), a NOS
cofactor essential for the coupling of L-arginine to NADPH
to generate NO (70, 442). Regardless of the exact O2•⫺
source, increased levels of NO (generated by iNOS) vis-à
-vis O2•⫺ results in the generation of the highly reactive
oxidant peroxynitrite (ONOO⫺) through a rapid direct
chemical reaction (Fig. 4). Peroxynitrite, either directly or
through complex chemical reactions with CO2, protons,
and other RS intermediate products, modifies thiol groups
(to promote S-nitrosylation and disulfides), side chains of
tyrosine or other amino acids (nitrotyrosine, histidinyl),
and can interact with transition metals centers. The consequential alteration in proteins’ function renders peroxynitrite a central mediator of NO-derived oxidative
stress and damage. The reader is referred to an excellent
recent review on the topic for further details (316).
Implicating NOSs as a significant source of RS contributing to the pathophysiology of diabetes and obesity is
complex. First, because direct measurements of NO products (nitrate and nitrite) are not clearly increased in obesity and diabetes (270, 366), possibly since they represent
a complex measure of NO generation and elimination.
⫺
Nevertheless, increased NO⫺
2 and NO3 have been reported, though mainly in persons with late diabetes complications (58, 200, 270). Second, since NO has vital physiological functions (vasodilatation), decreased NO generation and/or bioavailability may in fact be the pathological
link between NO and obesity/diabetes, rather than excessive production (146, 459). This would potentially contribute to endothelial dysfunction and hypertension, which
frequently associate with both conditions. The main putative mechanisms for lower NO bioavailability in obesity
and/or diabetes-related conditions include 1) decreased
NOS expression (396); 2) increased levels of endogenous
inhibitors (99, 277); 3) increased cogeneration of O2•⫺,
either by uncoupled NOS activity in diabetes (222, 223,
388) or through non-NOS-dependent mechanisms for
O2•⫺ generation (104); and 4) a manifestation of insulin
resistance itself, as insulin increases eNOS-mediated NO
generation (232; further discussed in sect. III).
Complementing the above is the evidence of dysregulated expression of NOSs in various tissues of human and
model diabetes and obesity. Genetic and nutritional ani-
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endothelium-derived vasodilatation. Moreover, given its
high reactivity with O2•⫺ (to form peroxynitrite, ONOO⫺),
it is even considered to exert “antioxidant effect” and
anti-inflammatory effects by limiting O2•⫺-induced NF␬B
activation and the ensuing state of chronic inflammation
(207). In contrast, NO is also an important mediator of
inflammation, and protein nitrosative modifications can
alter protein function, in some instances leading to irreversible protein damage. Moreover, peroxynitrite and
other nitrogen-containing RS are potent oxidants and are
major factors in mediating oxidative stress. As will be
discussed in following sections, all these translate into a
dual role in insulin action, with NO exerting a physiological as well as a pathophysiological role (i.e., in insulin
resistance). How is this duality in function that is so
typical of the entire oxidant field possible? In the case of
NO biology, much of this can be attributed to the family of
NOS, their expression profile, regulation, and enzyme kinetics properties, as detailed next.
NOSs catalyze the generation of NO from L-arginine,
relying on the cofactors flavin mononucleotide (FMN),
flavin adenine dinucleotide (FAD), heme, reduced nicotine adenine disphosphononucleotide (NADPH), and
(6R)-5,6,7,8-tetrahydrobiopterin (BH4) (4). Compared
with other RS, NO has a rather high diffusion distance in
biological systems given its lipophylic nature, neutral
charge, and relative low reactivity (316). Its major route of
elimination is the conversion to nitrate through the reaction with oxyhemoglobin, resulting in a half-life of 1 s. The
best-characterized physiological functions of NO, and
those which participate in the physiological response to
insulin, are attributed to the constitutively expressed endothelial NOS (eNOS, NOS3) and the neuronal NOS
(nNOS, NOS1). nNOS is in fact expressed in all major
insulin target tissues (except for adipose tissue), including liver (105) and skeletal muscle (220, 298). eNOS,
though, may be found in some nonendothelial cell types,
is probably mainly expressed in the endothelial cell component of various insulin target tissues including liver
(383), skeletal (220), and cardiac muscles (15), and in
adipose tissue (220). Its prime physiological function is
the generation of NO to promote vasodilatation. In accordance, and as will be further detailed in section III, the
vasodilatatory action of insulin is mediated through the
activation of NO production by eNOS. Both isoforms of
NOS are regulated by intracellular Ca2⫹ levels and by
calmodulin, generating NO in relatively short bouts to
levels reaching nanomolar concentration range (316).
In contrast to the constitutive NOS isoforms and their
role in normal physiology, the inducible NOS (iNOS,
NOS2) is responsible for NO generation reaching micromolar range. iNOS is expressed in inflammatory cells and
is robustly induced in most cell types including adipocytes and muscle cells by inflammatory signals (303, 304).
This response is mediated through the activation of
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BASHAN ET AL.
3. Other pathways and mechanisms contributing
to ROS production in insulin resistance states
Several additional pathways have been proposed to
contribute to ROS generation in insulin resistance-related
conditions (Fig. 4). Of particular interest in the context of
hyperglycemia is the generation of ROS related to autooxidation of glucose (190) and to the generation of nonenzymatically glycated proteins (Amadori products and
AGEs)(189). Receptors for AGE (RAGE) have been identified and shown to mediate ROS generation through the
interaction with extracellular AGE proteins (376, 468).
Interestingly, it was suggested that ROS generation in
response to activated RAGE is mediated via the NADPH
oxidase complex (452), and inhibition of NADPH oxidases prevented AGE-mediated damage in diabetes nephropathy (427).
Xanthine oxidase (XO) catalyzes final steps in purine
nucleotide degradation, with generation of superoxide.
Hence, theoretically, elevated activity of this enzyme
could be a source of increased ROS generation. Indeed,
some evidence links XO as a source of increased ROS
generation in diabetes and obesity. High-fat feeding was
Physiol Rev • VOL
shown to increase muscle arterioles XO activity and protein level, and allopurinol (an inhibitor of XO) improved
endothelial function (104). Increased XO activity was also
reported in livers of a rodent model of type 1, and allopurinol treatment diminished the increase in lipid hydroperoxide levels in plasma, liver, and heart of these
mice (91).
Finally, in addition to the relative contribution each
mechanism for ROS/RNS generation exerts, in the living
cell in which all or some mechanisms may operate simultaneously they frequently interact, raising various feedforward loops culminating in further oxidant production.
For example, S-nitrosylation of proteins of the mitochondrial electron transport chain (particularly complex I and
IV) may exaggerate mitochondrial ROS generation (77).
Evidence for mitochondrial NOS activity exists (85),
which may switch from NO to superoxide generation in
response to hyperglycemia (41). ROS and lipid peroxides
may also stimulate mitochondrial ROS (56, 296), constituting an additional amplification loop of ROS generation.
B. The Antioxidant Systems
Oxidative stress is frequently defined as the consequence of an imbalance between pro-oxidant processes
(oxidants generation, described in sect. IIA) and antioxidant defense mechanisms. The latter likely evolved to
counterbalance and limit the availability and potential
hazardous effects of excessive ROS or RNS generation,
and is thus an important component of the redox balance.
Yet, as mentioned above, when assessed in isolation, the
antioxidant side of the redox balance somewhat indirectly reflects on the role of oxidants, the topic of this
review. This is because low levels of antioxidants may be
the primary cause of oxidative stress, or rather represent
its consequence (reflecting increased consumption). Likewise, upregulation of antioxidant enzymes may reflect
increased antioxidant capacity, but may in fact represent
the biological response to increased RS generation. Nonetheless, antioxidant supplementation is a common potential therapeutic strategy applied in conditions associated
with “increased oxidative stress.” The shortcomings inherent to this approach lead to frequently disappointing
results in clinical trials related to insulin resistance, and
will be described in more detail in section V. Here, for the
sake of completeness, we briefly describe the antioxidant
defense systems, with emphasis on its components currently available for intervention trials described later. The
reader is referred to other reviews on the subject for more
detail (64, 153, 154, 250, 382).
The antioxidant defense capacity largely depends on
small molecules with antioxidant activity and on enzymes
catalyzing oxidant-modifying reactions (Fig. 6). Since antioxidant activity is broadly defined as the capacity to
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mal models of obesity demonstrate increased iNOS expression in skeletal and cardiac muscle, in adipose tissue
(329, 476), and in ob/ob mice also in liver (120). Yet, in
Zucker rats, total NOS activity and eNOS immunoreactivity in skeletal muscle were decreased compared with lean
controls (396). In humans, eNOS and iNOS, but not nNOS,
were shown to be more highly expressed at mRNA levels
in adipose tissue and isolated adipocytes of obese persons
(102, 103), although Western blot analysis confirmed increased protein levels only for eNOS (102). Consistently,
in diabetic kidneys, eNOS was increased due to elevated
expression in endothelium, whereas iNOS was increased
due to higher presence of inflammatory cells (178).
Jointly, it appears that dysregulated NOS activity accompanies diabetes and obesity, contributing to altered
ROS/RNS generation, though the proposed mechanisms
involved are variable. The increased abundance of nitrosative protein modification in diabetes and obesity (discussed in sects. IC and IVD) underscores the notion that
NOS contribute to dysregulated ROS/RNS generation in
insulin resistance-associated disorders. Consistent with
this view are studies with iNOS knockout mice. These
mice are protected against insulin resistance, attributed
to improved skeletal muscle, but not adipose tissue, insulin action, and glucose disposal (329). iNOS was also
proposed to play a role in hepatic insulin resistance in
ob/ob mice (120). Since iNOS is induced by inflammatory
signals, frequently in conjunction with increased superoxide generation, it is conceivable that in these tissues
peroxynitrite is elevated, contributing insulin resistance
potentially through nitrative protein modification.
ROS, RNS, AND INSULIN ACTION
41
antagonize oxidants, both “designated” antioxidants
and other endogenous metabolites (like uric acid and
bilirubin) contribute to the low-molecular-weight-derived antioxidant capacity. Hence, antioxidants are diverse compounds classically divided into preventive
and chain-breaking antioxidants based on their mode of
biochemical action (154). The preventive antioxidants
prevent the initiation of radical chain reactions through
direct scavenging of ROS, or through the binding of transition metals. The chain-breaking antioxidants are capable of interrupting the propagation of radical chain reactions by forming a stable product. This is based on the
potential for conversion reaction between the reduced
and oxidized forms. The hydrophilicity versus hydrophobicity of the compound determines the major milieu in
which a particular antioxidant will be more efficient. A
classical example for this concept is the ability of the
lipid-soluble vitamin E to primarily prevent lipid peroxidation. Yet, the oxidation-reduction potential of each antioxidant determines its ability to recycle other antioxidants, thereby contributing to “total antioxidant capacity.” Described below are the major antioxidants and their
state in conditions of insulin resistance.
1. Dietary (exogenous) small molecule antioxidants
In mammals, dietary consumption of both the watersoluble vitamin C and the lipid-soluble vitamin E contribPhysiol Rev • VOL
utes to the antioxidant potential (328). ␣-Lipoic acid, a
cofactor of the ␣-keto-acid dehydrogenases, is an important antioxidant that participates in redox cycling of vitamins C and E as well as glutathione by virtue of its
hydrophilic and lipophilic properties and its redox potential (317–319). The trace element selenium is also a dietderived factor associated with antioxidant defense, as the
major selenoproteins are key antioxidant enzymes (glutathione peroxidase, thioredoxin reductase), or exert antioxidant properties (selenoprotein P, W) (21, 44).
The circulating level of dietary antioxidants is the
most frequently used measure of the antioxidant status in
clinical trials, although how well it represents the concentration of these factors in tissues relevant for insulin
action is questionable. Furthermore, using this approach
has yielded inconsistent and occasionally conflicting results when comparing diabetic patients with controls (48,
443, 458; reviewed in Ref. 33). This possibly reflects differences in the populations studied (type of diabetes,
severity and duration of the diseases, late diabetic complications, alterations in nutritional intake of vitamins), as
well as methodology used for the various measurements.
Intracellular levels have mostly been assessed in red
blood cells, platelets, and leukocytes (33). These studies
somewhat more consistently demonstrated decreased
levels of dietary antioxidants, but again, it is unknown
how these results reflect the intracellular concentrations
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FIG. 6. Major ROS and RNS detoxifying systems. The major enzymes of ROS detoxification are shown in blue, and the major low-molecularweight antioxidant molecules are listed according to their lipophilicity or hydrophilicity. GPx, glutathione peroxidase; GR, glutathione reductase;
GSH, reduced glutathione; GSSG, oxidized glutathione; SOD, superoxide dismutase.
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BASHAN ET AL.
2. Endogenous low-molecular-weight antioxidants:
glutathione (GSH)
GSH is a tripeptide (␥-Glu-Cys-Gly) present in tissues
in the millimolar range. It is a potent reductant by virtue
of its thiol moiety, acting as the major intracellular watersoluble antioxidant (165). Through the action of enzymes
that catalyze various oxidation-reduction processes, GSH
participates in the detoxification of toxic peroxides (like
H2O2 by the activity of glutathione peroxidases, Fig. 6), in
the maintenance of protein SH groups (Fig. 5), and in
conjugation with xenobiotics to enable their elimination
(through the action of glutathione-S-transferase). After
participation in redox reactions, GSH is regenerated from
GSSH (its oxidized form) by the enzyme GSSG reductase,
using reduced NADPH as a cofactor. It has been demonstrated in various systems that depletion of GSH renders
cells or tissues more susceptible to oxidative injury while
loading cells and particularly the mitochondria with GSH
or the thiol antioxidant N-acetylcysteine can limit such
damage (115, 297, 338, 347).
In diabetes, most human studies do demonstrate decreased blood (plasma, blood cells) GSH levels, and animal studies also reveal decreased levels of reduced glutathione in heart, liver, and muscle (reviewed in Ref. 351).
Some studies suggest that decreased GSH levels precede
the occurrence of diabetes (308), but GSH as a biomarker
of diabetes risk has not been established by large-scale
clinical trials. Studies in animal models demonstrate that
N-acetylcysteine, a cell-permeable thiol antioxidant remPhysiol Rev • VOL
iniscent of GSH, has been shown to protect against the
development of diabetes (218). Yet, this effect likely represented the prevention of pancreatic beta-cell dysfunction rather than maintenance of insulin action. In contrast, GSH depletion in rats by pharmacological inhibition
of ␥-glutamylcysteine synthetase induced impaired glucose tolerance (229) and defective insulin action on its
metabolic target tissues (311). This is further discussed in
section IV.
3. Small protein antioxidants: thioredoxins
and glutaredoxins
Thioredoxins (Trx1-2; cytosolic and mitochondrial,
respectively) and glutaredoxins (Grx1,2,5; cytosolic and
two mitochondrial, respectively) have an increasingly appreciated array of functions (182, 259). Best characterized
is their role in preserving the reduced state of SH groups
in proteins that undergo sulfhydryl oxidation to form
intramolecular disulfide bonds, or undergo glutathionylation (reaction of protein-SH with GSH) or S-nitrosylation
(reaction of protein-SH with NO). Grx can also reduce
dehydroascorbate, thereby participating in small-molecule external antioxidant regeneration (455). This reducing capacity is facilitated by a typical dicysteine motif
(Cys-X-X-Cys) in the active site of all the members of this
family (take out Grx5, which has only one) and can be
regulated by the oxidation-reduction of additional, nonactive site cysteines (259). The regeneration of the reduced form of Trxs is typically catalyzed by thioredoxin
reductases (TrxR1-3, cytosolic, mitochondrial, and testisspecific, respectively), whereas Grxs are reduced mostly
by GSH.
The importance of the Trx-Grx system as a major
component of the antioxidant defense system, and its
involvement in diverse pathologies, has been demonstrated using a variety of cellular and animal experimental
models. Surprisingly, despite their involvement in multiple biological processes including signal transduction,
metabolism, and gene expression, a direct connection
with insulin action is not very strong. Worth mentioning
are early studies that demonstrated the ability of Trx to
reduce the disulfide bonds of the insulin molecule itself,
thereby having the potential capacity to affect its bioactivity (183). A more recently discovered potential connection may be through the role of Trx and Grx in
regulating the activity and expression of apoptosis signal-regulated kinase (ASK1) (363). This MAP kinase
kinase kinase (MAP3K) is activated by oxidants through
the oxidation of Trx and/or Grx and the subsequent dissociation of the ASK1-Trx complex. By this means, ASK1
mediates the oxidation-induced activation of the MAP
kinase pathways JNK and p38MAPK, two MAP kinases
proposed to be involved in the induction of insulin resistance, particularly of adipose tissue (419). Activation of
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in muscle, liver, fat, and the vasculature. Selenium levels
have not been consistently shown to be decreased in
diabetics and may even be somewhat elevated (31, 32).
An additional approach to look for an association
between decreased circulating levels of dietary antioxidants (as a potential cause or reflection of increased
oxidative stress) and impaired insulin action leading to
type 2 diabetes has been using prospective-observational
or intervention trials. Low plasma levels of vitamin E
were suggested to be associated with increased risk of
incident type 2 diabetes (282, 291, 344, 365). Inconsistent
results were reported for vitamin C (114, 291). Intriguingly, vitamin E supplementation was not protective (263,
282). This could potentially suggest either that only dietary source of vitamin E is effective, or that a related
food-derived factor other than vitamin E (114, 117) is in
fact responsible for the protective effect seen in observational trials. Selenium supplementation has been recently
shown to have no protective effect (and even potentially
increase the risk) for type 2 diabetes (31, 406). These
studies are further discussed in section V. Collectively,
there is only circumstantial and indirect evidence linking
dietary antioxidants with the regulation of insulin action
in healthy humans.
ROS, RNS, AND INSULIN ACTION
43
antioxidant state, further discussion of antioxidant enzymes is beyond the scope of the present review.
4. Antioxidant enzymes
C. Key Message From Section II
Maintaining endogenous antioxidants relies on enzymes which catalyze their synthesis and regeneration
(from the oxidized to the reduced form). For example,
these include ␥-glutamylcysteine synthetase and glutathione reductase, respectively, in the case of glutathione,
and the protein synthesis machinery and thioredoxin reductases for thioredoxin. Yet, the enzymes classically
referred to as “antioxidant enzymes” are those that directly act on oxidants, converting them into complete
nonoxidant moieties (like catalase), or at least to less
reactive species (like SOD, Fig. 6) (451). Catalase converts H2O2, mostly when in high concentrations based on
its high Km, to H2O2, whereas the three isoforms of SOD
(cytosolic, mitochondrial, and extracellular: SOD1-3, respectively) all convert O2•⫺ to H2O2 and molecular oxygen. The selenoproteins glutathione peroxidases (GPx)
also “neutralize” H2O2 using GSH, and GPx-1 may be an
important front-line defense against the effects of this
oxidant in the extracellular environment.
Data implicating changes in antioxidant enzymes in
diabetes are conflicting (33, 351). Moreover, it is difficult
to interpret, since, as mentioned above, both increase and
decrease in their expression or activity can be rationalized to indicate oxidative stress. In long-term diabetes, the
activity and expression of catalase, GSH reductase, GSH
peroxidase, and SOD is decreased in various tissues (2,
140, 310, 381, 392), but whether this indicates a primary or
secondary phenomenon is largely unclear. Given these
limitations, and the fact that different from dietary and
low-molecular antioxidants, manipulating antioxidant levels is not a readily available tool to modulate the oxidant-
Complex pro-oxidant and antioxidant systems are
operational in biological systems. These include designated systems for RS generation and detoxification (enzymes, external and endogenous small molecules), as
well as processes and molecules whose contributions to
the oxidant-antioxidant balance are indirect or are not
their primary function. A delicate and highly dynamic
balance governs the availability of ROS and RNS in normal physiology, a balance which is likely perturbed at
multiple levels in diabetes and obesity. This underlies the
common notion that these conditions are associated with
“increased oxidative stress.” Yet, when specific components of the oxidant-antioxidant balance are assessed, the
emerging result is highly complex and variable, with differences between various models, tissues, cell types, and
severity of the pathophysiological states. It seems fair to
speculate that there are numerous “types of oxidative
stress,” each representing different combinatorial perturbation of various component(s) of the complex oxidantantioxidant balance. Even if such perturbation is accepted to occur, assessing whether it plays a functional
role in physiology and/or pathophysiology, rather than
representing their consequence, is a separate question.
Section III describes how ROS and RNS participate in
normal insulin action. Sections IV and V deal with mechanisms and evidence for perturbed oxidant-antioxidant
balance playing a functional role in the pathophysiology
of insulin resistance-related conditions. The major concepts used to support a role for ROS/RNS participation as
positive versus negative regulators of insulin action, as-
FIG.
7. Major lines of evidence for ROS/RNS as positive and negative regulators of insulin signaling.
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ASK1 was indeed proposed to mediate TNF-induced insulin resistance (194), suggesting a putative link between
Trx/Grx and insulin action.
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BASHAN ET AL.
signing them a physiological versus pathophysiological
roles, respectively, are summarized in Figure 7.
III. PHYSIOLOGICAL ROLES OF ROS AND RNS
IN INSULIN ACTION
A. Oxidants Have Insulin-Mimicking Effects
FIG. 8. Physiological role of ROS in insulin signaling cascades. A: ROS are second messengers in insulin singaling, as they are reversible
inhibitors of protein and lipid phosphatases (in blue) that function as negative regulators in the insulin signaling cascades. This can affect both the
“metabolic arm” and the “mitogenic arm” of the insulin signaling cascade (in green or gray symbols, respectively). B: ROS generation in response
to insulin via Nox4. In addition to generation of extracellular superoxide by Nox complex at the plasma membrane, Nox4 may be also localized on
internal membrane pools, generating superoxide intraluminally. To reach the cytoplasm, superoxide anion would require a transporter, whereas
H2O2, the product of superoxide dismutation by SOD, can cross the membrane. IR, insulin receptor; IRS, insulin receptor substrates; PI3K,
phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PDK, phosphoinositidedependent kinase; PKB, protein kinase B (also Akt); PTEN, phosphatase and tensin homolog; PP2A, protein phosphatase 2A; MAPKAP-1,
mitogen-activated protein kinase-associated protein 1; PTP1B, protein tyrosine phosphatase 1B; ERK, extracellular signal-regulated kinase; NOX4,
NADPH oxidase 4.
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One of the earliest connections between ROS and
insulin has been the documentation that high (millimolar)
concentrations of H2O2 activate insulin signaling and/or
induce typical metabolic actions of insulin (75, 164, 169,
238, 280). The activation of the insulin signaling cascade
was shown to arise from the insulin-independent tyrosine
phosphorylation of the insulin receptor ␤-chain (164),
suggesting that H2O2 employs the same pathway as insulin. Propagation of the signal downstream yielded typical
metabolic actions of insulin. In adipocytes and muscle,
H2O2 increased glucose uptake (174, 238, 431), and in
adipocytes also stimulated GLUT4 translocation (273)
and lipid synthesis (280).
The initial steps in insulin signal transduction include
phosphorylation on tyrosine residues of the insulin receptor and its direct substrates. The phosphorylation state of
a protein reflects the balance between kinases and phosphatases, such that increased phosphorylation can result
from an increase in the activity of a kinase, and/or inhibition of a respective phosphatase. Thus, theoretically,
inhibiting phosphatase activity may have a similar effect
as enhancing activity of a related kinase. Millimolar concentrations of H2O2 modulate the tyrosine kinase-phosphatase balance. The insulin receptor kinase activity itself
is susceptible for oxidative regulation, as binding of ATP,
which is required for the autophosphorylation process of
the receptor, is modulated by hydrogen peroxide (374,
377). Yet, the main underlying cellular mechanism for the
insulinomimetic effect of millimolar H2O2 concentrations
is the inhibition of the catalytic activity of various protein
and lipid phosphatases (Fig. 8A). For the insulin receptor
and for the insulin receptor substrate 1 (IRS1) protein, the
intracellular single-domain enzyme PTP1B, a member of
an extensive family of protein tyrosine phosphatases
(PTPs), is the major tyrosine phosphatase. Hence, it is a
physiological negative regulator of the insulin signaling
cascade, a notion supported by the finding that mice with
PTP1B gene deletion exhibit increased insulin sensitivity
(88, 101, 234). Numerous studies have characterized the
biochemical regulation of PTPs by oxidation of their catalytic cysteine thiol moiety (90, 252, 274). The activity of
ROS, RNS, AND INSULIN ACTION
B. Insulin Induces ROS and RNS Production
Already in the late 1970s, insulin itself was shown to
elicit the generation of H2O2 in adipocytes (281). An early
characterization of the enzymology of this process revealed that insulin activated a plasma membrane-associated enzyme system with the properties of a Nox, resulting in the downstream production of H2O2 (242, 243, 293).
Differentiated 3T3-L1 adipocytes that were loaded with a
redox indicator dye generated cellular oxidants (likely
H2O2) within 1 min of stimulation by 0.1–10 nM insulin
(273). The oxidant signal peaked at 5 min and began to
dissipate by 10 min. To verify the participation of Nox4 in
insulin-induced generation of H2O2, its presence was first
demonstrated at both the mRNA and protein levels in
mature primary adipocytes and differentiated 3T3-L1 cells
(272). Furthermore, adenovirus-mediated expression of
Nox4 deletion constructs acting in a dominant-negative
fashion attenuated insulin-stimulated generation of oxidants. Similar results were obtained using siRNA gene
silencing approach (272). Complementarily, when overexPhysiol Rev • VOL
pressed, enhanced Nox4-mediated oxidant generation
was associated with enhanced insulin receptor autophosphorylation and with the inhibition of PTP1B catalytic
activity. Collectively, these findings suggest that Nox4
provides a link between the insulin receptor and ROS
generation by a mechanism that enhances insulin signal
transduction, at least in part via the oxidative inhibition of
cellular PTPs, specifically PTP1B (Fig. 8A).
How does insulin stimulate Nox4-mediated ROS generation? One intriguing observation has been that insulinmediated stimulation of NADPH oxidase activity may not
require the kinase activity of the insulin receptor (241),
yet this possibility was not reported by other studies.
Otherwise, the regulation of Nox4 activity by insulin remains largely unknown. It was suggested that Rac and the
small G protein G␣i2 may play a role in the regulation of
Nox4 in insulin-sensitive cells (Fig. 8B) (134, 243), possibly mediating the critical role proposed for these G proteins in insulin action (55, 210, 292).
In addition to ROS, RNS production is also stimulated in response to insulin. In vascular endothelium,
insulin-stimulated NO production is mediated by increasing expression and/or activation of eNOS (see detailed
description of NOS in sect. IIA2). This RNS-generating
effect of insulin underlies the vasodilatatory response to
insulin. Insulin-stimulated activation of eNOS is mediated
via the PI 3-kinase branch of the signaling cascade (294).
PKB phosphorylates eNOS on Ser1179 (or Ser1177 in
humans), leading to enhanced production of NO, an effect
that was diminished in cells expressing a mutant eNOS
with a disrupted PKB phosphorylation site (93, 290). Consistently, overexpression of a dominant inhibitory mutant of PKB in human umbilical vein endothelial cells
(HUVEC) nearly completely inhibited production of NO
in response to insulin (472). Knocking out the predominant PKB isoform in the vasculature, PKB␣ (Akt1) resulted in significantly lower levels of active eNOS in endothelial cells (54). On the basis of these data, it is likely
that PKB␣ (Akt1) isoform mediates insulin-induced activation of eNOS. However, although necessary, PKB-dependent phosphorylation of eNOS is not sufficient for full
eNOS activation, which can be achieved only when eNOS
binds calmodulin (294). Intriguingly, insulin stimulates
the protein complex formation consisting of eNOS, calmodulin and the heat shock protein (Hsp)90 (417, 473).
This complements the activation step facilitated by PKBmediated phosphorylation of eNOS on Ser1177 (290).
Similar to its effect on the endothelium, insulin stimulates NO production in vascular smooth muscle cells
(VSMCs) in a PI 3-kinase-dependent manner (23, 432).
However, since in VSMC iNOS and nNOS are expressed in
addition to eNOS (39, 168), the relative contribution of
each NOS isoform to insulin-induced NO production is
unknown (23, 43). In addition to the short-term effect of
insulin on NO production, long-term (⬎4 h) insulin stim-
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PTP1B is dependent on the oxidation state of its Cys215
residue: reduced Cys215 is required for catalytic activity
via the formation of a phosphoenzyme intermediate,
whereas oxidized Cys215 renders the enzyme inactive and
has been recognized as a critically important mode of PTP
regulation in vivo (19, 283).
Downstream of the tyrosine phosphorylation of IR
and IRSs, additional phosphorylation steps of proteins
(on Ser/Thr residues) and of phospholipids participate in
propagating the insulin signaling cascade. Reminiscent of
the effects of oxidants on PTP1B, a number of additional
cellular enzymes were suggested as potential targets for
oxidative inhibition by oxidants. A serine-threonine phosphatase, protein phosphatases 2A (PP2A), is a major negative regulator of PKB (by dephosphorylating its Ser473),
p70 S6-kinase, and ERK (438). PP2A has a redox-sensitive
cysteine residue that is potentially susceptible to inhibition by H2O2 (150). Likewise, the dual-specificity phosphatase MAP kinase phosphatase-1, which attenuates insulinstimulated MAP kinase activation, also depends on a reduced thiol for activity (247). The lipid phosphatase PTEN
is another important negative regulator of insulin signaling, as it catalyzes the dephosphorylation of 3⬘-phosphoinositides, lipid second messengers essential for insulin
signal transduction (299). Similar to the aforementioned
protein phosphatases, it is also inactivated by oxidation of
an essential cysteine residue in its active site (253, 254).
Collectively, external oxidants applied onto cells may
activate the insulin signaling pathways at several points
through the inactivation of protein and lipid phosphatases, all of which are negative regulators and off-mechanisms of insulin signaling.
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ulation also increases eNOS expression via mammalian
target of rapamycin (mTOR)-dependent activation of AP-1
and Sp-1 transcription factors (141, 245).
C. RNS and ROS Are Second Messengers in Insulin
Signal Transduction
Physiol Rev • VOL
D. Insulin-Like Effects of ROS and RNS Through
Alternative Pathways
In addition to this direct activation of the insulin
signaling cascade leading to typical metabolic effects of
the hormone, RS may participate in the activation of
“insulin-like” metabolic effects by activating other, noninsulin-initiated signaling pathways. One of the best physiologically relevant examples is the stimulation of glucose
transport in skeletal muscle during exercise (67, 266).
Like insulin, skeletal muscle contraction stimulates glucose transport by up to 50-fold during maximal exercise in
humans (226). Adding exogenous ROS to skeletal muscle
in vitro stimulates glucose transport (174), whereas the
thiol antioxidant N-acetylcysteine (NAC) was shown to
diminish contraction-mediated glucose uptake by ⬃50%
(367). This effect of NAC was associated with a similar
degree of inhibition of contraction-induced activation of
AMP-activated protein kinase (AMPK). AMPK is a central
signaling kinase mediating increased glucose uptake in
muscle under conditions of high AMP/ATP ratio, like
hypoxia and muscle contraction, constituting a non-insulin-dependent pathway to increase muscle glucose utilization (119, 159, 162, 181, 213, 246). Yet, ROS did not seem
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Are insulin-stimulated RS required for insulin action,
or in other words, do RS act as secondary messengers in
the insulin signaling cascade? Theoretically, RS are ideal
second messengers: they are short-lived, hence having an
“intrinsic shut-off mechanism” and functionality that is
limited in time and space, they can initiate chain reactions, and they can be generated upon request through
regulated mechanisms, as detailed above for insulin. Different RS may have different properties and chemical
reactivity, providing an additional aspect of specificity
which characterizes the potential second messenger’s
function of ROS and RNS (302). To prove a second messenger function for RS, it would be required to show that
in response to insulin-stimulated RS generation rises prior
to measured biological effects, that RS can potentiate
insulin action when administered at submaximal concentrations, and even better, that insulin action is diminished
if ROS or RNS generation is inhibited.
Studies in adipocytes and in muscle cells have shown
the ability of ROS to enhance insulin signal. Physiologically relevant concentrations (⬍0.1 mM) of H2O2 enhance
the response to 100 nM insulin, indicating a coregulatory
function of ROS in insulin receptor activation (375). In
addition, various antioxidants, such as NAC, were found
to inhibit the induction of insulin receptor kinase activity,
potentially by antagonizing the effects of insulin-induced
oxidants (374). Blocking the generation of cellular H2O2
with catalase or diphenyleneiodonium (DPI), a commonly
used inhibitor of cellular Nox activity, reduced insulinstimulated autophosphorylation of the insulin receptor
and tyrosine phosphorylation of IRS proteins by up to 48%
(273, 274). Moreover, expression of deletion constructs of
Nox4 led to an inhibition of insulin receptor and IRS-1
tyrosine phosphorylation (272). Similar results were obtained using siRNA approach. Decreasing Nox4 was also
associated with decreased insulin-stimulated PKB phosphorylation, suggesting that the diminution of insulin receptor and IRS tyrosine phosphorylation caused by inhibiting ROS generation may be of physiological significance.
Intriguingly, patients with Leprechaunism (severe insulin
resistance caused by rare mutations in the insulin receptor) exhibit diminished Nox4-mediated ROS generation
(323). While this may explain more generalized diminution of the biological response of various growth factors,
it also supports the role of Nox4-mediated ROS generation in insulin action.
As discussed in section IIIB, ROS generation by Nox4
in adipocytes is associated with oxidative inhibition of
cellular PTP1B activity (274). Overexpression of recombinant PTP1B was shown to inhibit insulin-stimulated
tyrosine phosphorylation of the insulin receptor, and this
effect was reversed when the cells also overexpressed
active Nox4. The proof that insulin-induced ROS production precedes biological outcomes is somewhat less established, but in one study ROS generation was shown to
peak at 1 min after insulin stimulation and gradually
return to prestimulus levels within 10 min (273). Collectively, these studies provide significant evidence in support of assigning a second messenger, or at least a positive modulator role for ROS generated by Nox4 in the
insulin signaling cascade (134).
The role of NO as a second messenger or a positive
modulator of insulin signaling was demonstrated in various systems. In isolated rat heart muscles and in cultured
human vascular smooth muscle cells, insulin-stimulated
glucose transport and GLUT4 recruitment were blocked
by an inhibitor of NO synthesis (26, 256). Moreover, using
in vivo models, intravenous administration of NG-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of all
NOS isoforms, acutely induced insulin resistance in rats
(18, 362). In addition, eNOS knockout mice were shown
to be insulin resistant at the level of the liver and peripheral tissues, whereas the nNOS knockout mice displayed
insulin resistance in peripheral tissues, but excluding the
liver (384). Taken together, these results indicate that
insulin-induced NO production is necessary for successful propagation of the metabolic actions of insulin.
ROS, RNS, AND INSULIN ACTION
E. Key Message From Section III
In normal physiology, ROS and RNS play a coregulatory second messenger function necessary for achieving
the full extent of biological effect of insulin. In addition,
they mediate other stimuli that culminate in “insulin-like”
metabolic effects, such as muscle contraction. Clearly,
poorly understood spatial and temporal coordinates, as
well as other “biological context” factors, ensure that
such intentional ROS and RNS generation yields physiological effects rather than harmful oxidative stress. As
will be discussed in subsequent sections, this physiological role of ROS and RNS poses a major challenge when
seeking to alleviate the pathological effects of oxidants
using antioxidant agents.
IV. CELLULAR AND MOLECULAR MECHANISMS
FOR IMPAIRED INSULIN ACTION INDUCED
BY OXIDANTS
As presented above, the insulin signaling cascade
constitutes a complex signaling network, culminating,
Physiol Rev • VOL
when adequately activated, in the induction of diverse
biological functions. Insulin resistance is the reduced capacity of insulin to induce its biological actions in its
target organs. Identifying which signaling step(s) mediate
insulin resistance induced in response to exposure to
oxidants is a daunting task, mainly because of the following two reasons.
1) Even when a signaling step is found to be defective, it may be difficult to prove that it functionally mediates the impairment in insulin-stimulated action. This is
because each signaling step functions within a complex
network rather than in a linear pathway. Moreover, even
assuming a linear pathway model, a certain degree of
decreased activity may still encompass residual function
that is sufficient to propagate the signal to the next step.
2) As discussed earlier, oxidants are commonly generated by various potential inducers of insulin resistance
(Fig. 3). Yet, each inducer may produce a unique “oxidant
stimulus” characterized by the specific oxidant species
generated, their temporal and spatial determinants, and
the specific biological context. These would result in a
highly diverse array of biological effects. Moreover, the
production of oxidants is frequently only one part of
several effects various stimuli induce, compromising the
ability to dissect out their respective role in a complex
biological setting. Thus it is very difficult to make generalizations about the effects of oxidants on the insulin
signaling cascade.
The reductionist’s approach to circumvent these difficulties is to investigate how exposure of cells or tissues
to oxidants affects insulin action, then to identify the
impaired signaling steps induced, and ultimately, to establish a cause-and-effect relationship between the oxidantinduced alteration and the impaired insulin action. Most
frequently, oxidants, oxidant donors, or enzymatic oxidant-generating systems are directly applied to generate
an exogenous oxidizing environment. To modulate endogenous oxidants production, strategies to manipulate the
pro-oxidant-antioxidant balance need to be applied.
These most frequently include the use of antioxidants, or
regulating the expression and activity of antioxidant enzymes pharmacologically or genetically. Clearly, these
approaches should be interpreted cautiously, since they
generate an artificial environment: exposing cells to exogenously applied oxidants suffers from taking the oxidants out of their true biological context when produced
in vivo in response to an oxidant-generating signal. Moreover, altering a single component of the oxidant-antioxidant balance may create paradoxical effects, since most
antioxidants have pro-oxidant activities in their oxidized
form, depending on the biological context. Nevertheless,
these approaches can assist in identifying specific mechanism(s) that then should be confirmed in the more complex and pathophysiological in vivo setting.
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to mediate AMP-mediated activation of AMPK, as glucose
uptake induced by AICAR, which mimics AMP-mediated
activation of AMPK, was not affected by NAC (367). Thus
the proposed role of ROS in mediating the stimulation of
glucose transport in response to skeletal muscle contraction is as follows: skeletal muscle contraction increases
O2•⫺ production via mitochondrial respiration. O2•⫺ is
then converted to H2O2 by SOD, resulting in direct activation of AMPK, Glut4 translocation to the plasma membrane, and a subsequent increase in glucose transport
(225). Interestingly, in skeletal muscle, but not in adipocytes, NAC did not affect insulin-mediated glucose uptake
(367). Thus, in muscle, NAC specifically antagonized the
second messenger role of ROS in mediating the increase
in glucose uptake in response to contraction, but not to
insulin. This differential effect of NAC unravels a largely
poorly understood specificity in the second messenger
function of ROS.
H2O2 functioning upstream of AMPK seems to represent only one role of RS in exercise-induced glucose
uptake. A role for RNS was suggested in murine muscle
cell line, in which NOS was activated after stimulation of
AMPK by AICAR (118). Cell treatment with NOS inhibitors completely blocked the increase in glucose transport
after activation of AMPK. Furthermore, as assessed in
human subjects in vivo, NOS inhibition reduces leg glucose uptake during cycling exercise (40), and similar
observations were made with electrical muscle stimulation in rats (350). Hence, activation of NOS and subsequent NO production is required for contraction-mediated
glucose transport downstream of AMPK activation.
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Baring in mind the limitations described above, the
following sections describe mechanisms offered to explain how oxidants impinge on the insulin signal transduction cascades, thereby inducing insulin resistance.
A. IR and IRS1 Serine Phosphorylation
and Enhanced Degradation
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When 3T3-L1 adipocytes and Fao hepatoma cells
were exposed to micromolar concentrations of H2O2 for
3 h or longer, IRS1 protein content was decreased (339).
Pharmacological inhibition of PI 3-kinase and mTOR indicated that this effect was largely mediated by “intrinsic”
kinases. However, unlike the effect of chronic exposure
to insulin (which theoretically could also be mediated by
insulin-induced oxidants generation, see sect. IIIB), the
decrease in IRS1 protein content could not be prevented
by inhibitors of the proteasome degradation machinery.
Furthermore, the degree of IRS1 decrease did not correspond to metabolic insulin resistance (339): when IRS1
decrease was prevented with rapamycin, insulininduced glycogenesis was not protected. Conversely,
the antioxidant lipoic acid protected against the decrease in insulin induction of PKB phosphorylation and
of glucose transport (339, 358) without inhibiting oxidation-induced decrease in IRS1 (339). These data suggest that H2O2-induced decrease in IRS1 protein content may not constitute a functional limiting step in
insulin action. A different notion was proposed in response to other oxidants: 3T3-L1 adipocytes exposed to
the lipid peroxidation product 4-HNE exhibited enhanced IRS1 and IRS2 degradation attributed to increased phosphorylation of IRS1 on Ser307 and elevated levels of HNE-modified IRS molecules (89). As
with H2O2, this was not mediated by enhanced proteasomal degradation. Yet, overexpression of fatty aldehyde dehydrogenase, which reverses HNE modification
of proteins partially protected IRS from degradation,
along with protection against insulin resistance (89). In
muscle cells, an NO donor (GSNO) induced enhanced
proteasomal degradation of IRS1 (411). In vivo relevance was proposed by demonstrating that whereas in
ob/ob mice IRS levels were diminished in skeletal muscle, this was not observed when iNOS was knocked
down on the ob/ob background, and these mice exhibited improved insulin sensitivity (411). Enhanced proteasomal degradation of IRS1 was also demonstrated in
vascular smooth muscle cells in response to angiotensin II (422). Oxidants were implicated by demonstrating
that this could be blocked by overexpressing catalase
or by pharmacological antioxidants. Thus enhanced IRS
protein degradation remains a potential mechanism for insulin resistance in response to some oxidants. The protein
degradation machinery (proteasomal or lysosomal) utilized
may be cell-type or tissue-specific.
2. Role of oxidants-induced serine phosphorylation in
decreasing IRS tyrosine phosphorylation (Fig. 9, C
and D)
Decreased insulin-stimulated tyrosine phosphorylation of IRS molecules also related to enhanced Ser/
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The IRS proteins are immediate substrates for the
tyrosine kinase activity of the IR upon insulin binding,
which constitutes a critical step in the insulin signal transduction cascades. Various knock-down approaches have
demonstrated that the members of this group of proteins,
particularly IRS1 and IRS2, are critical for normal insulin
action, although the relative expression and contribution
of each isoform varies among different cells and tissues,
as is the degree of functional overlap between them (301,
428, 456). In addition to multiple tyrosine residues accessible to the IR upon insulin stimulation, IRS molecules
undergo phosphorylation on multiple Ser residues. Multiple kinases have been demonstrated to exert IRS Ser/Thr
kinase activity. Some are “intrinsic” to the insulin signaling cascade, i.e., are activated by insulin as part of its
known signaling pathways. These include PI 3-kinase,
PKB, PKC-␰, GSK3, and p70 S6K (147, 258, 264, 326, 385,
423). Indeed, insulin stimulation results also in Ser phosphorylation of IRS1, exerting both positive-feedback
loops as well as negative termination signals (477). In
addition, kinases “extrinsic” to the classical insulin signaling cascades are bona fide IRS Ser/Thr kinases, including the stress-activated kinase JNK and IKK␤ (3, 125).
The most frequently described consequences of IRS
Ser phosphorylation are a decrease in its capacity to
undergo tyrosine phosphorylation, and enhanced targeting for degradation (477). Phospho-Ser residues can be
easily envisioned to prevent phosphorylation on an adjacent tyrosine. In addition, conformational changes in the
IRS molecule rendering it a poorer substrate for a tyrosine
kinase including the IR can be induced also by Ser residues distant from a specific critical phosphotyrosine site.
Ser307, Ser636, and others have been described to mediate reduced tyrosine phosphorylation of IRS1 (3, 34, 360,
424). In addition, enhanced IRS1 Ser phosphorylation was
proposed to release IRS1 from internal membrane pools
and to result in its increased proteasomal degradation (66,
339, 422).
Both “intrinsic” (p70 S6-kinase, ERK) and “extrinsic”
(JNK, IKK) IRS Ser/Thr kinases may be activated by oxidants (reviewed in Refs. 108, 109). Moreover, as discussed
in section III, various phosphatases are a target for inhibition
by oxidants, further contributing to enhanced phosphorylation. What is the functional consequence of oxidant-mediated IRS Ser/Thr phosphorylation on insulin action? Both
enhanced degradation and impaired insulin-induced tyrosine phosphorylation have been implicated.
1. Oxidant-induced IRS protein degradation (Fig. 9C)
ROS, RNS, AND INSULIN ACTION
49
Thr phosphorylation appears to involved kinases both
“intrinsic” and “extrinsic” to the insulin signaling cascade. In Fao cells exposed to H2O2, pSer307 and
pSer636 appeared to identify minimally overlapping
pools of IRS1 molecules (34): Ser636-phosphorylated
IRS1 exhibited peripheral distribution and exhibited
intact tyrosine phosphorylation, interaction capacity
with PI 3-kinase, and cellular redistribution upon insulin stimulation. In contrast, Ser307-phosphorylated
IRS1 molecules were retained in the cellular membrane
fraction and exhibited diminished capacity to undergo
Physiol Rev • VOL
tyrosine phosphorylation and to interact with PI 3-kinase following insulin stimulation. The combinatorial
nature of the oxidant-mediated regulation of IRS molecules by phosphorylation was exemplified by the fact
that partial prevention of insulin resistance was
achieved only using a combination of JNK and IKK
inhibitors (34). Furthermore, even this combination did
not fully restore IRS1 Ser/Thr phosphorylation state in
control cells, suggesting additional determinants linking oxidants to impaired insulin action through IRS1
phosphorylation.
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FIG. 9. Cellular mechanisms for ROS-induced insulin resistance. A: in normal, unstimulated state, insulin signaling molecules are distributed
between the cytosol (PI 3-kinase, PKB, and PDK) and internal membrane pools (like the majority of IRS molecules). The actin cytoskeleton is mainly
organized in muscle cells in stress fibers. B: upon insulin stimulation, tyrosine residues on the IR and IRS are phosphorylated through the actived
insulin receptor kinase, recruiting PI 3-kinase to the plasma membrane and in internal membrane pools. Subsequent generation of PIP3 recruits PDK
and PKB to the membrane fractions and activates the small GTPase Rac, which induces cytoskeletal reorganization. These propagate the insulin
signals, finally culminating in typical metabolic effects of insulin, like increased glucose uptake. IRS proteins not phosphporylated on tyrosine
residues are released into the cytosol. C: under conditions of increased oxidative stress (reactive species generation), stress-responsive signaling
cascades like the MAP kinase cascades are being activated, leading to increased Ser/Thr phosphorylation of IRS molecules. In addition, RNS induce
additional protein modifications of IR, IRS, PI3K, PKB, and actin (red triangles). Modified IRS molecules are released from internal membrane pools
and are subjected to increased protein degradation. D: under these conditions, insulin fails to normally elicit metabolic effects. This is because IRS
molecules are decreased in content and cannot be normally tyrosine phosphorylated when hyperphosphorylated on certain Ser/Thr residues.
Alternatively, PI3K fails to be activated in internal membrane pools which are required for subsequent propagation of the signal to PKB and Rac.
IR, insulin receptor; IRS, insulin receptor substrates; PI3K, phosphatidylinositol 3-kinase; PDK, phosphoinositide-dependent kinase; PKB, protein
kinase B (also Akt).
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BASHAN ET AL.
B. Impaired Signal Transmission From PI 3-kinase
to PKB
Physiol Rev • VOL
C. Disruption of spatial organization of the insulin
signal: potential role for the
actin cytoskeleton
Insulin signaling components undergo spatial redistribution upon insulin stimulation (Fig. 9, C and D). Best
described is the recruitment of PH-containing proteins,
like PKB and its upstream kinase PDK1 from the cytosol
to the sites of PI(3,4,5)P3 production by PI 3-kinase, a site
most frequently identified as the plasma membrane (187,
330). In addition, upon acute insulin stimulation, cellular
redistribution of IRS1 and PI 3-kinase occurs in adipocytes: a significant proportion of IRS1 is associated in the
unstimulated basal state with internal structures sedimenting at high velocity, consisting of internal membranes (“low density microsomes,” LDM) and/or cytoskel-
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Section IVA discussed the evidence that oxidants impaired insulin signal transduction by affecting IRS proteins. In fact, H2O2 were shown to impair insulin signal
transduction at steps as proximal as the IR: insulin-stimulated IR activation was decreased by H2O2 in HEK293
cells and NIH3T3 overexpressing the insulin receptor and
IRS1 (157) and in vascular smooth muscle cells and in
whole rat muscles exposed to micromolar H2O2 (94, 127).
Yet, other experimental models implicated more distal
oxidation-sensitive steps in the insulin signaling cascade.
In those, when assessed in total cell lysates (i.e., when
subcellular compartments are disregarded; these will be
discussed in sect. IVC), intact IR and IRS proteins tyrosine
phosphorylation were observed, as well as an intact insulin-stimulated interaction between IRS proteins and PI
3-kinase (211, 357, 429). In fact, these steps following
insulin stimulation were even reported to be exaggerated
in response to preexposure to oxidants, possibly reflecting the oxidant-induced inactivation of cellular phosphatases, particularly PTP1B (see sect. IIIA for further details), or impaired feedback inhibition by downstream
signaling components of the system. In contrast to this
effect on proximal insulin signaling events, both 3T3-L1
adipocytes and L6 muscle cells exposed to submillimolar
stable concentrations of H2O2 resulted in impaired PKB
activation (211, 429). Metabolic end points downstream of
PKB were all shown to be affected, including impairment
in insulin-stimulated glucose uptake and GLUT4 translocation, insulin-induced protein and glycogen synthesis, all
consistent with the induction of metabolic insulin resistance (211, 356, 430). Interestingly, this effect of oxidants
was stimulus specific, since under the same conditions
the metabolic effects of platelet-derived growth factor
were preserved (430). The oxidative mechanism of this
inhibition was further supported by demonstrating that
enhancing the antioxidant capacity of the cells using the
antioxidant lipoic acid or N-acetylcysteine protected
against this signaling effect (211, 358). Finally, it is noteworthy that a signaling defect between PI 3-kinase and
PKB is not a mechanism for insulin resistance unique to
oxidative stress, as it was observed in cultured cells in
response to FFA (378), ceramide (449), growth hormone
(418), and others (305). Both pharmacological antioxidants and overexpression of antioxidant enzymes provided partial protection against TNF- and dexamethasone-induced defects in PKB phosphorylation, suggesting
that oxidants mediated their effect (188). Thus insulin
signal transmission from PI 3-kinase to PKB may constitute an oxidation-sensitive step in the insulin signaling
cascade, which is impaired by various inducers of insulin
resistance through oxidant generation.
Closer analysis of the steps through which the insulin
receptor-initiated signal is transmitted from PI 3-kinase to
PKB reveals a complex process: phosphatidylinositol 3,4,5trisphosphate [PI(3,4,5)P3] needs to be generated, “sensed”
by the PH domains of PKB and one of its upstream kinases
PDK1, thereby promoting their translocation from the cytosol to the membranes where PDK1 can phosphorylate
PKB on Thr308 (Fig. 9B). An additional phosphorylation
step on Ser473 of PKB is required for full activation, but
although this step is also PI 3-kinase dependent, the exact
kinase responsible has not been unequivocally revealed (95,
368). Which of these steps is oxidation sensitive is largely
unknown. The human immunodeficiency virus (HIV) protease inhibitor nelfinavir induces insulin resistance characterized by normal insulin-induced PI 3-kinase activity but
impaired PKB activation (25, 359), and a recent study suggests that production of reactive oxygen species mediate
this effect (24, 248, 448), which can be relieved by cotreating
the cells with an SOD/catalase mimetic agent or ascorbic
acid (24, 448). Insulin-induced PI(3,4,5)P3 production was
intact in nelfinavir-treated cells, and a constitutively active
PI 3-kinase could not rescue impaired insulin action, suggesting that the oxidation-induced defect was downstream
of these steps (unpublished results). In contrast, membranetargeted PKB was normally phosphorylated, suggesting intact PKB-kinase activity. Furthermore, membrane-targeted
PKB promoted GLUT4 translocation, confirming that nelfinavir-induced oxidative stress impaired PKB sensing of
PI(3,4,5)P3, leading to defective insulin-stimulated GLUT4
recruitment to the plasma membrane. Whether this is the
result of oxidation-induced posttranslational modification of
PKB, as was suggested to occur in response to ceramide
(340), or due to its binding to a protein that prevents its
translocation, as was demonstrated with TRB3 (97), is unknown. The direct oxidative or nitrative modification of
PKB, as a cellular mechanism for oxidation-induced insulin
resistance, will be detailed in section IVD.
ROS, RNS, AND INSULIN ACTION
Physiol Rev • VOL
key proteins was associated with diminished PKB activation and impaired insulin-induced metabolic actions
(stimulation of glucose uptake, glycogenesis, and lipogenesis). In Fao cells, Ser636-phosphorylated IRS1 was still
released from the membrane fraction upon insulin stimulation, but not IRS1 molecules phosphorylated on
Ser307, which demonstrated decreased tyrosine phosphorylation and interaction with PI 3-kinase (34). Thus
oxidation-induced disruption of cellular redistribution of
signaling molecules in response to insulin stimulation was
associated with impaired insulin action. An in vivo model
of oxidative stress provided support for this notion. In
this model, oxidative stress was induced in rats using
buthionine sulfoximine (BSO), an inhibitor of ␥-glutamylcysteine synthetase, the rate-limiting enzyme in the glutathione biosynthesis. The consequential drop in tissue levels of glutathione, a major cellular antioxidant, was
shown to result in increased markers of oxidative stress
in both cells and animals, and in vivo induced impaired
glucose homeostasis. Whereas one study failed to demonstrate impaired insulin responsiveness in adipose tissue
and cultured adipocytes (229), another study using higher
doses (and different administration route) of the inhibitor
demonstrated impaired insulin signaling and metabolic
actions in both adipose tissue and skeletal muscle (311).
Remarkably, the underlying mechanism proposed was the
impaired ability of insulin to induce the translocation to
and activation of PI 3-kinase in intracellular compartments, fully consistent with the observations in the adipocyte cell line (429).
Recent studies suggest that insulin-induced actin remodeling is an oxidative stress-sensitive step underlying
the abnormalities in the cellular distribution of its signaling components and in signal transmission (211) (Fig.
9D). Actin has been shown to be oxidatively modified, as
will be discussed in section IVD. In addition, in L6 muscle
cells exposed to H2O2, insulin-induced actin remodeling
was diminished, as was the GTP loading of Rac1 (211).
Dose-response analysis of the effects of H2O2 demonstrated a closer association between the impairment in
GLUT4 translocation and the diminution of insulin-induced Rac1 activation, than the inhibition of PKB activation. Since Rac1 and PKB are parallel PI 3-kinase downstream substrates, these results suggest that in muscle
cells, insulin-induced Rac activation (and the resulting
actin remodeling) constitute an oxidation-sensitive point
between insulin stimulation and GLUT4 translocation.
D. Direct Modification of Insulin Signaling
Components by Oxidants
ROS/RNS can react and chemically modify various
cellular components, including proteins. Protein oxidative/nitrative modifications are largely classified into two
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etal elements (65, 196, 429). Insulin induces the release of
IRS1 from this fraction to the cytosol, potentially with
preference to IRS1 molecules phosphorylated on Ser residues, which are then available to the proteasome for
protein degradation as mentioned in section IVA (Fig. 9B).
In contrast, tyrosine-phosphorylated IRS1 accumulates in
the LDM, promoting the recruitment of PI 3-kinase from
the cytosol through the interaction between the SH2 domains of PI 3-kinase with phosphorylated tyrosine residues of IRS1. These effects of insulin, initially supported
by biochemical analyses (subcellular fractionation studies)(65, 196, 429), were later supported by cellular imaging approaches of L6 myoblasts (325). In these cells,
PI(3,4,5)P3 is produced in the plasma membrane in response to insulin stimulation, but also in intracellular
locale, most likely internal membranes captured within
actin cytoskeleton structures beneath the plasma membrane, reminiscent of lamellopodia (325, 354). These insulin-induced actin structures gather IRS1, the p110␣ subunit of PI 3-kinase (but not the ␤-isoform), and PKB (but
not PKC-␰, another PI 3-kinase-dependent enzyme). Thus
insulin-induced actin remodeling appears as a means of
generating “signaling microdomains” spatially segregating
some signaling components from others.
How does insulin promote actin remodeling, and is it
important for insulin action? Actin dynamics are regulated by small GTPases, and a few have been demonstrated to be activated (GTP-loaded) in response to insulin, and to mediate actin dynamics. The small GTPase
TC10 was suggested when overexpressed in adipocytes to
be activated in a class I PI 3-kinase-independent pathway
and to promote actin dynamics in adipocytes (219). This
signaling pathway was suggested to be required for insulin-induced GLUT4 translocation to the plasma membrane, potentially through the activation of PI3P generation (271). In contrast, in muscle cells, although overexpressed TC10 could be activated by insulin, it was not
required for the induction of actin structures (210).
Rather, another small GTPase, Rac1, was activated by
insulin in a PI 3-kinase-dependent (but PKB-independent)
pathway and was required for insulin-induced actin structures (210, 211). Furthermore, Rac activation was required for GLUT4 translocation to the plasma membrane
in response to insulin.
Several studies suggest that actin-facilitated redistribution of insulin signaling components is an oxidation-sensitive event in insulin signaling. 3T3-L1 adipocytes exposed to micromolar H2O2 concentrations exhibited normal PI 3-kinase activation when assessed in
total cell lysates, but markedly decreased activation of
PI 3-kinase in the intracellular high-speed pellet/LDM
fraction (357, 429). Furthermore, insulin-induced recruitment of PI 3-kinase into this fraction and IRS1
release from it were diminished (429). This failed ability to promote the cellular redistribution of these two
51
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BASHAN ET AL.
main categories (reviewed in Refs. 96, 131 and summarized in Table 2). 1) Readily reversible modifications can
be used also as a strategy to regulate protein function in
normal physiology, somewhat reminiscent of the wellcharacterized regulation by reversible phosphorylation/
dephosphorylation. In this group, oxidation-reduction
processes (redox regulation) are the prototypes, involvTABLE
2.
ing protein moieties such as vicinal sulfhydryls (133,
205). Only excessive accumulation of this type of modification and an ensuing “pathological” functional consequence signifies “oxidative/nitrative stress”. 2) Irreversible modifications are those for which cellular reversal mechanisms are limited or absent. Accumulation
of this group of modifications is considered a marker of
Examples of oxidative and nitrative protein modifications
Oxidative/Nitrative Protein
Modification
Modification Mechanism
Protein Target
S-nitrosylation
(172, 278)
General: redox
regulation (also in
normal physiology)
Interaction of nitric
oxide with cysteine
thiol (NO ⫹ CysSH) to form S-NO
Cys residues between acidic
and basic residues
关X(K/R/H)C(D/E)兴 (397)
Regulatory function
(increased or
decreased activity)
Nonenzymatic (S-NO is a
rather labile covalent
modification). Enhanced
by DTT and ascorbate
(470).
Enzymatic, mainly of low
molecular S-NOs (128)
Regulatory function
Glutaredoxin (mixed
disulfides) (182)
Cys in hydrophobic regions
Glutathionylation
(71, 235)
GSH ⫹ H2O2
(sulfhydryl
oxidation)
GSSG (disulfide
exchange)
Free sulfhydryls
(Cys residues)
Response to oxidative
stress
Protective strategy
against irreversible
modifications (235)
Irreversible
modifications
Tyrosine nitration
(3-NT) (198, 434)
General: loss of function,
“oxidative damage”
Peroxynitrite
(ONOO⫺)
Tyr side chain
Simultaneous
generation of NO
and superoxide
(activity of NOS)
Myeloperoxidase
(129)
Carbonylation
(78, 309)
Lipid adducts
(4-HNE) (333, 437)
Reversibility (R) Prevention (P)
3-NT modification
(reversal) activity has
been reported (14, 215)
Increased/decreased
tyrosine
phosphorylation
Direct metal
catalyzed oxidative
(MCO) attack
Secondary reaction
with reactive
carbonyls (RC)
关advanced
glycation end
products (AGEs),
lipid peroxides
(4-HNE)兴
4-HNE, an
electrophile
produced by
polyunsaturated
lipid peroxidation
Considered irreversible
protein damage
Protein backbone
Protein fragmentation
Generally considered
irreversible modification
MCO: Arg, Pro, Lys, Thr side
chains
Degradation, aggregation
RC: Lys, Cys, His side chains
May also be the
consequence (rather
than the cause) of
protein dysfunction (98)
R ⫽ various reductases
(carbonyl reductase) and
glutathione transferases
(336)
P ⫽ aminoguanidine,
ACE inhibitors,
N-acetylcysteine (336)
1,2-Michael additions: Lys
residues (unclear
physiological role)
Cys-4-HNE may be a
signaling mechanism in
normal physiology
1,4-Michael addition:
Cys ⬎⬎ His ⬎ Lys residues
His-4-HNE and Lys-4HNE are stable and
signify oxidative
damage
Definitions are as in Table 1. MCO, metal catalyzed oxidative; RC, reactive carbonyl.
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R ⫽ spontaneous for Cys-4HNE. Enzyme-catalyzed
by various aldoketo
reductases (10-30%), GST
(to GS-4-HNE, 30-60%)
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Reversible
modifications
Functional Consequence and
Biological Significance
ROS, RNS, AND INSULIN ACTION
Physiol Rev • VOL
The irreversible modification of nitrosylation of
tyrosine residues was also shown to impair the function
of insulin signaling components at different levels. 3T3-L1
adipocytes exposed to peroxynitrite exhibited impaired
insulin action (glucose uptake), along with normal insulin-stimulated tyrosine phosphorylation of IR, but decreased IRS1 tyrosine phosphorylation, interaction with
PI 3-kinase, and activation of PKB (307). Mass spectrometry analysis of IRS1 revealed tyrosine nitration of at least
five Tyr residues, one of which, Tyr939, is known to be
critical for the interaction with PI 3-kinase (307). Tyrosine
nitration of the regulatory p85 subunit of PI 3-kinase was
shown to cause dissociation from the catalytic p110 subunit of the enzyme (100), providing an additional molecular mechanism for nitration-mediated impairment in insulin signal propagation.
Lipid peroxidation adducts on proteins is potentially
the major carbonyl oxidative modification (447). 3T3-L1
adipocytes exposed to the lipid peroxidation product
4-HNE exhibit increased protein HNE modification and
impaired insulin actions (89). These were attributed to
enhanced degradation of IRS proteins secondary to their
HNE modification, as overexpressing fatty acid aldehyde
dehydrogenase, which reverse protein HNE modification,
prevented the decrease in IRS expression and in insulin
responses (89). A recent proteomic approach to identify
carbonylated proteins in adipose tissue of HFF mice identified the fatty acid binding protein as being one of the
proteins modified by 4-HNE (144). Fatty acid binding
protein was linked to the development of insulin resistance (knockout mice were protected from diet-induced
insulin resistance; Refs. 171, 186) and was shown to activate hormone-sensitive lipase when in the fatty acidbound state (212). Thus, if HNE modified fatty acid binding protein mimics the fatty acid-bound state, then these
data provide an additional putative mechanism linking
protein oxidative modification to insulin resistance (144).
E. Alterations in Gene Regulation
Reactive oxidants and radicals are prominent regulators of gene expression, as they can directly (through
oxidative modifications) and/or indirectly (by activating
stress-sensing kinases) regulate the activity of many transcription factors families (382). Direct modification,
mostly oxidation of cysteines critical for DNA binding
capacity, usually lead to decreased transcriptional activity, similar to the effect oxidants exert on protein phosphatases (discussed in sect. IIIA). In contrast, phosphorylation by kinases regulates the transcriptional capacity in
a more diverse manner, as reviewed in References 80, 382,
and 435. Oxidants therefore induce alterations in the expression of multiple genes, including gene products that
are related to insulin action, thereby providing an additional mechanism for oxidant-induced insulin resistance.
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oxidative/nitrative damage as it usually results in protein
dysfunction, with affected proteins frequently destined to
degradation and/or to form dysfunctional protein aggregates. Of these, best known is protein carbonylation, a
large group of oxidative/nitrative modifications resulting
in the introduction of various carbonyls (aldehydes and
ketones) into the protein (57, 78, 309). Carbonyls can
form by direct attack of the protein backbone by various
reactive species resulting in protein fragmentation. Alternatively, they occur by interaction of various amino acid
side chains (Arg, Lys, Pro, Thr) with either ROS/RNS or
secondarily with other active carbonyls. The latter include AGEs or lipid peroxidation products like 4-HNE.
One of the most studied irreversible modification is tyrosine nitration, the result of peroxynitrite reaction with
the OH moiety in the tyrosine side chain (198, 342, 434).
Along the discussion in section IC, increased total
serum protein carbonylation and nitrotyrosine levels have
been reported in diabetes (37, 50, 426), potentially implicating both ROS, RNS, and active carbonyls (like 4-HNE)
in systemic protein modification associated with this disorder. In cells, altered protein function occurred in response to in vitro oxidation/nitration. This was demonstrated on several specific proteins with potential relevance for insulin action [PKC-␰ (61), GAPDH (46, 158,
288), and actin (5, 79, 450)], though a connection to
impaired insulin action has not been reported for those.
Nevertheless, different types of modifications affecting
multiple proteins could impinge in a combinatorial fashion on the insulin signaling cascade, thereby contributing
to insulin resistance. To this end, several studies provide
direct evidence tying oxidative/nitrative modifications of
insulin signaling components to impaired insulin signaling
and actions.
The reversible S-nitrosylation of IR, IRS1, and PKB
have been shown to occur in response to NO donors in
cells in culture and in isolated muscle, as well as in
muscle of genetic and nutritional mouse models (217).
S-nitrosylation of Cys224 (470) or of Cys296 (265) of PKB
results in impaired activity, the latter because it prevents
an essential disulfide bridge with Cys310. Intriguingly,
nitrosylation of PKB may uncouple the enzyme’s phosphorylation state from its activity, suggesting that modified PKB may be dysfunctional even if normally phosphorylated (470). Support for a causative role of S-nitrosylation of insulin signaling components in insulin resistance
was suggested by the finding that the insulin sensitizer
rosiglitazone (49) and exercise (84) decreased iNOS expression and S-nitrosylation of IR, IRS1, and PKB in muscle of rodent models of obesity. Furthermore, to direct
S-nitrosylation of insulin signaling proteins as a cause of
insulin resistance, nitrosative stress may mediate inflammatory responses and ER stress, which in turn can cause
impaired insulin action (217).
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BASHAN ET AL.
1. Oxidant regulation of GLUT4 gene expression
Physiol Rev • VOL
2. Oxidant regulation of adiponectin gene expression
Adiponectin is currently the adipokine (fat-derived
hormone) most clearly related to human health and disease. Secreted almost exclusively from adipocytes, it is
strongly inversely correlated with fat mass in obesity and
with its associated cardiovascular risk. Consistently, its
physiological effects all seem to be beneficial to health
and include anti-inflammatory actions, insulin-sensitizing
actions at the level of the liver and muscle, direct antiatherosclerotic effects on the vasculature, and possibly anorexigenic signals in the hypothalamus, limiting excessive
food intake. Obesity-associated decrease in adiponectin
gene expression is thus a potentially strong mechanism
linking obesity to its common comorbidities.
Plasma and urinary lipid peroxidation markers indicative of systemic oxidative stress (TBARS and 8-epiPGF2␣, respectively) correlated with lower circulating
adiponectin levels (122). Establishing a causative role for
oxidants as an isolated factor in the repression of the
adiponectin gene was performed in cells in culture:
3T3-L1 adipocytes expressed lower adiponectin mRNA
levels when exposed to oxidants generated by either glucose oxidase (214, 393) or hyoxanthine and xanthine
(122) added to the medium, when H2O2 was added directly to the medium (52, 214), or when cells were exposed to the lipid peroxidation derivative 4-hydroxynonenal (4-HNE) (393). The sulfhydryl antioxidant N-acetylcysteine (52, 122, 214) or catalase (52, 393) prevented the
drop in adiponectin. Remarkably, 10-min exposure to 500
␮M H2O2 induced this effect 16 h later without compromising cell viability (214), suggesting cellular “memory”
and a delayed biological response to a severe short-term
oxidative insult. A transcriptional repression mechanism
utilized by H2O2 was shown by a reporter gene assay
using the adiponectin promoter (122). In vivo, hypoadiponectinemia and decreased adiponectin mRNA in adipose tissue were observed in BSO-treated rats (199), a
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GLUT4 is expressed in cell types in which insulin
induces an increase in glucose uptake, mainly skeletal
and cardiac muscle and fat cells. GLUT isoform-selective
inhibitors demonstrated that at least 60 – 80% of glucose
uptake into these cells and tissues is facilitated by GLUT4
(295, 355). In both human and experimental insulin resistance conditions, GLUT4 protein and mRNA expression is
largely unaltered in muscle, suggesting that the decrease
in insulin-stimulated glucose uptake is largely mediated
by impaired signaling promoting GLUT4 mobilization
from internal membrane pools to the plasma membrane
(124, 354). In contrast, decreased expression of GLUT4 in
adipocytes is tightly associated with total body insulin
resistance. Furthermore, fat-specific GLUT4 knockout
mice (1), and heterozygote total-body GLUT4 null mice
that have decreased expression of GLUT4 in fat (400),
exhibit an insulin-resistant and type 2 diabetes-like phenotype. These suggest that adipose tissue gene regulation
of GLUT4 is an important determinant of total body glucose homeostasis. Recently, retinol binding protein 4
(RBP4) was suggested to mediate liver and muscle insulin
resistance in response to diminished levels of GLUT4 in
adipose tissue (142, 469).
In vitro, adipocytes exposed to oxidants exhibited a
decrease in GLUT4 protein and mRNA content (332, 357).
The GLUT4 promoter includes putative binding sites for
C/EBPs and for NF1, among other transcription factors,
and is also thought to be regulated by PPAR␥, although
the exact binding site for this factor in the GLUT4 promoter has not been clearly identified. Upon exposure of
adipocytes to micromolar H2O2 concentrations, EMSA
studies suggested that oxidants decreased DNA binding
of C/EBP␣-containing dimers while increasing that of
C/EBP␦ dimers (331). Intriguingly, DNA binding capacity
of the C/EBPs transcription factor family is not known to
be particularly sensitive to redox regulation (62) [though
the ␰ isoform (also known as GADD153 or CHOP10) is
known to be upregulated by oxidative stress (151)]. This
suggested that expression level, rather than direct alteration in DNA binding capacity, altered C/EBP’s regulation
of GLUT4 expression. Consistent with this proposition,
C/EBP␣ protein and mRNA levels were downregulated,
whereas those of C/EBP␦ upregulated in response to oxidative stress (331). Moreover, the antioxidant lipoic acid
could prevent the decrease in C/EBP␣ mRNA and in
GLUT4 mRNA, consistent with an oxidation-mediated
mechanism. Intriguingly, similar observations were observed in response to TNF (206, 401, 402), which was
proposed to exert part of its actions by inducing ROS
generation (276, 467).
Despite the above, reporter gene assays suggest that
the C/EBP binding sites in the GLUT4 promoter cannot
fully mediate oxidation-induced GLUT4 gene repression
(331). An additional region proposed to mediate this effect was located ⬃700 bp upstream of the transcription
initiation site of the GLUT4 gene, an area that includes the
insulin responsive element (IRE)(68, 332). Although this
sequence was shown to mediate GLUT4 repression also in
response to chronic insulin (68), the mechanism exerted
by H2O2 differed, being associated with decreased DNA
binding of NF1 to IRE. Different from the C/EBPs, DNA
binding capacity of this transcription factor is known
to be redox sensitive, and indeed, partial recovery of its
DNA binding to the IRE sequence from the GLUT4 promoter could be demonstrated in nuclear extracts from
oxidized cells treated with DTT (332).
Thus, collectively, oxidants appear to repress GLUT4
gene expression by direct oxidation of NF1 as well as via
suppression of C/EBP␣ expression.
ROS, RNS, AND INSULIN ACTION
F. Key Message From Section IV
Multiple molecular mechanisms have been proposed
to contribute to the induction of cellular and whole body
insulin resistance by reactive species. These include oxidant-induced alterations in phosphorylation state and
protein modifications, altered cellular localization and
half-life, and changes in gene regulation. Affected proteins are either direct insulin signaling molecules, oxidant-sensitive signaling pathways which impinge on the
insulin signaling cascade, transcription factors, or hormones and cytokines indirectly affecting whole body insulin sensitivity.
V. MANIPULATION OF THE OXIDANT/
ANTIOXIDANT BALANCE TO ENHANCE
INSULIN ACTION
As detailed in the above sections, ROS and RNS
generation affects insulin action in seemingly opposing
modes: on one hand, their production is required for
insulin action, as RS play a second messenger role in
insulin signaling (see sect. III). On the other hand, they are
likely mediators in the development of impaired insulin
action (see sect. IV). This dual role creates a paradoxical
situation when attempting to manipulate ROS/RNS generation for therapeutic purposes; decreasing oxidant load
would be accepted to prevent or improve insulin resistance, but theoretically could induce insulin resistance by
decreasing the action of required second messengers.
Physiol Rev • VOL
This paradox likely contributes to the overall disappointing results of antioxidant intervention trials so far (discussed further in sect. VC).
This section describes the available information on
the possibility to prevent or improve insulin resistance by
manipulating the oxidant-antioxidant balance. Studies using in vitro, cell line systems are described first (sect. VA),
followed by studies in animal models and in humans
(sect. V, B and C, respectively). In cellular and animal
studies, attempts were made to inhibit ROS/RNS generation, as well as to facilitate the antioxidant defense. In
humans only the latter is currently available.
A. Cellular Systems
1. Preventing ROS/RNS generation
Cellular evidence for the ability to prevent insulin
resistance by decreasing ROS/RNS generation is available
for each of the three major ROS/RNS generating systems:
mitochondria, NADPH oxidases, and NOS (described in
sect. IIA). Inhibiting mitochondrial ROS generation by
overexpression of uncoupling proteins 1 (UCP1) in hepatoma cells prevented TNF-induced increase in the serine
phosphorylation of IRS1 and the attenuation of insulinstimulated tyrosine phosphorylation of this protein (194).
Consistently, preventing mitochondrial ROS generation in
3T3-L1 adipocytes partially prevented insulin resistance
induced by TNF, dexamethasone, and high glucose and
increased adiponectin levels (188, 261).
ROS production by NADPH oxidases was implicated
in the induction of insulin resistance (impaired insulinstimulated IRS1 tyrosine phosphorylation, activation of
PKB and GLUT4 translocation) by angiotensin II in L6
muscle cells (193). This was supported by demonstrating
that both pharmacological inhibitors of NADPH oxidases,
as well as siRNA-based gene silencing of p47Phox prevented these effects. Yet, a recent paper questioned the
possibility that NADPH oxidase is significantly expressed
in this cell line (191).
Inhibiting iNOS-induced NO generation in muscle
cells was proposed as a mode of action of the insulinsensitizing drugs metformin and troglitazone. Muscle cells
exposed to an inflammatory stimulus (TNF, interferon-␥,
and lipopolysaccharide) increased iNOS expression and
NO generation, which were associated with impaired insulin-stimulated activation of PI 3-kinase. Metformin and
troglitazone prevented these effects through their capacity to activate AMPK (both an activator of AMPK and a
pharmacological inhibitor of iNOS prevented inflammation-induced insulin resistance) (337). In contrast, in cultured human adipocytes, iNOS mRNA was activated only
in a transient manner in response to inflammatory cytokines, but the protein was undetectable (although readily
observable in 3T3-L1 adipocytes) (262). Consistently,
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model for oxidative stress, which also exhibits insulin
resistance (311). Collectively, the studies discussed in this
section suggest that oxidative stress in adipose tissue
mediates obesity-induced hypoadiponectinemia by regulating adiponectin gene expression, thereby contributing
to insulin resistance. Finally, a recent study demonstrated that adipose tissue hypoxia through ER stress
and activation of C/EBP␰/CHOP10/GADD153 directly
repressed the transcription of adiponectin (184). The
responses to both hypoxia (391) and ER stress (143, 166)
are associated with enhanced production of radicals, and
C/EBP␰ is a transcription factor known to be upregulated
by both ER and oxidative stress (151, 349). Thus, although
not supported by some reports (52), it is plausible that
reactive species also mediate hypoadiponectinemia induced by hypoxia and ER stress. Similarly, hypoadiponectinemia induced by fatty acids was proposed to be
mediated by increased production of reactive species secondary to decreased FOXO1 (409).
Thus oxidants may directly mediate the suppression
of the adiponectin gene in response to various adipose
tissue alterations in obesity, thereby contributing to
whole-body insulin resistance.
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BASHAN ET AL.
iNOS inhibitors could not prevent the decrease in insulinstimulated glucose uptake that was induced by exposure
to inflammatory agents (262). Regardless of this discrepancy between the two cell types, it appears based on in
vitro experimental systems that inhibiting either mitochondrial or NADPH-derived ROS generation, and at least
in muscle cells also iNOS-induced NO generation, can
relieve insulin resistance.
2. Enhancing antioxidant defense
Physiol Rev • VOL
B. Experimental Animal Studies
1. Preventing ROS/RNS generation in animals
Interfering with mitochondrial ROS production
in vivo to test its contribution in the induction of insulin
resistance has been difficult to accomplish. Although
overexpressing uncoupling proteins is indeed expected
to decrease mitochondrial ROS generation as shown in
cell culture systems (discussed in sect. VA1), it also
augments mitochondrial respiration and, hence, energy
expenditure. Hence, transgenic mice overexpressing
UCP1 or UCP3 in adipose tissue or muscle were found
to be protected from obesity and its metabolic consequences (63, 240, 255). Yet, these studies cannot prove
an isolated role of mitochondrial ROS production in
insulin resistance.
Interfering with ROS generation by the NADPH oxidase system frequently involves pharmacological inhibitors like DPI and apocynin. In vivo administration of DPI
decreased glucose levels (163, 180), but this was likely a
result of this inhibitors capacity to inhibit mitochondrial
NADH: coenzyme Q reductase, hence representing the
classical Pasteur effect (increased glucose utilization and
lactate generation in response to interference with mitochondria energy production). In vivo administration of
apocynin decreased adipose tissue ROS generation and
lipid peroxidation products in a genetic mouse model of
obesity and diabetes, decreased glucose and insulin levels, and elevated adiponectin (122). Although to the best
of our knowledge NOX4 knockout mice have not been
tested in the context of insulin resistance, these data
provide supporting evidence for an in vivo role of adipo-
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The most frequent strategy to facilitate antioxidant
defense is to supplement cells with antioxidant agents.
This is then followed by assessment of the ability to
induce insulin resistance in these cells, either in response
to direct exposure to oxidants or exposure to inducers of
insulin resistance thought to induce oxidants as part of
their mode of action. This line of research has yielded
numerous reports with positive results (109, 111 and references within).
The antioxidant lipoic acid partially prevented in
both 3T3-L1 adipocytes and in L6 muscle cells the induction of insulin resistance by exposure to a H2O2 enzymatic
generating system (used to generate stable, micromolar
concentrations of the oxidant) (269, 358). This thiol antioxidant has several possible modes of action: it directly
reacts with radicals, is involved in the recycling of other
antioxidants including vitamin C and E, enhances cellular
GSH by maintaining it in a reduced form and by increasing
its synthesis, and chelates transition metals (317, 319). In
addition, lipoic acid is a cofactor of the ␣-keto-acid dehydrogenases like pyruvate dehydrogenase, and thus may
directly affect the oxidative metabolism of glucose
through nonantioxidant mode of action. Thus comparison
of the ability of lipoic acid to prevent insulin resistance to
other antioxidants can be used to assess the likelihood of
its antioxidant mode of action. Supplementing 3T3-L1
adipocytes with vitamin C did not confer the protective
effect observed with lipoic acid upon exposure to H2O2generating system (358), whereas vitamin E supplementation protected L6 muscle cells (444). Yet, the thiol antioxidant NAC was shown to protect 3T3-L1 adipocytes
from insulin resistance induced by exposure to H2O2
(387). Moreover, this antioxidant provided partial protection against insulin resistance induced by angiotensin II in
vascular smooth muscle cells (422), or by TNF and dexamethasone in 3T3-L1 adipocytes (188). In the latter cells,
NAC prevented the H2O2-induced decrease in adiponectin
gene and protein expression (122). Collectively, these
studies suggest the potential beneficial effect of thiol
antioxidants in protecting against insulin resistance generated either in response to direct exposure to oxidants,
or to factors thought to induce insulin resistance at least
partly by provoking ROS generation.
An additional approach to enhance antioxidant defense has been to increase the activity of antioxidant
enzymes. This is most frequently achieved either by overexpression gene delivery approaches, or by chemical
agents mimicking the activity of antioxidant enzymes. The
SOD/catalase mimetic agent manganese (III) tetrakis(4benzoic acid) porphyrin (MnTBAP) provided 50 – 60% recovery of insulin-stimulated glucose uptake activity that
was decreased in 3T3-L1 adipocytes by TNF or dexamethasone (188). Insulin-stimulated phosphorylation of PKB
and of p70S6 kinase were similarly recovered by
MnTBAP. A similar partial protection by MnTBAP against
decreased insulin-stimulated PKB phosphorylation was
observed in 3T3-L1 adipocytes exposed to the HIV protease inhibitor nelfinavir (24), which are thought to involve ROS generation in the induction of insulin resistance (24, 248, 352). Increasing intracellular catalase activity, either by stable overexpression or by adding the
enzyme to the cells, prevented H2O2, TNF, dexamethasone, or angiotensin-induced insulin resistance (157, 188,
422).
ROS, RNS, AND INSULIN ACTION
cyte NADPH oxidase generation of ROS in whole body
insulin resistance.
2. Enhancing antioxidant defense in animals
aged rats (9), and insulin resistance induced by angiotensin II (30, 453, 465).
C. Human Studies
Observational studies (116, 282, 365) have provided a
rationale for conducting intervention trials to assess if
directly manipulating the oxidant-antioxidant balance can
improve insulin action in humans, thereby preventing the
occurrence of and/or improving metabolic control in diabetes. Such human intervention trials are restricted to
attempts to increase antioxidant defense by antioxidant
supplementation. (Theoretically, attenuating ROS/RNS
generation may occur secondary to other interventions,
like those that would improve metabolic control or inflammatory state, but approaches to directly inhibit the
major ROS/RNS generators are largely unavailable for
clinical applications.) Improved insulin sensitivity and/or
improved markers of glucose tolerance have been demonstrated with lipoic acid (110, 201, 202, 239), NAC (121),
vitamin E (45, 320 –322), and vitamin C (177) (and recently reviewed in Refs. 106, 107). Yet, this was mainly
observed in small-sized, short-term trials, and frequently,
reports exist demonstrating no measurable effects of the
same agents trialed on similar study populations (53, 76,
136, 263). Late diabetes complications known to associate
with metabolic control (407) also do not seem to be
positively affected by antioxidant therapy (reviewed in
Ref. 380). Consistently, despite some supporting evidence
for the ability to improve insulin action with antioxidants,
current clinical guidelines do not recommend antioxidant
supplementation for the general population of persons
with impaired insulin action, like type 2 diabetes (7). This
is not only because evidence for a beneficial effect is
anecdotal or too weak, but also due to evidence suggesting potential harm, including increase in all-cause mortality, with vitamin E, carotene, selenium, and other antioxidant supplementation (29, 32, 285). The recent demonstration that selenium supplementation may increase the
incidence of type 2 diabetes (406) potentially reflects
negative effects of selenium on insulin sensitivity. Yet,
whether this is mediated through an antioxidant effect is
unknown.
The careful assessment of evidence-based literature
conducted by clinical guideline committees reflects the
overall disappointing results in the ability to positively
affect insulin sensitivity-related health outcomes with
currently used antioxidants in placebo controlled intervention trials. Yet, it is difficult to determine whether this
current assessment rules out a basic mechanistic role for
ROS/RNS in the induction of insulin resistance in humans.
More likely, the extremely complex oxidant-antioxidant
systems and their intricate, occasionally opposing roles in
physiology and pathophysiology render currently existing
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Different antioxidants, including vitamins C and E,
lipoic acid, NAC, and flavanoids, have been shown to
attenuate different markers of systemic oxidative stress in
a variety of experimental animal models for obesity, type
1 or type 2 diabetes. Yet, whether such antioxidants can
prevent or improve insulin action by interfering with oxidation processes is still an open question. This is partially
because at the whole body level, an intricate relationship
between metabolic and endocrine control and the generation of oxidants complicates the interpretation of the
results and limits determining directionality in cause-effect relationships: Does the decrease in oxidative stress
cause the improved insulin action, or does it only reflect
(i.e., is the consequence of) lower glucose and insulin
levels? Nevertheless, several studies demonstrated that
lipoic acid supplementation positively affects glucose homeostasis and insulin action. With the use of the fatty
Zucker rat, a model of insulin resistance and obesity,
lipoic acid treatment was shown to improve markers of
insulin resistance including reduction in circulating insulin and FFA concentrations (203). At the skeletal muscle
level, isolated epitrochlearis muscles from rats treated
with lipoic acid exhibited improved insulin stimulated
glucose transport, glycogenesis, and CO2 production (170,
408). In general accordance with these findings, improved
insulin sensitivity both systemically as well as at the
skeletal muscle level was observed in the hypoinsulinemic streptozotocin (STZ)-induced diabetic rat model
(230). This was attributed to enhanced protein levels of
GLUT4 in muscle and was associated with improvement
in a variety of oxidative stress parameters (231). Further
supporting a thiol-antioxidant mode of action of lipoic
acid, NAC was also shown to protect against insulin resistance in ZDF rats or db/db mice (218, 421), or in normal
mice rendered insulin resistant and hyperglycemic in response to a 6-h infusion of high glucose (152). In accordance, in high-sucrose- or fructose-fed rats, increasing
dietary cysteine content either by high cysteine proteins
or by NAC markedly reduced diet-induced oxidative
stress and insulin resistance (36, 394). Thus most available information presents a striking agreement between
cell culture work (see sect. VA) and experimental animal
models in the potential therapeutic potential of thiolantioxidants to improve insulin action.
In addition to thiol antioxidants, enhancing antioxidant enzyme activity in vivo using the SOD/catalase
mimetic antioxidant MnTBAP improved insulin sensitivity in ob/ob mice (188). Tempol and other SOD mimetic antioxidants improved insulin action in various
rats with insulin resistance, including Zucker rats (16),
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D. Key Message From Section V
Studies in cell culture systems and in animal models
of obesity and diabetes have demonstrated the ability to
improve insulin action by modulating the pro-oxidant/
antioxidant balance. These provide the most compelling
evidence available implicating ROS/RNS in negatively affecting insulin signaling. However, human studies have
generally failed to yield evidence sufficiently powerful to
include currently available means of pharmacologically
manipulating the antioxidant system for improving health,
and have even raised safety concerns. Yet, consumption
of an antioxidant-rich diet is an accepted approach to
promote health. The complexity of the oxidant-antioxidant systems and their positive and negative influences on
the insulin signal transduction cascades likely underlie
the inconclusive state of this field, despite the enormous
investigative effort invested.
VI. SUMMARY AND CONCLUSIONS
ROS/RNS act as a double-edged sword in modulating
insulin signaling. On one hand, they are generated in
response to insulin and are required for insulin to exert its
full physiological actions. On the other hand, ROS/RNS
can impair insulin action, and since increased ROS/RNS
generation has been proposed to occur in obesity and
diabetes, they have been implicated in the pathogenesis of
insulin resistance. This seemingly paradoxical function of
Physiol Rev • VOL
bioactive agents is actually more the rule than it is the
exception in biology. Insulin itself, like most hormones,
would impair its own bioactions when present in excess.
However, in “free radical biology,” understanding physiological versus pathophysiological functions appears more
perplexing. The reasons for this are likely multiple:
1) ROS/RNS are a vast group of agents, each with unique
chemical-physical characteristics, interacting with each
other to generate yet additional ROS/RNS. Thus, unlike
insulin, this is not a single agent whose activity is governed by timing and concentration alone. 2) ROS/RNS are
short lived and are difficult to measure. Most existing
means of assessing their generation are indirect, and their
accuracy is questionable. 3) Biological systems developed
complex, interconnected antioxidant machineries that exist in normal physiology in a carefully maintained homeostatic balance. We can assume that physiological function
of ROS/RNS is possible as long as homeostasis is maintained, whereas pathophysiology occurs when perturbed,
but we currently have at hand limited tools to quantitatively measure the state of the oxidant-antioxidant balance. Hence, ROS/RNS biofunction is influenced by a
multidimensional matrix of factors constituting a “biological context.” Its individual components can be partially
defined (in which organ, subcellular location, timing and
duration, etc.), but its overall output is difficult to assess.
Despite this, it is possible to conclude that physiological function of ROS/RNS in insulin signaling includes
the generation of superoxide and/or H2O2 by NADPH
oxidase systems, and NO by eNOS. Likely, this regulated
RS generation is confined in time and space. Molecular
targets for such ROS generation are protein and lipid
phosphatases acting within the insulin signaling cascades
as shut-off mechanisms. Hence, their temporary, reversible inactivation propagates the insulin signal transduction cascades. Pathological ROS/RNS generation involves
multiple ROS/RNS generation systems, RS types, organs
and organelles, and is compounded by alterations in the
antioxidant defense systems. Molecular targets of such
ROS/RNS generation are multiple and include modifications of insulin signaling components (oxidative modifications, phosphorylation as a consequence of the activation of ROS/RNS-sensitive kinases), transcription factors
modulating the gene regulation of signaling components
and/or regulatory proteins (adipokines), and more generic
components of cellular function (cytoskeleton, protein
degradation machineries).
Translational research in this field, i.e., the ability to
improve insulin action by modulating the oxidant/antioxidant balance, has been disappointing so far. The fundamental features of the field outlined above, particularly,
the dual role of ROS/RNS in insulin action, the complexity
of the system, and our limited ability to assess it are part
of the reason. In addition, high availability of agents perceived as “nontoxic natural antioxidant” at low cost has
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modes aimed at manipulating the oxidant-antioxidant balance clinically ineffective. This could be either because
supplemented antioxidant is efficiently balanced (buffered) by other components of the system that are in
equilibrium with it, because excessive antioxidants can be
converted to pro-oxidants, and/or because a specific antioxidant simply does not affect the specific mechanism(s) responsible for excessive oxidant generation. Reflecting this notion is the recommendation endorsed by
clinical guidelines (7), to consume sufficient amounts of
naturally occurring dietary antioxidants by balanced eating habits. Whether consuming diets proposed to be high
in antioxidants exerts its beneficial effects by providing
carefully balanced, bioavailable antioxidant mix, and/or
includes novel, yet uncharacterized antioxidants, or simply includes yet unidentified dietary components unrelated to an antioxidant mode of action altogether, is
largely unknown. Clearly, addressing these possibilities
experimentally will enhance current understanding of the
role of ROS/RNS in insulin action and resistance, the
relevant mechanisms by which these effects occur, and
hopefully, would eventually lead to interventions to prevent insulin resistance and its enormous toll on human
health.
ROS, RNS, AND INSULIN ACTION
led to numerous studies, and on the other hand, to lack of
financial incentive to support large-scale, high-quality research. But has this field exhausted itself? Our hope is
that this review, by discussing both the achievements of
research in the field as well as the shortcomings in our
current understanding of this complex system, will aid in
directing research effort to prove otherwise. One can
envision (and hope) that better understanding of components of the biological context of RS function will assist in
developing “context-specific” modulators of RS, that
would help alleviate the health burden of insulin resistance-associated conditions.
ACKNOWLEDGMENTS
GRANTS
Our own work cited herein was supported by grants from
the Israel Science Foundation, the Israeli Ministry of Health,
D-Cure Diabetes Care in Israel, and the Leslie and Susan Gonda
(Goldschmied) Center for Diabetes Research and Education. N.
Bashan is Chair of the Fraida Foundation in Diabetes Research.
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