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[Cell Cycle 7:7, 874-879; 1 April 2008]; 2008 Landes Bioscience
874 Cell Cycle 2008; Vol. 7 Issue 7
The Nrf2 transcription factor is a crucial regulator of the cellular
redox homeostasis through its capacity to induce the expression of
enzymes, which detoxify reactive oxygen species, and of other
antioxidant proteins. Therefore, it plays an important role in the
protection from carcinogenesis induced by various insults. In addi-
tion, recent results identified a novel role of Nrf2 in tissue repair.
In the liver, regeneration after partial hepatectomy was strongly
delayed in the absence of Nrf2. This defect was shown to result
from transient resistance to insulin and insulin-like growth factor
1 that was caused by chronic oxidative stress in hepatocytes. These
results demonstrate a link between Nrf2 deficiency, oxidative stress
and insulin resistance, and suggest that activation of this transcrip-
tion factor could be a novel strategy to improve liver regeneration
in patients with acute or chronic liver injury. In addition, it may
help to alleviate oxidative stress-induced insulin resistance in the
liver and potentially also in other organs.
Liver Regeneration
In contrast to other organs, the liver has a unique capability to
fully regenerate after injury (reviewed in ref. 1). There are many
conditions that require this regenerative potential, such as resection
of liver tissue in patients with primary or metastatic liver tumors
or cell loss caused by viruses, autoimmune diseases and toxins,
including alcohol or commonly used anti-inflammatory, anticonvul-
sant, or chemotherapeutic drugs. Due to the crucial function of the
liver in metabolic regulation and detoxification of various substances,
rapid and effective liver repair is essential. If the extent of injury is
not too high, full regeneration of the liver can occur. However, the
regenerative capacity is insufficient after chronic injury as observed
for example in chronic viral hepatitis or after long-term alcohol abuse
(reviewed in ref. 2). These conditions often cause liver cirrhosis,
which is characterized by replacement of functional epithelial tissue
by non-functional connective tissue. While such conditions affect an
enormous part of the population world-wide, therapeutic approaches
are still unsatisfactory and mainly supportive. Therefore, the devel-
opment of specific therapies to enhance the regenerative capacity
of the liver is essential for the improvement of human health. This
requires a thorough understanding of the mechanisms underlying
liver homeostasis and regeneration and the identification of factors
that control and accelerate this process.
A particularly useful model to study liver regeneration in rodents
is partial hepatectomy. In this procedure, the large and median lobes
of the liver, which comprise approximately two third of the organ,
are surgically removed. As a consequence, the normally quiescent and
highly differentiated liver cells proliferate, and the original liver mass
is restored within a few days (reviewed in ref. 1).
The regeneration process after hepatectomy occurs in two
phases: priming, and cell cycle progression (reviewed in ref. 1).
The pro-inflammatory cytokines tumor necrosis factor- (TNF)
and interleukin-6 (IL-6) play an important role in the priming
step (reviewed in ref. 1). These cytokines activate nuclear factor B
(NFB), activator protein 1 (AP-1), and signal transducer and acti-
vator of transcription 3 (STAT3), resulting in G
0
to G
1
transition
and survival of liver parenchymal cells.
3-5
In the second phase of
liver repair, growth factors are required for progression of hepatocytes
through the cell cycle (reviewed in ref. 1). Mitogens involved in this
process include hepatocyte growth factor (HGF),
6,7
ligands of the
epidermal growth factor receptor,
8,9
and fibroblast growth factors.
10

In addition, insulin-like growth factor 1 (IGF-1) was shown to be
involved in this process.
11
Upon completion of the repair process
and restoration of the original liver mass, hepatocyte proliferation is
inhibited and further growth of the liver does not occur. The mecha-
nisms that are involved in this thorough growth control are not fully
understood, but signaling through transforming growth factor
(TGF) and activin
12
was shown to inhibit hepatocyte proliferation
upon completion of repair.
Oxidative Stress Impairs Liver Regeneration
Although the regeneration process is highly efficient, it can
be impaired by chronic oxidative stress caused by toxins, such as
ethanol or haloalkanes. This causes severe damage of hepatocytes
through lipid peroxidation and alkylation of cellular macromolecules
(reviewed in refs. 13 and 14). In turn, apoptotic and in particular
necrotic death of hepatocytes leads to activation of hepatic stellate
cells (HSC). The latter are responsible for enhanced deposition of
fibrillar collagen, and expression of various cytokines such as TGF-,
*Correspondence to: Sabine Werner; Institute of Cell Biology; ETH Zurich;
Honggerberg, Zurich CH-8093 Switzerland; Tel.: +41.44.633.3941; Fax:
+41.44.633.1174; Email: Sabine.werner@cell.biol.ethz.ch
Submitted: 01/15/08; Accepted: 01/18/08
Previously published online as a Cell Cycle E-publication:
http://www.landesbioscience.com/journals/cc/article/5617
Perspective
The cytoprotective Nrf2 transcription factor controls insulin receptor
signaling in the regenerating liver
Tobias A. Beyer and Sabine Werner*
Institute of Cell Biology; Department of Biology; ETH Zurich; Zurich, Switzerland
Key words: Nrf2, insulin, IGF-1, liver regeneration, oxidative stress, PI3K, ROS

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Nrf2 and insulin resistance
www.landesbioscience.com Cell Cycle 875
resulting in liver fibrosis and cirrhosis (reviewed in ref. 15). Oxidative
stress is generated in the liver by reactive oxygen species (ROS).
These harmful molecules are mainly produced by activated inflam-
matory cells through NAD(P)H oxidases, but they are also metabolic
intermediates of xenobiotics, and they are generated as by-products
in several metabolic reactions, in particular in the respiratory chain
(reviewed in ref. 16).
The Nrf2 transcription factor
To avoid damage by ROS, a tight regulation of the cellular
redox balance is required, and this is achieved at least in part by the
action of NF-E2 related factor 2 (Nrf2). Nrf2 is a member of the
capn collar family of proteins, which also includes the related
Nrf1 and Nrf3 transcription factors, as well as p45 NF-E2, Bach1
and Bach2. These proteins, in particular Nrf2, bind to antioxidant
response elements (ARE) in the promoters of their target genes.
Expression of many antioxidant proteins and enzymes that detoxify
ROS or various drugs are controlled by Nrf2, including glutathione
S-transferases (GST), NAD(P)H quinone oxidoreductase 1 (NQO1),
glutamate-cysteine ligase catalytic and modulatory subunits (GCLC
and GCLM), peroxiredoxin-1, and heme oxygenase-1 (reviewed in
refs. 17 and 18).
Under normal conditions, Nrf2 is retained in the cytoplasm
via binding to the actin-binding protein Keap-1 (reviewed in ref.
19), which also mediates its degradation via the ubiquitin-protea-
some pathway.
20,21
Upon addition of electrophilic substances,
which directly interact with Keap1 through Michael addition, Nrf2
becomes liberated and shuttles to the nucleus, where it activates
its target genes. In addition, phosphorylation of Nrf2 by different
kinases may also result in liberation from Keap1 and subsequent
translocation to the nucleus. This mechanism has been suggested
to regulate the activation of Nrf2 in response to oxidative stress
(reviewed in refs. 18 and 19).
The important role of Nrf2 in the cellular stress response is
reflected by the phenotype of Nrf2-deficient mice. Whereas young
animals are healthy under normal laboratory conditions,
22
older
mice develop symptoms resembling those of patients with the auto-
immune disease systemic lupus erythematosus, most likely due to
enhanced oxidative tissue damage.
23
Even young Nrf2 knockout
mice are highly susceptible to treatment with butylated hydroxy-
toluene, which caused death in these animals from acute respiratory
distress syndrome.
24
They are also highly sensitive to acetamino-
phen-induced hepatotoxicity
25,26
and to liver carcinogenesis induced
by benz(a)pyrene.
27
Expression and Function of Nrf2 during Tissue Repair
Although the roles of Nrf2 in toxin detoxification and cancer
prevention have been well established, the functions of this tran-
scription factor in tissue repair are still poorly characterized. In
previous studies, our laboratory demonstrated a strong induction of
Nrf2 expression in inflammatory cells and keratinocytes after skin
injury. Most importantly, Nrf2 knockout mice were characterized
by prolonged inflammation during wound healing, although the
repair of the injured epidermis and wound closure in general were
not affected by the loss of Nrf2.
28
Following these studies on skin
wounding, we now addressed the role of Nrf2 in liver regenera-
tion. In contrast to the skin, upregulation of Nrf2 expression was
not observed at any stage of liver regeneration following partial
hepatectomy. Since Nrf2 activity is predominantly regulated at the
posttranslational level (see above), we used ARE reporter mice to
monitor activation of this transcription factor during liver regenera-
tion. These mice carry a transgene in their genome, which includes
the ARE of the rat nqo1 gene, followed by a minimal promoter
and the coding sequence for human placental alkaline phosphotase.
Therefore, they allow detection of Nrf2 activation through alkaline
phosphatase staining.
29
Although a small population of hepato-
cytes, in particular around blood vessels, clearly expressed the ARE
reporter gene, most hepatocytes did not show activity of the reporter.
Nevertheless, it is still possible that the basal activity of Nrf2 in these
cells is required for liver regeneration. The important role of the
basal Nrf2 activity in tissue homeostasis was demonstrated in a skin
carcinogenesis study, which was recently performed in our labora-
tory. Thus, mice expressing a dominant-negative mutant of Nrf2 in
the epidermis showed a strongly enhanced incidence and multiplicity
of chemically-induced skin tumors, although activation of the ARE
reporter gene could not be observed at any time point during the
development of the tumors.
30
Therefore, the basal activity, which
cannot be detected by the available reporter mice, is obviously
important for skin cancer prevention and this may also be the case
for liver regeneration.
Role of Nrf2 in Redox Homeostasis of the Liver
To determine the role of Nrf2 in liver regeneration, we first analyzed
if the loss of Nrf2 disturbs the cellular redox balance in normal liver
and after hepatectomy.
31
Indeed, cultured primary hepatocytes from
Nrf2 knockout mice had enhanced levels of intracellular ROS as
determined biochemically. A similar finding was obtained in vivo
using dihydroethidine, a dye that intercalates into DNA upon oxida-
tion, and therefore allows monitoring of oxidative stress. Nuclear
staining was observed in livers of Nrf2 knockout mice, in particular
after hepatectomy, whereas livers of wild-type animals showed only
weak fluorescence. These results identify Nrf2 as an important regu-
lator of the cellular redox balance in the liver and demonstrate that
deficiency in Nrf2 results in chronic oxidative stress in hepatocytes,
which is further aggravated upon liver injury. Reduced expression
of ROS-detoxifying Nrf2 target genes in normal and particularly in
injured liver was identified as the underlying mechanism. However,
this did not result in an obvious defect in liver function as revealed by
the normal serum levels of aspartate and alanine aminotransferases.
Excessive release of these liver-specific enzymes into the serum is a
hallmark of liver dysfunction and damage (reviewed in ref. 2), and
this was not observed in the Nr2 knockout mice.
Recent studies revealed that the related Nrf1 transcription factor
is also involved in ROS detoxification in the liver. Cultured hepato-
cytes of mice lacking Nrf1 in liver parenchymal cells had enhanced
levels of ROS. These animals also developed severe steatosis and
spontaneous liver cancer,
32
a phenotype not observed in Nrf2-defi-
cient mice. Therefore, Nrf1 and Nrf2 seem to regulate overlapping
but also distinct target genes, and in particular the latter remain to
be identified.
These results shed new light onto the regulation of the cellular
redox balance in hepatocytes. They are complemented by studies on
the role of NFB in the liver. Luedde et al.,
33
deleted the IB kinase
subunit NEMO/IKK, which is essential for activation of NFB, in

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Nrf2 and insulin resistance
876 Cell Cycle 2008; Vol. 7 Issue 7
liver parenchymal cells. Interestingly, these mice developed sponta-
neous steatosis and hepatocellular carcinoma. This phenotype was at
least in part mediated by enhanced oxidative stress in the liver, since
it could be alleviated by treatment of the animals with antioxidants.
Enhanced oxidative stress was also seen in the absence of IKK,
although phenotypic abnormalities were only seen in these mice
upon challenging of the liver.
34,35
The enhanced levels of ROS seen
upon reduction of NFB activity are consistent with the upregula-
tion of antioxidative defenses by this transcription factor.
36
Interestingly, our recent results suggest a cross-talk between Nrf2
and NFB, since the activity of the latter was strongly enhanced in
the regenerating liver of Nrf2 knockout mice.
31
This is most likely
due to the enhanced oxidative stress in Nrf2-deficient cells, since
elevated levels of ROS cause NFB activation.
37
The latter in turn
enhances survival of hepatocytes after hepatectomy.
38
Therefore, its
activation is most likely a compensatory effect to reduce the extent of
oxidative stress and cell death in Nrf2 knockout mice.
Impaired Liver Regeneration in Nrf2 Knockout Mice
When partial hepatectomy was performed with Nrf2 knockout
mice, we found a significant delay in liver regeneration.
31
In mice of
both genotypes only very few proliferating hepatocytes were detected
in non-injured liver and 624 h after hepatectomy. However, a five-
fold increase in the number of apoptotic cells was observed early (6 h)
after injury in the knockout animals compared to wild-type controls,
whereas necrosis could never be observed in mice of both genotypes.
At later stages after hepatectomy, only extremely few apoptotic cells
were detectable. Proliferation peaked 48 h after hepatectomy, but the
peak was much less pronounced in Nrf2 knockout mice compared
to wild-type controls. 1 day later, the number of proliferating cells
remained lower in the knockout mice compared to wild-type animals.
At day 5 after hepatectomy, proliferation was almost completed in
the wild-type mice, whereas Nrf2-deficient hepatocytes continued to
proliferate at this time point. In spite of these differences, liver repair
was completed in mice of both genotypes at day 7 after injury as
demonstrated by the almost complete absence of proliferating cells.
Furthermore, the liver/body weight ratio had almost returned to the
levels seen before injury. These findings demonstrate that repair can
occur in the absence of Nrf2, but with a strong delay.
Role of Insulin/IGF-1 Signaling in the Liver Regeneration
Phenotype of Nrf2 Knockout Mice
In a search for the mechanisms underlying the delayed regenera-
tion of Nrf2-deficient liver, a defect in insulin/IGF-1 signaling in
the regenerating liver was identified
31
(Fig. 1). Consistent with data
obtained with adipocytes,
39
the chronic oxidative stress seen in hepa-
tocytes of Nrf2-deficient mice resulted in resistance to exogenous
insulin or IGF-1 in vitro. In vivo, tyrosine phosphorylation of the
insulin receptor substrates 1 and 2 (IRS-1 and -2) in response to
activation of the insulin receptor was strongly reduced in the regen-
erating liver of Nrf2-deficient mice. The activation of the insulin
receptor shortly after hepatectomy most likely occurs through release
of IGF-1 from its binding proteins upon injury.
Previous studies had shown that the inhibitory effect of ROS
on insulin/IGF-1 receptor signaling is mediated through activation
of serine/threonine kinases by ROS, which in turn phosphory-
late IRS-1 and IRS-2. This causes insulin/IGF-1 resistance, since
Ser/Thr-phosphorylated IRS proteins dissociate from the insulin
receptor. Therefore, activation of IRS proteins through tyrosine
phosphorylation does no longer occur. As a consequence, IRS-1 and
IRS-2 can no longer associate with and activate phosphatidylinositide-
3-kinase (PI3K) (reviewed in refs. 40 and 41). Consistent with these
data, association of PI3K with IRS-1 in response to insulin was
reduced in Nrf2-deficient hepatocytes in vitro, and reduced associa-
tion of these proteins was also seen in hepatectomized liver of these
mice in vivo. One of the inhibitory IRS serine/threonine kinases
may be JNK, which is known to be activated by ROS
35,42
and which
showed enhanced and prolonged activation in the injured liver of
Nrf2-deficient mice.
31
The Ser phosphorylation of IRS-1 (at Ser307
in the mouse/Ser312 in human IRS-1) inhibits its phosphoryla-
tion at Tyr608 (Tyr612 in human IRS-1) by the insulin or IGF-1
receptor,
43
thereby causing insulin/IGF-1 resistance.
44
The same
effect can be achieved by other IRS kinases, including IKK-
45
or
protein kinase C,
40
and the role of these kinases in the injured liver
of Nrf2-deficient mice remains to be identified.
Loss of Nrf2 Impairs the Activation of Pro-Mitogenic and Anti-
Apoptotic Signaling Pathways in the Regenerating Liver
Insulin/IGF-1 signaling is known to activate several pro-mitogenic
and anti-apoptotic signaling pathways, including the mitogen-acti-
vated kinase pathway and the PI3K-Akt pathway.
46
These pathways
were also activated in the liver of wild-type mice, but activation of the
p38 MAPK pathway and in particular of the PI3K-Akt pathway was
strongly impaired in the absence of Nrf2.
31
Most importantly, the
time-course of their activation correlated with the time-course of IRS
activation, whereas activation of the epidermal growth factor receptor
and the hepatocyte growth factor receptor, two major regulators of
liver repair (see above) could not be observed at this time point, and
there was also no difference in activation of these receptors between
wild-type and Nrf2 knockout mice.
The reduced p38 activation may well be involved in the regen-
eration defect of Nrf2-deficient mice, since cell cycle entry of
hepatocytes after partial hepatectomy was strongly inhibited by
systemic treatment of rats with a p38 inhibitor.
47
However, enhanced
and prolonged p38 activation was seen in c-Jun deficient mice and
this caused reduced hepatocyte proliferation and enhanced mortality
of these animals after hepatectomy.
48
Thus, the role of this pathway
in liver regeneration in general and in the phenotype of Nrf2
knockout mice in particular remains to be determined.
Of particular importance, however, is the strongly reduced
activation of the PI3K/Akt signaling pathway in Nrf2-deficient hepa-
tocytes after hepatectomy in vivo and after insulin treatment in vitro.
Thus, the strong insulin-mediated activation of Akt, which was seen
in cultured primary hepatocytes of wild-type mice, was no longer
observed in cells from Nrf2-deficient mice. In vivo, phosphoryla-
tion of Akt, which results in its activation and which occurs within
36 h after hepatectomy in the liver of wild-type mice, was strongly
reduced in the absence of Nrf2. In turn, reduced phosphorylation of
the Akt targets glycogen synthase kinase-3, p70/S6K and Bad was
observed. Since these proteins are crucial for cell proliferation and
survival (reviewed in refs. 49 and 50), the defect in PI3K signaling is
likely to be responsible for the delayed hepatocyte proliferation and
for the increased apoptosis of these cells after hepatectomy of Nrf2-
deficient mice.

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Nrf2 and insulin resistance
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Possible Therapeutic Consequences
The results described above reveal a crucial role of Nrf2 in liver
regeneration through its capacity to regulate the cellular redox
balance and thus efficient insulin/IGF-1 signaling in the injured
liver. The basal rather than the inducible activity of Nrf2 is likely
to be responsible for this effect, since ARE activation was only
observed in very few hepatocytes after hepatectomy, and since the
defect in insulin/IGF-1 signaling occurred at an early stage, where
upregulaton of Nrf2 target genes was not yet observed. Therefore,
further enhancement of Nrf2 action may help to speed up the liver
regeneration process. In addition, it may well be that activation
of Nrf2 is insufficient in diseased liver. In this case pharmacologic
activation of this transcription factor may be particularly useful and
could possibly prevent the fibrosis, which frequently occurs after
chronic injury. Therefore, activation of Nrf2 by natural substances,
such as sulforaphane or avicins, or by synthetic pharmaceuticals, e.g.,
by synthetic triterpenoids,
51-54
could be a promising new strategy to
improve regeneration in patients with acute or chronic liver damage.
However, long-term activation may also be deleterious, in particular
in patients with liver cancer, since this may enhance malignant
progression and resistance to chemotherapy. This hypothesis is
based on the finding that inactivating mutations in the Keap1 gene
were found in non-small-cell lung cancer, resulting in Nrf2 hyper-
activation. As a consequence, expression of antioxidant proteins,
detoxification enzymes and drug transporters was enhanced, resulting
in higher malignancy and resistance to chemotherapy.
55
Thus, future
studies will reveal if timely controlled activation of Nrf2 in the liver
is beneficial. In addition, it will be interesting to determine if Nrf2
deficiency is also associated with insulin resistance in other organs.
Figure 1. Schematic representation of insulin/IGF-1 receptor signaling in the regenerating liver and its impairment in the absence of Nrf2. Nrf2-mediated
expression of ROS-detoxifying enzymes prevents accumulation of ROS in the liver. Lack of Nrf2 causes oxidative stress and activation of serine-/threonine
kinases, which phosphorylate IRS-1 and IRS-2. These kinases include JNK and possibly others, such as protein kinase C. Ser/Thr phosphorylation of IRS-1
and IRS-2 causes dissociation of these proteins from the insulin receptor, resulting in reduced tyrosine phosphorylation of IRS-1 and IRS-2 by the receptor
kinase. Since tyrosine phosphorylation of IRS-1 is required for PI3K activation, phosphorylation of Akt and downstream targets as well as p38 activation are
reduced. The deficiency in these signaling pathways cause a delay in hepatocyte proliferation and enhanced apoptosis of these cells after liver injury. As a
consequence, liver regeneration is delayed in the absence of Nrf2.

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Nrf2 and insulin resistance
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Acknowledgements
The Nrf2 research in the laboratory of S.W. is supported by
a grant from the Swiss National Science Foundation (3100A9-
109340/1 to S.W.) and by the ETH Zurich.
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