MOLECULAR AND CELLULAR BIOLOGY, Nov. 2011, p. 4286–4297
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 31, No. 21
IRE1-Dependent Activation of AMPK in Response to Nitric Oxide?
Gordon P. Meares,1,6* Katherine J. Hughes,2Aaron Naatz,3Feroz R. Papa,4Fumihiko Urano,5
Polly A. Hansen,3Etty N. Benveniste,6and John A. Corbett3*
The University of Alabama at Birmingham, Department of Medicine, Division of Endocrinology, Diabetes and Metabolism,
Birmingham, Alabama 352941; Saint Louis University, Edward A. Doisy Department of Biochemistry, St. Louis,
Missouri 631042; The Medical College of Wisconsin, Department of Biochemistry, Milwaukee Wisconsin 532263;
The University of California, San Francisco, Department of Medicine, San Francisco, California 941434;
The University of Massachusetts Medical School, Program in Gene Function and Expression and
Program in Molecular Medicine, Worcester, Massachusetts 016055; and The University of
Alabama at Birmingham, Department of Cell Biology, Birmingham, Alabama 352946
Received 19 May 2011/Returned for modification 3 June 2011/Accepted 24 August 2011
While there can be detrimental consequences of nitric oxide production at pathological concentrations,
eukaryotic cells have evolved protective mechanisms to defend themselves against this damage. The unfolded-
protein response (UPR), activated by misfolded proteins and oxidative stress, is one adaptive mechanism that
is employed to protect cells from stress. Nitric oxide is a potent activator of AMP-activated protein kinase
(AMPK), and AMPK participates in the cellular defense against nitric oxide-mediated damage in pancreatic
?-cells. In this study, the mechanism of AMPK activation by nitric oxide was explored. The known AMPK
kinases LKB1, CaMKK, and TAK1 are not required for the activation of AMPK by nitric oxide. Instead, this
activation is dependent on the endoplasmic reticulum (ER) stress-activated protein IRE1. Nitric oxide-induced
AMPK phosphorylation and subsequent signaling to AMPK substrates, including Raptor, acetyl coenzyme A
carboxylase, and PGC-1?, is attenuated in IRE1?-deficient cells. The endoribonuclease activity of IRE1
appears to be required for AMPK activation in response to nitric oxide. In addition to nitric oxide, stimulation
of IRE1 endoribonuclease activity with the flavonol quercetin leads to IRE1-dependent AMPK activation.
These findings indicate that the RNase activity of IRE1 participates in AMPK activation and subsequent
signaling through multiple AMPK-dependent pathways in response to nitrosative stress.
Nitric oxide, an important mediator of both physiological
and pathological processes, has been implicated in the devel-
opment of a number of inflammatory diseases. When produced
at low concentrations, nitric oxide can promote cell growth and
survival. At high concentrations, such as those produced dur-
ing inflammation by inducible nitric oxide synthase (iNOS),
nitric oxide induces extensive cellular injury that includes DNA
damage, inhibition of oxidative metabolism, and induction of
endoplasmic reticulum (ER) stress (5, 12, 39). Pancreatic
?-cells are exquisitely sensitive to oxidative damage, as glu-
cose-stimulated insulin secretion requires the oxidation of glu-
cose to CO2, resulting in the accumulation of ATP. Nitric
oxide, produced in micromolar concentrations in response to
interleukin 1 (IL-1) and gamma interferon (IFN-?), mediates
the damaging effects of these cytokines on ?-cell function (3,
33). While nitric oxide stimulates cellular damage, it also ac-
tivates a number of signaling pathways that limit additional
cellular damage and repair existing damage. In pancreatic
?-cells, the protective responses activated by nitric oxide in-
clude (i) JNK-dependent induction of GADD45? (growth ar-
rest and DNA damage-inducible protein 45?) and DNA re-
pair, (ii) activation of AMP-activated protein kinase (AMPK),
resulting in enhanced metabolic recovery, and (iii) activation
of the unfolded-protein response (UPR) (25, 34, 38, 54, 57,
AMPK is a conserved heterotrimeric (?, ?, and ? subunits)
serine/threonine kinase involved in sensing and responding to
the energetic demand within eukaryotic cells (15). AMPK is
activated by phosphorylation at threonine 172 in the catalytic ?
subunit (19) in a constitutive fashion by the upstream kinase
LKB1; however, this phosphorylation is rapidly removed by a
phosphatase to maintain low basal activity (18, 43). AMPK is
activated under conditions that decrease cellular ATP levels,
such as hypoxia, DNA damage, glucose deprivation, and free
radical generation (2, 24, 32, 42). This includes nitric oxide-
induced activation of AMPK (2). Activation of AMPK from
disruption of energy homeostasis is due to the increased AMP/
ATP ratio, leading to binding of AMP to the regulatory ?
subunit; this binding of AMP causes a conformational change
in the AMPK complex that attenuates dephosphorylation (43).
The LKB1-dependent activation of AMPK can also be repli-
cated using AMP mimics such as 5-aminoimidazole-4-carboxy-
amide ribonucleoside (AICAR) (49). While LKB1 is a domi-
nant AMPK kinase, AMPK can also be phosphorylated and
activated independent of the cellular energy status. LKB1-
independent activation of AMPK can be mediated by the
Ca2?-sensitive calmodulin-dependent protein kinase kinase
(CaMKK) (20) and TGF?-activated kinase-1 (TAK1) (35).
AMPK regulates many cellular processes through the phos-
phorylation of target substrates. The mammalian target of
* Corresponding author. Mailing address for John A. Corbett: The
Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee,
WI 53226. Phone: (414) 955-8768. Fax: (414) 955-6510. E-mail: jcorbett
@mcw.edu. Mailing address for Gordon P. Meares: The University
of Alabama at Birmingham, MCLM 386, 1918 University Blvd.,
Birmingham, AL 35294. Phone: (205) 934-7668. Fax: (205) 975-5648.
?Published ahead of print on 6 September 2011.
rapamycin complex 1 (mTORC1) is a multisubunit kinase
composed of at least mTOR, FKBP12, mLST8, and Raptor
that is negatively regulated by AMPK. Under favorable growth
conditions, mTORC1 is active and promotes protein synthesis
through an inhibitory phosphorylation of the negative regula-
tor 4E-binding proteins and through an activating phosphory-
lation of p70 ribosomal S6 kinase 1 (S6K1) (4). Raptor acts as
a scaffold to recruit these substrates to the mTOR complex (36,
47). In response to cellular stress, AMPK inhibits mTORC1
signaling, in part through the phosphorylation of Raptor, lead-
ing to the dephosphorylation and inactivation of S6K1 (13).
Nitric oxide can cause ER stress and activate the highly
conserved UPR (38). The UPR includes three trans-ER mem-
brane proteins—activating transcription factor 6 (ATF6), eu-
karyotic translation initiation factor 2-alpha kinase 3 (PERK),
and inositol-requiring enzyme 1 (IRE1)—which transmit sig-
nals from the ER lumen to the cytosol and nucleus (40). ATF6
is a transcription factor that is released from the ER by pro-
teolytic cleavage and translocates to the nucleus to stimulate
the expression of UPR-associated genes (51, 59). PERK is a
serine/threonine kinase that phosphorylates eukaryotic trans-
lation initiation factor 2? (eIF2?) under ER stress conditions.
This response attenuates protein synthesis in an effort to re-
duce the protein burden on the ER (17). IRE1 is both a kinase
and an endoribonuclease. In response to ER stress, IRE1 is
activated by dimerization and transautophosphorylation and
splices the mRNA of XBP1 (6). Active IRE1 can also form a
complex with the adaptor protein TRAF2, facilitating the ac-
tivation of apoptosis signaling kinase 1 (ASK1) and subsequent
activation of JNK, thus coupling ER stress to MAPK signaling
We recently reported that IL-1 stimulates AMPK activation
in pancreatic islets in a nitric oxide-dependent fashion (34);
however, the mechanisms of this activation are currently un-
known. The purpose of this study was to identify the mecha-
nisms by which nitric oxide activates AMPK. We show that
kinases known to activate AMPK are dispensable for AMPK
activation in response to nitric oxide. In contrast, a novel sig-
naling role for the UPR transducer IRE1? in the activation of
AMPK in pancreatic ?-cells in response to nitric oxide has
MATERIALS AND METHODS
Materials. INS832/13 cells were obtained from Chris Newgard (Duke Univer-
sity, Durham, NC), IRE1?-deficient mouse embryo fibroblasts (MEFs) were
provided by Fumihiko Urano (University of Massachusetts Medical School),
PERK-deficient MEFs were provided by Ronald Wek (University of Indiana),
and INS1 cells stably expressing wild-type, kinase-deficient (K599A), or RNase-
deficient (K907A) IRE1? mutants under the control of a tetracycline (Tet)-
inducible promoter were provided by Feroz Papa (University of California, San
Francisco, CA). Plasmids containing inactive mutants of IRE1? and INS832/13
cells expressing RNase-deficient (K907A) and kinase-deficient (K599A) IRE1?
have been described previously (37). Dominant negative AMPK adenovirus was
provided by Christopher Rhodes (University of Chicago). RPMI 1640 and Dul-
becco’s modified Eagle medium (DMEM) tissue culture medium, L-glutamine,
streptomycin, and penicillin were from Mediatech, Inc. (Manassas, VA). Fetal
calf serum and quercetin were from Sigma (St. Louis, MO). (Z)-1(N,N-Diethyl-
amino)diazen-1-ium-1,2-diolate (DEANO), (Z)-1-[N-(3-ammoniopropyl)-N-(n-
propyl)amino]diazen-1-ium-1,2-diolate (PAPA NONOate, referred to here as
PAPA), thapsigargin, and tunicamycin were purchased from Axxora (San Diego,
CA). Antibodies used in this study include those specific for phospho-Thr172-
AMPK, phospho-Ser79-ACC, phospho-Ser473-Akt, phospho-Thr308-Akt, phos-
pho-Ser51-eIF2?, total AMPK, phospho-RAPTOR, phospho-p70S6K1, phos-
pho-PERK, IRE1, TAK1, LKB1 (Cell Signaling, Danvers, MA), and GAPDH
(Ambion, Foster City, CA). Horseradish peroxidase-conjugated donkey anti-
rabbit and donkey anti-mouse immunoglobulins were from Jackson Immuno-
Research Laboratories, Inc. (West Grove, PA). Predesigned Silencer Select
small interfering RNAs (siRNAs) were purchased from Ambion (Austin, TX).
Cell culture and treatments. INS832/13 cells were cultured in RPMI 1640
supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 1 mM
sodium pyruvate, 55 ?M ?-mercaptoethanol, 10 mM HEPES, 100 U/ml penicil-
lin, and 100 ?g/ml streptomycin. MEFs were cultured in DMEM supplemented
with 10% FBS, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin and 100
?g/ml streptomycin. Cells were maintained at 37°C under an atmosphere of 95%
air and 5% CO2. For cells treated with nitric oxide donors or AICAR, a stock
solution of 10 mM or 50 mM, respectively, was prepared in the appropriate
medium immediately before addition to the cells. Mutants of IRE1? were tran-
siently transfected into INS832/13 cells using Lipofectamine 2000 (Invitrogen,
Carlsbad, CA) according to the manufacturer’s protocol. Stable Tet-inducible
INS-1 cell lines expressing either wild-type (WT) or RNase-deficient (K907A)
IRE1? have been described (14). IRE1? expression was induced by the addition
of 1 ?g/ml doxycycline followed by overnight culture. Treatments were per-
formed 12 to 16 h after induction. Adenoviral transduction was performed as
described previously (34).
RNA interference. Cells were reverse transfected with siRNA and Lipo-
fectamine 2000 in 6-well plates according to the manufacturer’s protocol: all
transfections contained 100 pmol siRNA. To knock down AMPK?, both iso-
forms were targeted and cells were cotransfected with 50 pmol of each siRNA.
The siRNAs (Silencer Select; Ambion) used were LKB1#1 (S163339), LKB1#2
(S163340), TAK1 (S162205), AMPK?1#1 (S134808), AMPK?2#2 (S134963),
IRE1? (S176364), and PKC? (S130022).
XPB1 splicing. Total RNA was isolated using an RNeasy kit (Qiagen, Valen-
cia, CA) or TRIzol reagent (Invitrogen). RNA (1 ?g) was reverse transcribed to
cDNA using oligo(dT) and Moloney murine leukemia virus reverse transcrip-
tase. cDNA was analyzed by quantitative real-time PCR (qRT-PCR) using a
Light Cycler 480 (Roche) and primers spanning the splice junctions of XBP-1
(6a). Data were normalized to ?-actin. XBP-1 splicing was also analyzed by
RT-PCR using following primers: XBP-1 forward, ACACGCTTGGGAATGGA
CAC; XBP-1 reverse, CCATGGGAAGATGTTCTGGG. PCR products were re-
solved on a 3% agarose gel stained with ethidium bromide. Quantitative PCR of
PGC-1? was performed using a TaqMan gene expression assay according to the
manufacturer’s instructions in an ABI Prism 7500 analyzer (Applied Biosystems,
Foster City, CA). Reactions were carried out in 20 ?l and analyzed using the ??CT
Immunoblotting. Cells were washed twice with phosphate-buffered saline
(PBS) and then lysed with immunoprecipitation lysis buffer (20 mM Tris [pH
7.5], 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.5% NP-40, 1 mM sodium
orthovanadate, 100 ?M phenylmethanesulfonyl fluoride, 50 mM sodium fluo-
ride, and protease inhibitor cocktail; Sigma, St. Louis, MO). The lysates were
sonicated and centrifuged at 20,800 ? g for 15 min. Protein concentrations were
determined by a Bradford assay. Samples were mixed with Laemmli sample
buffer (2% sodium dodecyl sulfate [SDS]) and placed in a boiling water bath for
5 min. Proteins were resolved in SDS-polyacrylamide gels and transferred to
nitrocellulose, and the membranes were incubated with primary antibody over-
night at 4°C. Primary antibodies were used at a 1:1,000 dilution except the
GAPDH antibody, which was used at a 1:25,000 dilution. Incubations with
horseradish peroxidase-conjugated donkey anti-mouse, donkey anti-rabbit, or
donkey anti-rat IgG (1:7,000 dilution) secondary antibodies were performed for
1 h at room temperature, followed by detection with enhanced chemilumines-
Aconitase activity. INS832/13 cells were isolated by centrifugation, and mito-
chondrial aconitase activity was determined as described previously (34, 45).
Briefly, aconitase was assayed at 340 nm in a reaction mixture containing 20 mM
citrate, 0.2 mM NADP, 6 mM MnCl2, 50 mM Tris-Cl (pH 7.4), 0.6 U of isocitrate
dehydrogenase, and 50 ?l of cell extract in a total volume of 200 ?l at room
temperature. Aconitase activity was quantified as 1 pmol of NADP (reduced)
formed per minute per microgram of protein.
Statistical analysis. Statistical analysis was performed using Student’s t test or
Student Newman-Keuls post hoc analysis of variance (ANOVA).
Nitric oxide induces AMPK activation and inhibition of
mTORC1 signaling. Treatment of insulinoma INS832/13 cells
with the nitric oxide donor (Z)-1(N,N-diethylamino)diazen-1-
VOL. 31, 2011NO-ACTIVATED AMPK CONNECTS IRE1 TO mTOR SIGNALING4287
ium-1,2-diolate (DEANO) for 10 to 60 min results in the rapid
activation of AMPK, as measured by the activation-associated
phosphorylation of AMPK at threonine 172 and the subse-
quent phosphorylation of the AMPK substrates acetyl-coen-
zyme A carboxylase (ACC) and Raptor (Fig. 1A). To verify the
regulatory role of AMPK in mTOR signaling, INS832/13 cells
were treated with the AMPK activator, AICAR. AICAR en-
hanced the phosphorylation of AMPK and the AMPK-medi-
ated phosphorylation of Raptor (Fig. 1B). These effects were
associated with a concomitant decrease in the phosphorylation
of the mTORC1 substrate S6K1. Recent studies have shown
that S6K1 phosphorylates the scaffold protein Rictor in the
rapamycin-insensitive mTOR complex (TORC2), leading to
attenuation of TORC2 substrate phosphorylation (28). One
substrate of TORC2 is Akt, which is phosphorylated at serine
473 (44). Consistent with the decrease in S6K1 phosphoryla-
tion observed in response to AICAR, there is an increase in
the phosphorylation of Akt at serine 473 and threonine 308
The simultaneous siRNA knockdown of both isoforms (?1
and ?2) of the catalytic subunit of AMPK? was used to confirm
that under basal (unstimulated) conditions AMPK regulates
S6K1 and Akt. As expected, AMPK knockdown increased
basal S6K1 phosphorylation and decreased Akt phosphoryla-
tion (Fig. 1C, lanes 1 and 2). Under these conditions, the
modest level of AMPK knockdown (?50%) was not sufficient
to attenuate the stimulatory effect of nitric oxide on the phos-
phorylation of Raptor or the subsequent inhibition of S6K1
phosphorylation (Fig. 1C). These findings suggest either that
AMPK is present in such a large excess that even a 50%
decrease in protein is not sufficient to alter substrate phosphor-
ylation under stimulated conditions or that nitric oxide-in-
duced phosphorylation of Raptor is AMPK independent. To
examine these possibilities, INS832/13 cells were transduced
with adenovirus expressing a dominant negative mutant of
AMPK (DN-AMPK) that has been shown to effectively inhibit
AMPK in INS832/13 cells (34). As shown in Fig. 1D, the
stimulatory effects of nitric oxide on Raptor phosphorylation
are attenuated in cells expressing DN-AMPK. These findings
suggest that nitric oxide-induced Raptor phosphorylation is
mediated by AMPK.
The mechanism controlling Akt phosphorylation in response
to nitric oxide appears to differ from activation induced by
pharmacological activation of AMPK. In response to AICAR,
the phosphorylation of Akt at 473 and 308 is enhanced (Fig.
1B); however, nitric oxide treatment results in attenuation in
the phosphorylation of Akt (Fig. 1C). These findings, which
suggest that the regulation of Akt phosphorylation in response
to nitric oxide is independent of mTOR, are consistent with
previous reports showing that Akt is a direct target for inhib-
itory S-nitrosation by nitric oxide (58). Overall, these findings
confirm that nitric oxide activates AMPK (2, 34, 61) and that
AMPK activation correlates with Raptor phosphorylation and
the inhibition of mTORC1 signaling (13). These studies also
suggest that nitric oxide may alter mTOR signaling in part
through AMPK activation.
FIG. 1. Nitric oxide activates AMPK and modifies mTOR signaling. (A) INS832/13 cells were treated with the nitric oxide donor DEANO for
0 to 60 min (B) or the indicated concentrations of AICAR for 1 h. (C) INS832/13 cells were mock transfected or transfected with siRNA for
AMPK?1 and AMPK?2 (together) for 48 h followed by treatment with DEANO (1 mM) for the indicated times. (D) INS832/13 cells were
transduced with an adenoviral dominant negative AMPK using a multiplicity of infection (MOI) of 50 to 200 for 24 h followed by treatment with
1 mM DEANO for 30 min. Following treatments, the cells were harvested, and lysates were separated by SDS-gel electrophoresis followed by
Western blot analysis using the antibodies specific for the indicated target proteins. Results are representative of three independent experiments.
4288 MEARES ET AL.MOL. CELL. BIOL.
Nitric oxide-induced AMPK activation is independent of
known AMPK kinases. LKB1, CaMKK, and TAK1 have been
shown to function as activators of AMPK (18, 20, 35). LKB1 is
a primary kinase responsible for the activating phosphorylation
of AMPK (18, 49, 56). To determine if LKB1 is responsible for
nitric oxide-stimulated AMPK activation, two distinct siRNAs
were used to knock down endogenous LKB1 in INS832/13
cells. These siRNAs reduce steady-state levels of LKB1 pro-
tein, and this is associated with attenuation of H2O2-induced
AMPK phosphorylation (Fig. 2A). These findings are consis-
tent with previous reports showing that H2O2-induced AMPK
activation is LKB1 dependent (49). In contrast, LKB1 knock-
down does not influence DEANO-induced AMPK phosphor-
ylation (Fig. 2A). These data suggest that nitric oxide-induced
AMPK activation could be independent of LKB1.
In endothelial cells, low concentrations of nitric oxide have
been shown to stimulate AMPK activation by a pathway de-
pendent on soluble guanylate cyclase (sGC) and CaMKK (61).
To determine if this pathway mediates the activation of AMPK
with high levels of nitric oxide, INS832/13 cells were treated
with DEANO in the absence or presence of the CaMKK in-
hibitor STO-609. Inhibition of CaMKK did not influence
DEANO-induced activation of AMPK, although basal levels of
AMPK phosphorylation were reduced, indicating that the in-
hibitor was effective (Fig. 2B). Similarly, the sGC inhibitor
ODQ did not influence DEANO-induced AMPK phosphory-
FIG. 2. LKB1, CaMKK, and TAK1 are not essential for nitric oxide-induced activation of AMPK. (A) INS832/13 cells were mock transfected
or transfected with two distinct siRNAs for LKB1 for 48 h followed by treatment with 100 ?M H2O2or 1 mM DEANO for 30 min. (B) INS832/13
cells were pretreated for 30 min with 1 to 10 ?M concentrations of the CaMKK inhibitor STO-609 followed by treatment with 1 mM DEANO
for 30 min. (C) INS832/13 cells were mock transfected or transfected with siRNA for TAK1 for 48 h followed by treatment with 1 mM DEANO
for 30 min. (D) INS832/13 cells were transfected with siRNA for LKB1 and/or TAK1 for 48 h or pretreated for 30 min with 10 ?M STO-609 as
indicated. Cells were then treated with 1 mM DEANO for 30 min, and target protein levels were analyzed by Western blot analysis. (E) Summary
of the effects of target molecule inhibition on DEANO-induced AMPK phosphorylation (*, 30 ?M PKC? attenuated AMPK phosphorylation but
was toxic to cells, and the loss of AMPK phosphorylation was not confirmed by siRNA for PKC?). Results are representative of three independent
VOL. 31, 2011 NO-ACTIVATED AMPK CONNECTS IRE1 TO mTOR SIGNALING4289
lation (data not shown), nor did the Ca2?chelator 1,2-bis(2-
aminophenoxy)ethane-N,N,N?,N?-tetraacetic acid tetrabis(ace-
toxymethyl ester) (BAPTA-AM) (Fig. 2E). These results
suggest that sGC and CaMKK are not involved in the activa-
tion of AMPK by nitric oxide in ?-cells. However, the ability of
STO-609 to attenuate basal AMPK phosphorylation suggests
that CaMKK may function as a regulator of basal AMPK
TAK1 has also been reported to phosphorylate AMPK (35)
and to influence mTOR signaling through AMPK (22). siRNA
knockdown was used to examine the potential role of TAK1 in
nitric oxide-induced AMPK phosphorylation. Targeted siRNA
effectively reduced TAK1 expression but did not prevent
DEANO-induced phosphorylation of AMPK (Fig. 2C). Simi-
lar results were obtained using a second distinct siRNA specific
to TAK1 (not shown). These findings indicate that TAK1,
much like LKB1 and CaMKK, is not responsible for nitric
oxide-induced phosphorylation of AMPK. We also explored
the possibility that these AMPK kinases function in a compen-
satory fashion by examining the effects of simultaneous knock-
down of LKB1 and TAK1 and inhibition of CaMKK on nitric
oxide-induced AMPK and Raptor phosphorylation. The pres-
ence of a combination of siRNA against LKB1 and TAK1 and
the CaMKK inhibitor STO-609 did not prevent nitric oxide-
stimulated AMPK or Raptor phosphorylation (Fig. 2D). These
findings indicate that it is unlikely for compensatory AMPK
activation by these known AMPK activators to be involved in
the regulation of AMPK by nitric oxide. A number of addi-
tional potential target molecules were examined in our effort
to identify the mechanism of nitric oxide-induced AMPK phos-
phorylation. The findings from these studies are summarized in
Fig. 2E. The sum total of these studies suggests that nitric
oxide activates AMPK by a pathway that is independent of
known AMPK kinases.
Nitric oxide-induced AMPK activation is IRE1 dependent.
Nitric oxide is known to cause ER stress and activation of the
UPR in a number of cell types, including ?-cells (12, 38, 53).
Consistent with these previous studies, DEANO treatment of
INS832/13 cells for 10 to 60 min results in increased phosphor-
ylation of PERK and its substrate eIF2? (Fig. 3A). In response
to nitric oxide, UPR activation temporally parallels AMPK
activation and the inhibition of mTOR signaling (Fig. 1A).
These data raise the possibility of regulatory cross talk between
the UPR and AMPK/mTOR signaling pathways in response to
Considering the temporal correlation of AMPK and UPR
activation in response to nitric oxide, the potential role(s) of
the endoribonuclease/kinase IRE1? and the eIF2? kinase
PERK in regulating the response of AMPK to nitric oxide was
examined. AMPK phosphorylation in response to nitric oxide
(DEANO at concentrations from 0.1 to 1 mM) is attenuated in
INS832/13 cells depleted of IRE1? by siRNA knockdown (Fig.
3B). Under these conditions, the activity of IRE1? is impaired
as the stimulatory effects of nitric oxide on the splicing of
XBP1 are attenuated in INS832/13 cells transfected with
IRE1? siRNA (Fig. 3C). Importantly, the role of IRE1? in the
FIG. 3. IRE1 participates in the activation of AMPK by nitric oxide. (A) INS832/13 cells were treated with 1 mM DEANO for the indicated
times or left untreated, followed by Western blot analysis for phosphorylated PERK and eIF2?. (B) INS832/13 were mock transfected (?) or
transfected with siRNA for IRE1? for 48 h, followed by treatment with the indicated concentrations of DEANO for 30 min. The cells were
harvested, and AMPK phosphorylation and total IRE1? and AMPK levels were determined by Western blot analysis. (C) The effects of a 30-min
treatment with 1 mM DEANO on XBP1 splicing in INS832/13 cells transfected with IRE1? siRNA are shown. Splicing was analyzed by qRT-PCR.
(D) INS832/13 cells, mock transfected or transfected with IRE1? siRNA as for panel B, were treated with the indicated concentrations of AICAR
for 30 min. The cells were harvested, and levels of phosphorylated AMPK and ACC and total levels of AMPK and IRE1? were determined by
Western blot analysis. Results are representative of two or three individual experiments.
4290 MEARES ET AL.MOL. CELL. BIOL.
regulation of AMPK appears to be selective for nitric oxide.
IRE1? siRNA knockdown does not influence AICAR-induced
AMPK phosphorylation or AMPK-mediated ACC phosphor-
ylation (Fig. 3D). The reduction in nitric oxide-induced AMPK
phosphorylation observed in siRNA-treated IRE1?-deficient
cells was approximately 40%. While this level of reduction is
consistent with a partial knockdown of IRE1?, it is also pos-
sible that pathways in addition to IRE1? contribute to AMPK
activation in response to nitric oxide. Further, the results pre-
sented in Fig. 1 indicate that AMPK is present in excess, and
large changes in activity or level are needed to have discernible
affects on downstream signaling. Consistent with these find-
ings, the 40% reduction in AMPK phosphorylation in IRE1?
knockdown cells (as shown in Fig. 3B) did not alter the phos-
phorylation of AMPK substrates such as ACC and Raptor in
response to nitric oxide (data not shown).
MEFs deficient in IRE1? were used to confirm that IRE1?
participates in AMPK activation in response to nitric oxide.
Like ?-cells, MEFs respond to DEANO with an increase in the
phosphorylation of AMPK and Raptor (Fig. 4A). Compared to
wild-type cells, there is a marked reduction of nitric oxide-
induced AMPK phosphorylation in IRE1??/?MEFs. At 0.5
mM, DEANO induces maximal AMPK phosphorylation, and
there is little further increase in AMPK phosphorylation at
concentrations up to 2 mM (Fig. 4A). In IRE1?-deficient
MEFs, DEANO-induced AMPK phosphorylation is attenu-
ated at all concentrations examined. Consistent with an inhi-
bition in AMPK activation, DEANO-stimulated phosphoryla-
tion of ACC and Raptor is also attenuated in IRE1??/?cells
compared to wild type (Fig. 4A). In concert with Raptor-
dependent attenuation of mTORC1 signaling, the phosphory-
lation of S6K1 was significantly reduced in wild-type MEFs;
however, it remained near basal levels in IRE1??/?cells
treated with DEANO (Fig. 4A and B). Even though AMPK
activation is attenuated, IRE1??/?cells are responsive to ni-
tric oxide, as DEANO stimulates the phosphorylation of eIF2?
to similar levels in wild-type and IRE1?-deficient MEFs (Fig.
4A). As an additional control, we show that the total levels of
AMPK? are indistinguishable in the wild-type and IRE1??/?
cells (Fig. 4A and B), confirming that the defects in AMPK
signaling are not associated with reduced levels of this energy
sensor. The regulation of AMPK by nitric oxide was confirmed
using a second donor, PAPA, which also stimulates the IRE1-
dependent phosphorylation of AMPK in MEFs (Fig. 4C).
We also examined whether AMPK-dependent gene expres-
sion is negatively affected by loss of IRE1?. PGC-1? expres-
sion is increased in response to nitric oxide in an AMPK-
dependent fashion (30). Following exposure to exogenous
nitric oxide, PGC-1? is increased in wild-type (WT) MEFs,
while nitric oxide-induced expression of PGC-1? is absent in
IRE1??/?cells (Fig. 4D). These data indicate that IRE1 par-
ticipates in the regulation of nitric oxide-induced AMPK acti-
vation and subsequent signaling to targets, such as PGC-1?,
that participate in regulation of mitochondrial function and
biogenesis. Since AMPK participates in the recovery of mito-
chondrial function following nitric oxide-mediated damage
(34), the effects of IRE1 depletion on the recovery of mito-
chondrial aconitase activity were evaluated. INS832/13 or
INS832/13 cells depleted of IRE1 using siRNA were treated
for 1 h with DEANO or were treated for 1 h with DEANO,
washed, and then cultured for 3 additional hours, at which time
mitochondrial aconitase activity was examined. One hour of
incubation with DEANO results in ?75% reduction in aconi-
tase activity (Fig. 4E). Removal of the nitric oxide donor by
washing and continued culture for 3 h results in a nearly com-
plete recovery of aconitase activity in control cells, while the
recovery of aconitase activity is attenuated (by 20%) in cells
deficient in IRE1? (siRNA knockdown resulted in a 70% loss
of IRE1, as determined by Western blot analysis [data not
shown]). The depletion of IRE1 attenuates the recovery of
aconitase activity (Fig. 4E) to levels similar to those observed
under conditions in which AMPK is inhibited (34). These find-
ings indicate that the stimulatory effects of nitric oxide on
PGC-1? expression and the recovery of mitochondrial aconi-
tase activity following nitric oxide-induced damage are regu-
lated, at least in part, by the IRE-1?-dependent activation of
To determine if IRE1 deficiency causes a general defect in
signaling to AMPK, the effects of activators (H2O2and sorbi-
tol) that are known to enhance AMPK activity in an LKB1-
dependent fashion (49) were examined. The expression of
LKB1 (data not shown) and the phosphorylation of AMPK
and its downstream substrate ACC are similar in wild-type and
IRE1??/?cells treated with H2O2(change [fold] between
phosphorylated AMPK and AMPK, 1.86 ? 0.25 in WT versus
2.5 ? 0.76 in IRE1?/?cells; n ? 3; not significant [NS]) and
sorbitol (change [fold] between phosphorylated AMPK and
AMPK, 5.2 ? 1.3 in WT versus 6.1 ? 3.3 in IRE1?/?cells; n ?
3; NS). For these studies, the AMPK activator AICAR was
used as a positive control (Fig. 4G). To explore the potential
influence of additional UPR transducers, AMPK activation in
response to nitric oxide was also examined using PERK-defi-
cient MEFs. PERK?/?and wild-type MEFs respond to
DEANO in a similar manner, with an increase in the phos-
phorylation of AMPK and ACC (Fig. 4F), suggesting that
PERK does not influence AMPK activation in response to
nitric oxide. Together, these data support the identification of
a novel role for IRE1? in a pathway leading to the activation
of AMPK in response to nitric oxide. This pathway now pro-
vides a mechanistic association of one component of the UPR
with the mTOR signaling pathway and induction of PGC-1?
expression in response to nitric oxide.
ER stress alone is insufficient to activate AMPK. To deter-
mine if IRE1?-dependent activation of AMPK is a general
feature of ER stress, the phosphorylation of AMPK, Raptor,
and eIF2? was examined in wild-type MEFs and INS832/13
cells treated with UPR activators. While the glycosylation in-
hibitor tunicamycin and nitric oxide both stimulate ER stress,
as indicated by increased XBP-1 splicing (Fig. 5A) and phos-
phorylation of eIF2? (Fig. 5B), tunicamycin does not increase
the phosphorylation of AMPK or Raptor (Fig. 5B). In contrast,
DEANO stimulates AMPK and Raptor phosphorylation in
wild-type MEFs (Fig. 5B). As with MEFs, treatment of
INS832/13 cells with the Ca2?ATPase inhibitor thapsigargin,
or the reducing agent dithiothreitol (DTT), results in the in-
duction of ER stress, as evidenced by the increased phosphor-
ylation of eIF2?. Under these conditions, thapsigargin and
DTT failed to increase AMPK phosphorylation (Fig. 5C). In
contrast, DEANO stimulates both AMPK and eIF2? phos-
phorylation following a 30-min incubation (Fig. 5C). These
VOL. 31, 2011NO-ACTIVATED AMPK CONNECTS IRE1 TO mTOR SIGNALING 4291
FIG. 4. Nitric oxide-induced AMPK activation and signaling is attenuated in IRE1-deficient cells. (A) Wild-type and IRE??/?MEFs were
treated with the indicated concentrations of DEANO for 10 min, and the phosphorylation of AMPK, ACC, Raptor, S6K, and eIF2? was
determined by Western blot analysis. Total levels of IRE1? confirm its absence in the deficient MEFs, and total levels of AMPK and GAPDH are
shown as loading controls. (B) The phosphorylated forms of AMPK, Raptor, and S6K1 and total AMPK in wild-type and IRE1?-deficient MEFs
treated with 1 mM DEANO for 10 min were quantified. (C) Wild-type and IRE??/?cells were treated with the indicated concentrations of PAPA
4292MEARES ET AL.MOL. CELL. BIOL.
data suggest that the presence of IRE1 is required for nitric
oxide-induced AMPK activation; however, the induction of
general ER stress using classical activators is not sufficient to
induce AMPK phosphorylation.
The RNase activity of IRE1? is required for nitric oxide-
induced AMPK activation. Mutations in the kinase and RNase
domains of IRE1? were used to determine the mechanisms by
which IRE1? regulates AMPK. INS832/13 cells expressing WT
IRE1?, IRE1?-K599A (kinase deficient), or IRE1?-K907A
(RNase deficient) were treated with nitric oxide for 30 min,
and AMPK activation was examined. Mutation of the kinase
activity of IRE1? did not prevent but rather enhanced nitric
oxide-induced AMPK phosphorylation (Fig. 5D and E). In
contrast, mutation of the RNase activity of IRE1? significantly
attenuated nitric oxide-induced AMPK phosphorylation (Fig.
5D and E). IL-1? induces nitric oxide-dependent activation of
AMPK (34). Consistent with the previous results, mutation of
the RNase activity of IRE1? significantly attenuated IL-1?-
induced AMPK phosphorylation (Fig. 5F). These data suggest
that exogenously supplied and endogenously produced nitric
oxide stimulate the IRE1? RNase-dependent activation of
Recently, flavonols such as quercetin have been identified as
IRE1 ligands that, in addition to other biological actions, stim-
ulate IRE1 RNase activity independent of kinase activation
(55). Additionally, quercetin has been shown to activate
AMPK (1, 21). Therefore, to independently verify that IRE1?
RNase activation stimulates AMPK phosphorylation, the ef-
fects of quercetin on AMPK phosphorylation were examined.
While the phosphorylation of AMPK and ACC is stimulated in
a concentration-dependent manner by quercetin in WT MEFs
(Fig. 6A), this stimulatory effect is attenuated in IRE1?-defi-
cient cells (Fig. 6A). The effects of tunicamycin and quercetin
on IRE1?-dependent splicing of XBP1 were used to confirm
that quercetin stimulates IRE1 RNase activation (Fig. 6B). As
with the MEFs, quercetin stimulated the concentration-depen-
dent phosphorylation of AMPK and ACC in INS832/13 cells
(Fig. 6C and D). To confirm that quercetin stimulates IRE1?
for 15 min, and levels of phosphorylated AMPK and total AMPK were determined by Western blot analysis. (D) Wild type and IRE??/?cells were
treated with 1 mM DEANO for 3 h and PGC-1? mRNA accumulation was determined by qRT-PCR. (E) INS832/13 cells were transfected with
scrambled (control) or IRE1? siRNA for 24 h and then either treated for 1 h with DEANO or treated for 1 h with DEANO, washed, and cultured
for 3 additional hours. The cells were isolated, and mitochondrial aconitase activity was measured. (F) Wild-type or PERK?/?MEFs were treated
with the indicated concentrations of DEANO for 10 min, and levels of phosphorylated AMPK and ACC and total AMPK were determined by
Western blot analysis. (G) Wild-type and IRE??/?MEFs were treated with the AMPK activator H2O2(100 ?M) or sorbitol (0.6 M) for 30 min
or AICAR (2 mM) for 2 h. The MEFs were harvested, and levels of phosphorylated AMPK and ACC and total levels of IRE1?, AMPK, and
GAPDH were determined by Western blot analysis. Data are means ? standard errors of the means (SEM) (B, D, and E; n ? 3 to 4;*, P ? 0.05)
or are representative of three independent experiments (A, C, F, and G).
FIG. 5. IRE1? RNase but not ER stress activates AMPK. (A) Wild-type MEFs were treated with tunicamycin (5 ?M) or DEANO (1 mM) for
the indicated times, and XBP-1 splicing was measured by RT-PCR. (B) Wild-type MEFs were treated with 5 ?M tunicamycin for the indicated
times or left untreated, and phosphorylated AMPK, Raptor, and eIF2? were examined by Western blot analysis. Wild-type MEFs treated with
DEANO were used as a positive control. The asterisk indicates a variable cross-reactive band. (C) INS832/13 cells were treated with thapsigargin
(1 ?M) or DTT (2 mM) followed by examination of eIF2? phosphorylation; DEANO was used as a positive control. (D and E) INS832/13 cells
transfected with wild-type IRE1? or with IRE1?-K599A (kinase deficient) or IRE1?-K907A (RNase deficient) mutants were treated with DEANO
(1 mM) for 30 min, and AMPK phosphorylation was examined by Western blot. (F) Stable INS1 cell lines were treated for 16 h with doxycycline
(1 ?g/ml) to induce WT or K907A-IRE1?-myc, followed by treatment with IL-1? (10 U/ml) for 16 h. The level of phosphorylated AMPK was
quantified. Data are means ? SEM; n ? 3;*, P ? 0.05 versus WT control.
VOL. 31, 2011NO-ACTIVATED AMPK CONNECTS IRE1 TO mTOR SIGNALING 4293
RNase-dependent activation of AMPK, INS1 cells stably
expressing a Tet-inducible WT or RNase-deficient mutant
(K907A IRE1?-myc) were examined. Quercetin stimulated
AMPK phosphorylation in cells expressing WT IRE1?-myc,
and this phosphorylation was significantly attenuated in cells
expressing the RNase-deficient mutant K907A-IRE1?-myc
(Fig. 6E and F). These data indicate that nitric oxide and the
flavonol quercetin stimulate AMPK activation through IRE1?
Nitric oxide, produced in response to inflammatory cyto-
kines or supplied exogenously, causes widespread cellular
damage. Nonetheless, ? cells can survive this onslaught and
repair the damage if nitric oxide is removed from the cells
before a critical threshold of damage is reached (8, 25, 45). The
mechanisms and participants responsible for this recovery pro-
cess are only beginning to be elucidated. We showed previously
that AMPK is transiently activated in a nitric oxide-dependent
fashion in response to IL-1? or by the exogenous addition of
nitric oxide using donors, and this activation is associated with
improved metabolic function and an attenuation of ?-cell
death following nitric oxide treatment (34). This is particularly
important for ? cells because proper metabolic function (oxi-
dation of glucose and production of ATP) is essential for
glucose-stimulated insulin secretion. While these previous
studies outlined an important protective action of AMPK, the
mechanisms responsible for nitric oxide-mediated AMPK ac-
tivation in ? cells were unknown.
To explore the mechanisms by which nitric oxide modifies
AMPK signaling, we initially evaluated the effects of inhibitors
and siRNA knockdown for each of the known AMPK kinases,
LKB1, CaMKK, and TAK1. In all cases, inhibition or siRNA
knockdown did not inhibit nitric oxide-stimulated AMPK ac-
tivation, suggesting that each of these known AMPK kinases is
dispensable (Fig. 2) for the stimulatory actions of nitric oxide
on AMPK. Nitric oxide has also been shown to activate ER
stress responses in ? cells, and the temporal nature of this
activation correlates with the ability of nitric oxide to activate
AMPK. Specifically, nitric oxide stimulation of AMPK phos-
phorylation correlates temporally with PERK and eIF2? phos-
phorylation, suggesting that ER stress may influence the acti-
FIG. 6. Quercetin stimulates IRE1? RNase-dependent AMPK activation. (A) Wild-type and IRE??/?MEFs were treated with the indicated
concentrations of quercetin, and phosphorylated AMPK and ACC were measured by Western blotting. (B) Wild-type and IRE??/?MEFs were
treated with tunicamycin (5 ?M) or quercetin (200 ?M) for 2 h, and XBP-1 splicing was measured by RT-PCR. XBP-1u and XBP-1s indicate
unspliced and spliced XBP-1, respectively. (C and D) INS832/13 cells were treated with the indicated concentrations of quercetin, and phosphor-
ylated AMPK and ACC were measured by Western blotting. (E and F) Stable INS1 cell lines were treated for 16 h with doxycycline (1 ?g/ml) to
induce WT or K907A-IRE1?-myc, followed by treatment with quercetin for 1 h. The levels of phosphorylated AMPK and total AMPK and
IRE1?-myc were examined. Data are means ? SEM (D and F; n ? 3;*, P ? 0.05 versus control) or are representative of three independent
experiments (A, B, C, and E).
4294MEARES ET AL.MOL. CELL. BIOL.
vation of AMPK in response to nitric oxide. In examining this
hypothesis, we made the unexpected observation that the IRE1
pathway regulates AMPK activation in response to nitric oxide.
This signaling through AMPK leads to IRE1-dependent regu-
lation of mTOR. Interestingly, this appears to be selective for
nitric oxide, as cells lacking IRE1 respond normally to LKB1-
mediated activation of AMPK (e.g., H2O2) (Fig. 4G). PERK,
an eIF2? kinase, does not participate in the regulation of
AMPK in response to nitric oxide, nor does the general induc-
tion of ER stress using classical activators tunicamycin, DTT,
or thapsigargin (Fig. 5). These findings suggest a novel signal-
ing role for IRE1 in the activation of AMPK in response to
nitric oxide (Fig. 7). This signaling pathway is selective for
nitric oxide, as classical activators of ER stress do not stimulate
AMPK activation, and IRE1 deficiency does not lead to a
general loss of AMPK activation (Fig. 7).
Downstream of the IRE1-dependent activation of AMPK is
the attenuation of mTOR signaling through the phosphoryla-
tion of Raptor. The ability of nitric oxide to modify the phos-
phorylation status of Raptor is mediated by AMPK, as adeno-
virus-mediated expression of a dominant negative mutant of
AMPK attenuates nitric oxide-induced Raptor phosphoryla-
tion (Fig. 1D), consistent with previous findings (13). In re-
sponse to nitric oxide, AMPK, in an IRE1-dependent fashion,
inhibits mTORC1 signaling, in part, through the phosphoryla-
tion of Raptor leading to the dephosphorylation and inactiva-
tion of S6K1. These results are consistent with the effects of
AMPK activation and inhibition of mTORC1 signaling, in part
through the phosphorylation of Raptor, leading to the dephos-
phorylation and inactivation of S6K1 in response to cell stress
(13). While we have focused on delineating the mechanism of
AMPK activation in response to nitric oxide and the down-
stream influence on mTOR signaling in the context of Raptor,
this is only one aspect of a complex signaling network altered
by nitric oxide that influences mTOR signaling. In Fig. 1C, we
show that nitric oxide reduces the activation-associated phos-
phorylation of Akt. When active, Akt inhibits the negative
regulator tuberous sclerosis complex 2 (TSC2), and the inhi-
bition of TSC2 promotes the activation of mTOR (26, 31).
Thus, the ability of nitric oxide to remove the positive input
from Akt would serve to further reduce mTOR activity. Addi-
tionally, Akt is a negative regulator, through inhibitory phos-
phorylation, of glycogen synthase kinase 3 (GSK3) (9). GSK3
also phosphorylates TSC2, and this serves to inhibit mTOR;
however, this requires a priming phosphorylation by AMPK on
TSC2 (27). Therefore, following exposure to nitric oxide, IRE1
may also signal to TSC2 through AMPK to reduce mTOR
activity. One consequence of this signaling would be the inhi-
bition of protein synthesis, and it is well known that nitric oxide
is an effective inhibitor of protein synthesis in many cell types,
including ? cells (10, 11). Additional studies will clarify the role
for each of these pathways in response to nitric oxide and the
role of this regulation in cellular recovery and survival follow-
ing nitric oxide-mediated damage.
The kinase(s) responsible for the direct phosphorylation of
AMPK in response to nitric oxide has yet to be identified. The
expression of a kinase-deficient IRE1? mutant in insulinoma
cells did not prevent nitric oxide-induced AMPK activation,
indicating that IRE1 does not directly phosphorylate
AMPK. It is also unlikely that JNK phosphorylates AMPK
under these conditions, as the knockout of IRE1? does not
prevent nitric oxide-induced JNK phosphorylation (data not
shown), consistent with nitric oxide-induced JNK activa-
tion’s being dependent on sGC (46). Our inhibitor and
siRNA studies, summarized in Fig. 2, identified a number of
broad signaling pathways that were not responsible for AMPK
phosphorylation, including pathways dependent on calcium, mi-
tochondrion permeability transition, ubiquitin, phosphatidylinosi-
tol-3-kinase (PI3K), HSP90, and others. Additionally, it is possi-
ble that nitric oxide-induced inhibition of metabolic enzymes such
as cytochrome c oxidase (7) is necessary to elevate AMP levels
and promote the AMP-bound and phosphatase-resistant AMPK
Cells may modify the mechanism of AMPK activation de-
pending on the level of nitric oxide and/or cell type. At low
concentrations of nitric oxide, replicating those produced by
endothelial NOS (eNOS), AMPK is activated by CaMKK in a
guanylate cyclase-dependent fashion in endothelial cells (61).
However, in response to high levels of nitric oxide, replicating
those produced by iNOS, AMPK activation is unaffected by
CaMKK inhibition with STO-609, guanylate cyclase inhibition
with ODQ, and calcium chelation with BAPTA-AM (Fig. 2).
While these findings suggest concentration-dependent mecha-
nisms of action of nitric oxide, lower concentrations of nitric
oxide (DEANO, 100 ?M) also stimulate AMPK phosphoryla-
tion in ? cells in an IRE1-dependent fashion (Fig. 3B). These
findings suggest that the role of IRE1? in the regulation of
AMPK by nitric oxide may be cell type dependent. Somewhat
unique to pancreatic ? cells is the efficient folding, trafficking,
and secretory capacity that is needed for the synthesis and
secretion of insulin. In addition, there is a strict dependence of
? cells on oxidative metabolism (glucose to CO2) for function
(insulin secretion) and, because of this secretory demand, ?
cells have an adaptive response to oxidative stress that is as-
FIG. 7. Schematic diagram of nitric oxide-activated AMPK activa-
tion in ? cells. Nitric oxide induces IRE1-dependent phosphorylation
of AMPK and subsequent AMPK-dependent phosphorylation of Rap-
tor and ACC. The AMPK signaling node connects IRE1 to mTORC1
in response to nitric oxide (black arrows) and is independent of the
currently known AMPK kinases (gray arrows, indicating that they do
not participate in this pathway). The direct effects of nitric oxide on
IRE1 are currently unknown.
VOL. 31, 2011NO-ACTIVATED AMPK CONNECTS IRE1 TO mTOR SIGNALING4295
sociated with UPR induction (48). Thus, it seems physiologi-
cally plausible to couple the induction of protective responses
to cellular stress (IRE1? and the UPR) with regulators of
oxidative capacity (AMPK) to provide a mechanism by which
? cells coordinate the regulation of oxidative metabolism with
the response to cellular stress as mechanisms to afford protec-
tion from nitric oxide. In addition, the concentration- and cell
type-selective nature of these actions could also suggest that
the mechanism of nitric oxide-induced AMPK activation is
dependent on the physiological context, e.g., regulation of vas-
cular relaxation (eNOS) versus cell defense in response to
inflammation (iNOS) and injury.
The finding that ER stress alone is not sufficient to stimulate
AMPK activation (Fig. 5) suggests that IRE1 may be differen-
tially activated in response to nitric oxide compared to ER
stress-inducing agents such as tunicamycin and thapsigargin.
Whereas tunicamycin stimulates both kinase and RNase acti-
vation of IRE1 (60), stimuli such as nitric oxide and quercetin
may preferentially activate IRE1 RNase activity. Quercetin has
been shown to activate IRE1 RNase activity independent of
kinase activation (55), while nitric oxide induces RNase acti-
vation, but the impact on kinase activation is unknown and
requires further investigation. Our findings that quercetin in-
duces IRE1-dependent activation of AMPK and that cells ex-
pressing kinase-deficient IRE1 have enhanced nitric oxide-
induced AMPK activation suggest that IRE1 RNase activation
in response to these agents participates in a pathway leading to
AMPK phosphorylation and that this response may be atten-
uated by IRE1 kinase activation. This is in line with the recent
finding that the kinase and RNase activities of IRE1 can dif-
ferentially impact cell fate (14). Thus, the RNase-dependent
activation of AMPK may promote adaptive responses through
upregulation of genes such as PGC-1?, while IRE1 kinase
activation may suppress this response to promote elimination
of irreparably damaged cells.
From an evolutionary standpoint, it is logical for IRE1 to
play a regulatory role in the activation of AMPK signaling
pathways. Both of these highly conserved proteins are respon-
sive to cellular stress, and once active, they promote the res-
toration of cellular homeostasis and adaptation to environ-
mental changes (15, 29). Additionally, ER stress has newly
found roles in the regulation of lipid metabolism and gluco-
neogenesis (41, 52) pathways that have long been known to be
regulated by AMPK (16). The IRE1-AMPK pathway could
also provide a mechanism to couple the ER stress pathway
with the regulation of metabolism. This form of regulation may
be critical for cell survival or the loss of cell viability in re-
sponse to stress. By coupling these pathways, ? cells would
have an exquisite level of control over rapid posttranslational
modifications and long-term adaptive responses through reg-
ulation of gene expression.
This work was supported by grants from the NIH (F32 DK084645 to
G.P.M. and DK052194 and AI-44458 to J.A.C.) and a gift from the
Forest County Potawatomi Foundation.
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