From the Howard Hughes Medical Institute, Joslin
Diabetes Center, Harvard Medical School, Boston, Massachusetts
02215 and ¶ Howard Hughes Medical Institute, Program in Molecular
Medicine, Department of Biochemistry and Molecular Biology,
University of Massachusetts Medical School,
Worcester, Massachusetts 01605
Received for publication, August 15, 2002, and in revised form, October 24, 2002
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ABSTRACT |
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Activation of the c-Jun N-terminal kinase
(JNK) by proinflammatory cytokines inhibits insulin signaling, at least
in part, by stimulating phosphorylation of rat/mouse insulin receptor
substrate 1 (Irs1) at Ser307
(Ser312 in human IRS1). Here we show that JNK mediated
feedback inhibition of the insulin signal in mouse embryo fibroblasts,
3T3-L1 adipocytes, and 32DIR cells. Insulin stimulation of
JNK activity required phosphatidylinositol 3-kinase and Grb2
signaling. Moreover, activation of JNK by insulin was inhibited by a
cell-permeable peptide that disrupted the interaction of JNK with
cellular proteins. However, the direct binding of JNK to Irs1 was not
required for its activation by insulin, whereas direct binding was
required for Ser307 phosphorylation of Irs1.
Insulin-stimulated Ser307 phosphorylation was reduced 80%
in cells lacking JNK1 and JNK2 or in cells expressing a mutant Irs1
protein lacking the JNK binding site. Reduced Ser307
phosphorylation was directly related to increased insulin-stimulated tyrosine phosphorylation, Akt phosphorylation, and glucose uptake. These results support the hypothesis that JNK is a negative feedback regulator of insulin action by phosphorylating Ser307 in Irs1.
Insulin resistance is a common problem that is associated with
obesity and hypertension, infection and injury, and type 2 diabetes, in
which A number of mechanisms might contribute to the dysregulation of the
insulin-signaling pathway, including serine phosphorylation of the
insulin receptor or the Irs proteins, degradation of Irs proteins, or
altered activity of phosphoprotein or phospholipid phosphatases
(10-16). Irs1 and Irs2 contain many potential serine or threonine
phosphorylation sites that might play regulatory roles during the
insulin response. Various metabolites associated with insulin
resistance stimulate serine phosphorylation of Irs1, including free
fatty acids, diacylglycerol, fatty acyl CoAs, ceramides, and glucose
(17). Moreover, proinflammatory cytokines, especially interferon More than 100 potential serine phosphorylation sites exist in Irs1, and
many protein kinases phosphorylate Irs1, including JNK, protein
kinase C JNK is a member of the MAP kinase family of protein kinases, which also
includes ERK and p38 (25, 26). JNK is activated by proinflammatory
cytokines induced during microbial infection or thermal or mechanical
injury (27, 28). Three JNK isoforms, JNK1, JNK2, and JNK3, are
expressed in multiple splice variants (29). Disruption of these genes
in mice reveals that JNK1 and JNK2 mediate T cell activation and brain
development (30-32), and JNK3 mediates neuronal apoptosis in the
hippocampus (33). Various growth factors also activate JNK, including
prolactin, epidermal growth factor, nerve growth factor and
platelet-derived growth factor, insulin-like growth factor 1, and
ligands for some G protein-coupled receptors (34-40). Insulin
stimulates JNK in various cells and tissues, but the mechanisms
involved and the role of JNK during insulin action are poorly defined
(41-43). Here we show that insulin-stimulated JNK associates with Irs1
and phosphorylates Ser307, which inhibits insulin
signaling. Thus, JNK might serve a dual function as a heterologous
inhibitor of insulin action during acute and chronic inflammation and
as a feedback inhibitor during insulin stimulation.
Antibodies and Reagents--
Antibodies against JNK, phospho-JNK
(pThr183/pTyr185), Pkb/Akt, and phospho-Pkb/Akt
(pSer473) were purchased from New England Biolabs.
Monoclonal antibodies against JNK and phospho-c-Jun
(pSer63) were purchased from Santa Cruz. Phosphotyrosine
antibody (PY20) was purchased from Transduction Laboratories.
Antibodies against Irs1 and phosphorylated Ser307 in Irs1
were described previously (20, 24). Insulin was purchased from
Roche Molecular Biochemicals, and TNF Cell Culture--
Murine myeloid progenitor 32D cells were
maintained in RPMI 1640 medium supplemented with 10% FBS, 5%
WEHI conditioned medium (as a source of interleukin-3), and 5 mM histidinol and made quiescent by serum starvation for
4 h (20). 32D transfectants were generated by electroporation and
selected in histidinol as described previously (44); site-directed
mutagenesis of the JNK binding motif in Irs1 was described
previously (20). Mouse embryo fibroblasts from wild type or
JNK1::JNK2 knockout mice were grown in
DMEM with 10% FBS (45). 3T3-L1 preadipocytes were maintained at
37 °C in 10% CO2 in DMEM containing 2 mM
glucose and 10% calf serum. These cells were differentiated into
adipocytes by incubation for 3 days in DMEM supplemented with 25 mM glucose, 1 µM insulin, 0.5 mM
3-isobutylmethylxanthine, 1 µM dexamethasone, and 10%
FBS and 3 days in DMEM supplemented with 1 µM insulin and
10% FBS (46). More than 90% differentiation was achieved after 4-9
days in DMEM containing 25 mM glucose and 10% FBS with no
other additives.
Cell Lysis, Immunoprecipitation, and Western Blot
Analysis--
Cells were lysed in 20 mM Tris (pH 7.4)
containing 150 mM NaCl, 1% Nonidet P-40, 5 mM
EDTA, 10 mM NaF, 10 mM pyrophosphate, 100 µM NaVO4, 1 mM
phenylmethanesulfonyl fluoride, 5 µg/ml leupeptin, and 5 µg/ml
protinin. Lysates were resolved by SDS-PAGE, transferred to
nitrocellulose, and proteins were detected by immunoblotting and
chemiluminescence (Amersham Biosciences). To analyze the association of
Irs1 with JNK in 32D cells, immunoprecipitation was performed with Irs1
antibody immobilized on protein G-Sepharose using a SeizeTM X protein G
immunoprecipitation kit (Pierce) and analyzed by immunoblotting with
monoclonal JNK antibody. For 3T3-L1 adipocytes, immunoprecipitates with
monoclonal JNK antibody were analyzed by immunoblotting with Irs1 antibody.
2-Deoxyglucose Uptake in 3T3-L1 Adipocytes--
Fully
differentiated 3T3-L1 adipocytes were placed in DMEM containing 5 mM glucose and 0.1% bovine serum albumin for 2 h at 37 °C. Before glucose transport measurements, cells were washed with
KRH buffer (20 mM HEPES (pH 7.4) 1.25 mM
MgSO4, 1.25 mM CaCl2, 136 mM NaCl, 4.7 mM KCl, and 0.1% bovine serum
albumin) and incubated with synthetic peptides before insulin
stimulation. Glucose transport was determined by the addition of 0.1 mM 2-deoxyglucose containing 0.5 µCi of
2-[1,2-3H]-deoxy-D-glucose (PerkinElmer Life
Sciences) as described previously (47). Nonspecific uptake was assessed
in the presence of 10 µM cytochalasin B and subtracted
from all of the measured values. Glucose transport experiments were
terminated after 10 min by aspiration by four washes with ice-cold
phosphate-buffered saline. Cells were lysed in 0.1% SDS in
phosphate-buffered saline, and radioactivity was determined by
scintillation counting.
Irs1 Is Necessary for the Insulin-induced Activation of
JNK--
We used 32D cell lines stably expressing the human insulin
receptor alone or with rat Irs1 to determine the function of
insulin-stimulated JNK. 32D cells are murine myeloid progenitors that
express few endogenous insulin receptors and no Irs proteins and
require interleukin-3 for growth (48); however, they naturally express
JNK1 and JNK2 (data not shown). JNK1 and JNK2 are activated by tandem
Thr/Tyr phosphorylation (Thr183 and Tyr185 in
JNK1), which is detected in both isoforms by immunoblotting with a
phosphospecific-JNK antibody (
Before insulin stimulation, JNK phosphorylation was not detected by
JNK Promotes Ser307 Phosphorylation of Irs1 During
Insulin Stimulation--
We investigated the relationship between
insulin-stimulated JNK activity and Ser307 phosphorylation
of rat Irs1 in transfected 32DIR/Irs1 cells. During insulin
stimulation, tyrosine-phosphorylated Irs1 activates the PI 3-kinase
To confirm that tyrosine phosphorylation of Irs1 was required for
activation of JNK by insulin, 32DIR cells were transfected
with F18Irs1, a rat Irs1 mutant that lacks all the known
tyrosine phosphorylation sites, including those that activate the PI
3-kinase and ERK pathways (48). Consistent with the inhibitory effect
of LY294002 or PD98059 on JNK phosphorylation, insulin failed to
stimulate JNK phosphorylation in 32DIR/F18Irs1
cells (Fig. 2B). Insulin-stimulated phosphorylation of
Ser307 was also significantly reduced. However, like
inhibition by LY294002 or PD98059, a minor pathway independent of Irs1
tyrosine phosphorylation might be involved (Fig. 2B).
Insulin-stimulated Ser307 Phosphorylation of Irs1 Is
Reduced in Mouse Embryo Fibroblasts (MEFs) Lacking JNK1 and
JNK2--
To further establish the role of JNK in insulin signaling,
MEFs lacking both JNK1 and JNK2
(JNK1 The Role of the JNK-binding Motif in Irs1--
JNK binds
specifically to scaffold proteins that mediate its interaction with
upstream regulatory kinases and downstream substrates. The consensus
amino acid sequence motif that binds to JNK (JNK-binding peptide (JBP))
is best characterized by alignment of JNK-interacting protein 1 (Jip1)
and Jip2 (49). Previous studies reveal that two leucine residues within
the JNK-binding motif are essential for JNK binding (49). Irs1 contains
a similar JNK-binding motif, including both leucine residues at
positions 856 and 858 (20). We prepared 32DIR cells
expressing mutant Irs1 proteins that contain glycine substitutions for
the leucine residues (Irs1GSG) to determine the effect of
blocking JNK binding to Irs1 on the activation of JNK and ability to
phosphorylate Ser307 (Fig.
4A). Insulin stimulated the
binding of wild type Irs1 to JNK in 32DIR/Irs1 cells,
whereas insulin failed to stimulate this interaction between JNK and
Irs1GSG, confirming that the JNK-binding motif was required
(Fig. 4B).
During insulin stimulation, wild type Irs1 and Irs1GSG were
rapidly tyrosine phosphorylated, but phosphorylation of Irs1 rapidly declined, whereas that of Irs1GSG was sustained for at
least 20 min (Fig. 5A).
Although both Irs1 and Irs1GSG mediated insulin-stimulated
JNK activation, only Irs1 was susceptible to inhibition by
Ser307 phosphorylation (Fig. 5A). These results
reveal that direct binding of JNK to Irs1 was not required for insulin
stimulation, whereas direct binding was required for JNK-mediated
Ser307 phosphorylation. Moreover, Irs1GSG
displayed more sensitive and intense tyrosine phosphorylation than wild
type Irs1 at every concentration of insulin tested (Fig. 5B). Consistent with increased tyrosine phosphorylation,
Irs1GSG mediated more phosphorylation of Pkb/Akt during
insulin stimulation (Fig. 5B). By contrast, the time course
of JNK phosphorylation was not significantly affected by the
Irs1GSG mutant, whereas the sensitivity of JNK
phosphorylation to insulin might be slightly impaired (Fig.
5B). These results support the hypothesis that an
interaction between JNK and Irs1 was not essential for activation but
was required for insulin-stimulated Ser307
phosphorylation.
Cell-permeable JNK-binding Motif Inhibits Activation of JNK by
Insulin--
Insulin stimulated phosphorylation of the 46-kDa JNK
isoform in fully differentiated 3T3-L1 adipocytes, which increased its activity as revealed by phosphorylation of c-Jun (Fig.
6). Consistent with previous results,
insulin strongly stimulated Ser307 phosphorylation of Irs1
in these cells; Irs1 was also tyrosine-phosphorylated during insulin
stimulation (Fig. 6). The effect of Ser307 phosphorylation
on insulin signaling in 3T3-L1 adipocytes was investigated by
inhibiting JNK activation with a 12-amino acid peptide composed of the
JNK-binding peptide identified in Jip1 (50). The JNK-binding peptide
inhibits competitively the interaction of JNK with regulatory scaffolds
or substrates. To facilitate translocation of the JNK-binding peptide
(JBP) across the plasma membrane, the 12-amino acid transduction
domain from human immunodeficiency virus TAT protein (HIV-TAT) was
added to the N terminus (51). This chimeric peptide, TAT/JBP, was
incubated with 3T3-L1 adipocytes for 30 min to disrupt JNK binding
before insulin stimulation. TAT/JBP inhibited in a
dose-dependent way insulin-stimulated JNK activity as
assessed by c-Jun phosphorylation (Fig.
7A). TAT/JBP also blocked
insulin-stimulated binding of JNK to Irs1 (Fig. 7B).
Inhibition of JNK Activity Reduced Ser307
Phosphorylation of Irs1 and Enhanced Insulin Signal Transduction and
Glucose Uptake--
To investigate the relation between JNK and Irs1,
we treated 3T3-L1 adipocytes with the TAT/JBP and analyzed
insulin-signaling events. TAT/JBP (20 µM) inhibited
insulin-stimulated JNK activity and suppressed insulin-stimulated
Ser307 phosphorylation of Irs1. Consistent with the
inhibitory role of Ser307 phosphorylation,
insulin-stimulated tyrosine phosphorylation of Irs1 increased (Fig.
8A). TAT/JBP also increased
Akt phosphorylation during insulin stimulation (Fig. 8A). In
3T3-L1 adipocytes, insulin stimulates the uptake of 2-deoxyglucose by
translocation of Glut4 to the plasma membrane (52). As expected,
insulin stimulated uptake of 2-deoxyglucose into 3T3-L1 adipocytes.
Incubation of 3T3-L1 adipocytes for 30 min with TAT/JBP but not the
12-residue HIV-TAT peptide increased insulin-stimulated glucose uptake
(Fig. 8B). These results are consistent with the conclusion
that JNK-mediated phosphorylation of Irs1 at Ser307
inhibits insulin action.
Our cell-based experiments reveal that recruitment of active JNK
to Irs1 might be a common mechanism for feedback or heterologous inhibition of the insulin signal. JNK is a member of the MAP kinase family, which also includes the extracellular signal-regulated protein
kinases (ERK1/2), p38 MAP kinases, and the ERK5 pathway (25). MAP
kinases are activated by dual-specificity MAP kinase kinases that
phosphorylate adjacent tyrosine and threonine residues (29). During
insulin stimulation, ERK1 and ERK2 are strongly activated by the
dual-specificity kinases MEK1 or MEK2, which are activated when
Grb2/Sos bound to Irs1 or Shc activates the Ras JNK activation is best understood during cytokine stimulation, which
involves the recruitment of an upstream multilineage kinase, a
dual-specificity kinase (MKK4 or MKK7), and JNK into a regulatory
complex including JNK-interacting protein Jip1 or Jip2 (49, 55). By
contrast, previous studies suggest that insulin stimulates JNK through
PI 3-kinase, Ras, or Shp2 (56). These distinct pathways might converge
because the permeable JNK-binding peptide that disrupts
TNF Irs1 is required for insulin stimulation of JNK in 32DIR
cells, because insulin barely stimulates JNK in these cells lacking Irs1. In 32DIR/Irs1 cells, the PI 3-kinase and the Ras Our results with 32DIR cells, MEFs, and 3T3-L1 adipocytes
reveal that JNK is the principle kinase that mediates
Ser307 phosphorylation during insulin stimulation. However,
on inhibition of JNK through various strategies, some
insulin-stimulated Ser307 phosphorylation still occurs.
Many kinases are known to phosphorylate Irs1. Recent evidence indicates
that IKK JNK has many effects on cellular function because it phosphorylates and
activates various transcription factors, including ATF2 and ATFa, c-Jun
and JunD, and Elk1 and Sap1 (28). Although insulin stimulation of JNK
might play an important role in gene transcription as revealed in other
systems, our results also suggest that JNK-mediated phosphorylation of
Ser307 in rodent Irs1 mediates negative feedback of insulin
signaling. Insulin stimulates the binding of activated JNK to Irs1 in
3T3-L1 and 32DIR/Irs1 cells, which attenuates
insulin-stimulated tyrosine phosphorylation. Consistent with this
hypothesis, disruption of the consensus JNK-binding motif in Irs1
significantly reduces the phosphorylation of Ser307 during
insulin stimulation and increases insulin-stimulated tyrosine phosphorylation and Pkb/Akt activation. Moreover, inhibition of JNK
activation by incubating 3T3-L1 adipocytes with a permeable TAT/JNK-binding peptide promotes insulin-stimulated glucose uptake. This result is consistent with reduced insulin-stimulated JNK activity
and Ser307 phosphorylation and increased Irs1 tyrosine
phosphorylation and Pkb/Akt phosphorylation. Experiments with mouse
embryo fibroblasts lacking JNK1 and JNK2 also support these results.
The possibility is not excluded that the binding of JNK to Irs1 itself
might have some inhibitory effect in addition to Ser307
phosphorylation, such as competing with other proteins for binding to
Irs1. Although our attention for the moment focuses on
Ser307 phosphorylation, additional serine residues might
also be involved. Moreover, we have not distinguished between JNK1 or
JNK2 in our experiments, because both homologs bind to Irs1 (data
not shown).
Insulin resistance and compensatory hyperinsulinemia dysregulate many
physiological processes that contribute to life-threatening metabolic,
vascular, and cardiac diseases (58, 59). Although new drugs are
emerging to improve insulin sensitivity, the molecular mechanisms that
cause insulin resistance are difficult to establish. The idea that
inflammation causes insulin resistance has been known for a long time
(60) and is consistent with the idea that anti-inflammatory drugs like
high-dose aspirin promote insulin sensitivity (22, 61). The
physiological response to infection, physical or thermal trauma, or
obesity invariably involves the production of proinflammatory cytokines
like TNF In summary, JNK mediates feedback inhibition of insulin signaling by
phosphorylation of rat/mouse Irs1 at Ser307
(Ser312 in human Irs1). In addition to our experiments with
32DIR/Irs1 cells, MEF cells and 3T3-L1 adipocytes, insulin
is reported to stimulate JNK activity in Rat-1 fibroblast, Chinese
hamster ovary cells overexpressing human insulin receptors, L6
myotubes, and rat adipocytes (41-43). These results suggest that
activated JNK might be an important negative feedback regulator for
insulin signaling, and thus inhibiting JNK or interfering with JNK-Irs1 interaction might be a good therapeutic target to reduce insulin resistance.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cells fail to secrete sufficient insulin to compensate for
peripheral insulin resistance (1-3). Insulin signaling complexes are
assembled by insulin-stimulated tyrosine phosphorylation of scaffold
proteins, including the
Irs1 1 proteins, Shc, APS
and Shc, Gab1/2, Dock1/2 and cbl (4-6). Although the role of each of
these substrates merits attention, work with transgenic mice reveals
the importance of Irs1 and Irs2 for somatic growth and carbohydrate
metabolism (7, 8). Tyrosine phosphorylation sites in Irs1 and Irs2 bind
to the Src homology-2 domain in various signaling proteins that mediate
the insulin response, including PI 3-kinase, Grb2, Shp2, Crk, and
others (9).
,
interleukin 1
, or TNF
produced during infection, injury, or
chronic obesity stimulate serine phosphorylation of Irs1 and cause
insulin resistance (18, 19). Insulin itself induces serine
phosphorylation of Irs1, suggesting that chronic compensatory
hyperinsulinemia in response to stress-induced insulin resistance might
exacerbate the problem (20).
, IKK
, mammalian target of rapamycin, MAPK, and AMPK
(11-13, 21-23); however, the kinases and phosphorylation sites that
are physiologically important for Irs1 function are difficult to
resolve. Although many kinases phosphorylate Irs1, JNK is especially
interesting because it associates with Irs1 and phosphorylates
Ser307 (11, 20). Insulin also stimulates Ser307
phosphorylation in various cultured cell lines; and the orthologous site in human Irs1 (Ser312) is phosphorylated in muscle
during a hyperinsulinemic clamp (24). Ser307 is located
next to the phosphotyrosine-binding domain in Irs1, and its
phosphorylation inhibits the interaction of the phosphotyrosine-binding domain with the phosphorylated NPEY motif in the activated insulin receptor (20). Because the phosphotyrosine-binding domain is important
for efficient insulin-stimulated tyrosine phosphorylation of Irs1,
Ser307 phosphorylation might contribute to insulin
resistance during physiological stress.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was purchased from R&D
Systems. LY294002 and PD98059 were purchased from
Calbiochem-Novabiochem Corp. Peptides were synthesized by Boston
Biomolecules and purified by high pressure liquid chromatography, and
the sequences were confirmed by mass spectrometry.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
JNKPI). Both JNK homologs
are variably expressed as two alternative transcripts that yield a
46-kDa and a 54-kDa isoform that are detected by
JNKPI.
In this report we do not distinguish between the homologs.
JNKPI, suggesting that under basal conditions JNK was
not activated. However, the 46- and 54-kDa isoforms of JNK were
phosphorylated 5 min after insulin was added to the
32DIR/Irs1 cells (Fig.
1A). JNK phosphorylation was
maximal at 10 nM insulin in 32DIR/Irs1 cells,
whereas it was weakly phosphorylated during insulin stimulation of
32DIR cells (Fig. 1B). Thus, Irs1 was required
in 32DIR cells for maximal sensitivity of JNK to insulin
stimulation.
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Fig. 1.
Irs1 is necessary for the insulin-induced
activation of JNK in 32D cells. A, total cell lysates
from 32DIR cells stably transfected with empty vector or
Irs1-expressing vector treated with 10 nM insulin for the
indicated times were analyzed by immunoblotting with anti-phospho-JNK
( JnkPI) and anti-JNK (
Jnk) antibodies. B,
total cell lysates from 32DIR cells stably transfected with
empty vector or Irs1-expressing vector treated with the indicated doses
of insulin for 15 min were analyzed by immunoblotting with
anti-phospho-JNK and anti-JNK antibodies.
Pkb/Akt cascade that can be inhibited by LY294002 and activates the
Grb2/Sos/Ras
ERK1/2 cascade that can be inhibited by PD98059 (9).
Each compound inhibited insulin-stimulated phosphorylation of the 46- and 54-kDa isoforms of JNK in 32DIR/Irs1 cells and
inhibited JNK-mediated phosphorylation of c-Jun (Fig.
2A). Thus, PI 3-kinase and
MEK1 mediated JNK activation in insulin-stimulated
32DIR/Irs1 cells (Fig. 2A). Each drug also
inhibited by 75% the insulin-stimulated phosphorylation of
Ser307 in Irs1, suggesting that the majority of
Ser307 phosphorylation might be mediated by JNK. However,
other insulin-stimulated kinases appear to be involved (Fig.
2A).
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Fig. 2.
Insulin induces the activation of JNK and
Ser307 phosphorylation of Irs1 in 32D
cells. A, total cell lysates from 32DIR
cells stably expressing Irs1 treated with LY294002 or PD98059 for 30 min before 15 min of stimulation with 10 nM insulin
(Ins) were analyzed by immunoblotting with anti-phospho-JNK
( JnkPI), anti-phospho-c-Jun
(
JunPI), anti-phospho-Ser307
(
Ser307), and anti-Irs1 (
Irs1)
antibodies. B, total cell lysates from 32DIR
cells stably expressing wild type Irs1 or F18 mutant Irs1 in which all
18 tyrosine residues were mutated to phenylalanine, treated with 10 nM insulin (Ins) for 15 min were analyzed by
immunoblotting with anti-phospho-JNK, anti-phospho-Ser307,
and anti-Irs1 antibodies.
/
::JNK2
/
MEFs) were used (45). Insulin-stimulated JNK activity assayed by
immunoblotting c-Jun phosphorylation was completely absent in
JNK1
/
::JNK2
/
MEFs, whereas c-Jun phosphorylation was stimulated maximally in wild
type cells 10 min after insulin stimulation (Fig.
3). Insulin induced Ser307
phosphorylation of Irs1 in wild type MEFs, whereas
phosphorylation was significantly reduced in
JNK1
/
::JNK2
/
MEFs. However, transient Ser307 phosphorylation was
detected reproducibly after 20 min of insulin stimulation and might
represent the JNK-independent pathway (Fig. 3). Insulin-stimulated
tyrosine phosphorylation of Irs1 was increased in the
JNK1
/
::JNK2
/
MEFs. Tyrosine phosphorylation of Irs1 ordinarily declined after 20 min
of insulin stimulation in wild type cells; however, it remained high
for at least 40 min in
JNK1
/
::JNK2
/
MEFs. Thus, JNK is a major kinase responsible for insulin-induced Irs1
Ser307 phosphorylation that inhibits insulin-stimulated
tyrosine phosphorylation.
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Fig. 3.
Insulin-stimulated Ser307
phosphorylation of Irs1 is reduced in mouse embryo fibroblasts
lacking JNK1 and JNK2. Wild type (WT) or JNK1 and JNK2
double knock-out (Jnk1/2 /
) MEF
cells were deprived of serum for 4 h before stimulation with
insulin. Total cell lysates or immunoprecipitates (IP) of
Irs1 from MEF cells treated with 10 nM insulin for the
indicated times were analyzed by immunoblotting with
anti-phospho-Ser307 (
Ser307),
anti-phosphotyrosine (
PY), anti-Irs1
(
Irs1), and anti-phospho-c-Jun
(
JunPI) antibodies.
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Fig. 4.
Mutation at JNK-binding motif of Irs1
abrogates the interaction between Irs1 and JNK in 32D cells.
A, a schematic of the JNK-binding motif in the rat Irs1
sequence and the pair of leucine residues at positions 856 and 858 that
were mutated to glycine residues. B, immunoprecipitates
(IP) of Irs1 or total cell lysates from 32DIR
cells stably expressing wild type Irs1 or mutant Irs1GSG
treated with 10 nM insulin for 10 min were analyzed by
immunoblotting with monoclonal anti-JNK ( Jnk) and
anti-Irs1 (
Irs1) antibodies.
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Fig. 5.
Mutation at JNK-binding motif in Irs1 reduces
Ser307 phosphorylation and enhances Irs1
tyrosine phosphorylation in 32D cells. A, total cell
lysates from 32DIR cells stably expressing wild type Irs1
or mutant Irs1GSG treated with 10 nM insulin
for the indicated times were analyzed by immunoblotting with
anti-phosphotyrosine ( PY),
anti-phospho-Ser307 (
Ser307),
anti-Irs1 (
Irs1), and anti-phospho-JNK
(
JnkPI) antibodies. B, total cell
lysates from 32DIR cells stably expressing wild type Irs1
or mutant Irs1GSG treated with the indicated doses of
insulin for 10 min were analyzed by immunoblotting with
anti-phosphotyrosine (
PY),
anti-phospho-Ser307 (
Ser307),
anti-Irs1, anti-phospho-JNK (
JnkPI), and
anti-phospho-Pkb/Akt (
PkbPI)
antibodies.
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Fig. 6.
Insulin induces the activation of JNK and
Ser307 phosphorylation of Irs1 in 3T3-L1
adipocytes. Fully differentiated 3T3-L1 adipocytes were deprived
of serum overnight. Total cell lysates or immunoprecipitates
(IP) of Irs1 from 3T3-L1 adipocytes treated with 10 nM insulin for the indicated times were analyzed by
immunoblotting with anti-phospho-JNK ( JnkPI),
anti-phospho-c-Jun (
JunPI),
anti-phospho-Ser307 (
Ser307),
anti-phospho-tyrosine (
PY) and anti-Irs1
(
Irs1) antibodies.
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Fig. 7.
Cell-permeable peptide inhibitor of JNK
inhibits the interaction between Irs1 and JNK in 3T3-L1
adipocytes. A, total cell lysates from 3T3-L1
adipocytes treated with control (TAT-Peptide) or TAT/JBP at
the indicated concentrations for 30 min before 10 min of stimulation
with 10 nM insulin were analyzed with anti-phospho-c-Jun
( JunPI) antibody. B,
immunoprecipitates (IP) of JNK using monoclonal anti-JNK
antibody or total cell lysates from 3T3-L1 adipocytes treated with or
without 20 µM TAT/JBP for 30 min before 10 min of
stimulation with 10 nM insulin were analyzed by
immunoblotting with anti-Irs1 (
Irs1) and monoclonal
anti-JNK (
Jnk) antibodies.
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Fig. 8.
Inhibition of JNK activity reduces
Ser307 phosphorylation of Irs1 and enhances
insulin signal transduction and glucose uptake in 3T3-L1
adipocytes. A, total cell lysates from 3T3-L1
adipocytes treated with or without 20 µM TAT/JBP for 30 min before 10 min of stimulation with 10 nM insulin were
analyzed by immunoblotting with anti-phospho-Ser307
( Ser307), anti-phosphotyrosine
(
PY), anti-Irs1 (
Irs1), anti-phospho-c-Jun
(
JunPI), anti-phospho-Pkb/Akt
(
PkbPI), and anti-Pkb/Akt antibodies
(
Pkb). B, glucose uptake was measured in
3T3-L1 adipocytes treated with or without 20 µM JBP for
30 min before 10 min of stimulation with the indicated concentrations
of insulin.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Raf cascade
(53, 54); however, a molecular pathway linking the receptors for
proinflammatory cytokines or insulin to JNK is difficult to establish.
-stimulated JNK activation also inhibits insulin-stimulated JNK;
whether MLK and MKK4 or MKK7 are involved during insulin stimulation is
unknown. Perhaps other kinase cascades are employed during insulin
stimulation and are recruited with JNK by other scaffold proteins that
contain a JNK-binding motif.
MEK1/2
ERK1/2 cascades appear to be involved, because LY294002 or
PD98059 significantly inhibit insulin-stimulated JNK activity. Although
Irs1 contains a JNK-binding motif similar to that in Jip1 or Jip2, a
direct interaction between JNK and Irs1 is not involved in the JNK
activation during insulin stimulation. A mutant Irs1 protein lacking
the JNK-binding motif mediates JNK activation normally in
insulin-stimulated 32DIR cells. By contrast,
F18Irs1, which lacks all the tyrosine phosphorylation sites
but retains the JNK binding motif, fails to activate JNK during insulin
stimulation and fails to bind inactive JNK. Thus, Irs1-mediated
activation of the PI 3-kinase and Ras
MEK1/2 cascades is required
for JNK activation, and Irs1 only binds activated JNK.
can directly phosphorylate Irs1 Ser307 (57). In
addition, the possibility exists that mTOR phosphorylates Ser307. We showed previously that insulin stimulates
Ser307 phosphorylation in 3T3-L1 preadipocytes without
activating JNK (24). Apparently, unknown differences in insulin
signaling in preadipocytes disrupt the link between the insulin
receptor and JNK, whereas other cell lines including 32D cells, MEF
cells, and adipocytes show strong activation of JNK in response to
insulin. Differential coupling between the insulin receptor and JNK
might play an important role in feedback regulation of the insulin
signaling cascade.
that activate various serine kinases (28). Considerable
evidence suggests that serine phosphorylation of the insulin receptor
or the Irs proteins might inhibit insulin signaling and promote insulin
resistance (62). During obesity, adipocytes produce TNF
, which
promotes insulin resistance and stimulates serine phosphorylation of
Irs1, whereas disruption of TNFR1 partially restores insulin
signaling and glucose tolerance in obese mice (18, 63-66). The
signaling cascades regulated by TNF
are complex and involve many
branch points, including the activation of JNK, p38, and the IKK
(28). IKK
might also be important because high doses of salicylates that inhibit IKK
improve glucose tolerance in obese mice (22, 61).
Salicylates increase insulin-stimulated phosphorylation of IRS proteins
in the liver, revealing a potential mechanism for their effect on
insulin action; the effect might occur indirectly through other
downstream kinases, through NF
B-regulated gene expression. or
through direct phosphorylation of Irs1 (57). By contrast, a direct role
for JNK to regulate insulin signaling is compelling, because both Irs1
and Irs2 contain a JNK-binding motif. Other kinases might also bind to
this motif and with JNK explain, at least in part, insulin resistance
that occurs during trauma and obesity.
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FOOTNOTES |
---|
* This work was supported in part by Research Grants DK38712 (to M. F. W.) and DK63368 (to R. J. D.) and by a Diabetes and Endocrinology Center Grant DK32520 (to R. J. D.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by an American Diabetes Association postdoctoral fellowship.
Investigators of the Howard Hughes Medical Institute.
** To whom correspondence should be addressed: Howard Hughes Medical Institute, Joslin Diabetes Center, 1 Joslin Place, Boston, MA 02215. Tel.: 617-732-2578; Fax: 617-732-2593; E-mail: morris. white{at}joslin.harvard.edu.
Published, JBC Papers in Press, November 1, 2002, DOI 10.1074/jbc.M208359200
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ABBREVIATIONS |
---|
The abbreviations used are:
Irs, insulin
receptor substrate;
JNK, c-Jun N-terminal kinase;
PI, phosphatidylinositol;
TNF, tumor necrosis factor;
TNFR1, tumor necrosis
factor receptor 1;
IKK, IB kinase-
;
ERK, extracellular
signal-regulated kinase;
FBS, fetal bovine serum;
DMEM, Dulbecco's
modified Eagle's medium;
MEK, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase;
MEF, mouse embryo
fibroblast;
JBP, JNK-binding peptide;
Jip, JNK-interacting protein;
HIV, human immunodeficiency virus;
MAPK, mitogen-activated protein
kinase;
AMPK, AMP-activated kinase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Carter, E. A. (1998) Nutr. Rev. 56, S170-S176[Medline] [Order article via Infotrieve] |
2. | Mizock, B. A. (1995) Am. J. Med. 98, 75-84[CrossRef][Medline] [Order article via Infotrieve] |
3. | Taylor, S. I. (1999) Cell 97, 9-12[Medline] [Order article via Infotrieve] |
4. |
Pawson, T.,
and Scott, J. D.
(1997)
Science
278,
2075-2080 |
5. | Nelms, K., O'Neill, T. J., Li, S., Hubbard, S. R., Gustafson, T. A., and Paul, W. E. (1999) Mamm. Genome 10, 1160-1167[CrossRef][Medline] [Order article via Infotrieve] |
6. | Saltiel, A. R., and Kahn, C. R. (2001) Nature 414, 799-806[CrossRef][Medline] [Order article via Infotrieve] |
7. | Withers, D. J., and White, M. F. (1999) Curr. Opin. Endocrinol. Diab. 6, 141-145[CrossRef] |
8. | Withers, D. J., Gutierrez, J. S., Towery, H., Burks, D. J., Ren, J. M., Previs, S., Zhang, Y., Bernal, D., Pons, S., Shulman, G. I., Bonner-Weir, S., and White, M. F. (1998) Nature 391, 900-904[CrossRef][Medline] [Order article via Infotrieve] |
9. | Yenush, L., and White, M. F. (1997) BioEssays 19, 491-500[Medline] [Order article via Infotrieve] |
10. |
Rui, L.,
Fisher, T. L.,
Thomas, J.,
and White, M. F.
(2001)
J. Biol. Chem.
276,
40362-40367 |
11. |
Aguirre, V.,
Uchida, T.,
Yenush, L.,
Davis, R. J.,
and White, M. F.
(2000)
J. Biol. Chem.
275,
9047-9054 |
12. |
Liu, Y. F.,
Paz, K.,
Herschkovitz, A.,
Alt, A.,
Tennenbaum, T.,
Sampson, S. R.,
Ohba, M.,
Kuroki, T.,
LeRoith, D.,
and Zick, Y.
(2001)
J. Biol. Chem.
276,
14459-14465 |
13. |
Ozes, O. N.,
Akca, H.,
Mayo, L. D.,
Gustin, J. A.,
Maehama, T.,
Dixon, J. E.,
and Donner, D. B.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
4640-4645 |
14. | Clement, S., Krause, U., Desmedt, F., Tanti, J. F., Behrends, J., Pesesse, X., Sasaki, T., Penninger, J., Doherty, M., Malaisse, W., Dumont, J. E., Marchand-Brustel, Y., Erneux, C., Hue, L., and Schurmans, S. (2001) Nature 409, 92-97[CrossRef][Medline] [Order article via Infotrieve] |
15. | Salmeen, A., Andersen, J. N., Myers, M. P., Tonks, N. K., and Barford, D. (2000) Mol. Cell 6, 1401-1412[Medline] [Order article via Infotrieve] |
16. |
Klaman, L. D.,
Boss, O.,
Peroni, O. D.,
Kim, J. K.,
Martino, J. L.,
Zabolotny, J. M.,
Moghal, N.,
Lubkin, M.,
Kim, Y. B.,
Sharpe, A. H.,
Stricker-Krongrad, A.,
Shulman, G. I.,
Neel, B. G.,
and Kahn, B. B.
(2000)
Mol. Cell. Biol.
20,
5479-5489 |
17. |
Shulman, G. I.
(2000)
J. Clin. Invest.
106,
171-176 |
18. | Hotamisligil, G. S., Peraldi, P., Budvari, A., Ellis, R. W., White, M. F., and Spiegelman, B. M. (1996) Science 271, 665-668[Abstract] |
19. |
Kanety, H.,
Feinstein, R.,
Papa, M. Z.,
Hemi, R.,
and Karasik, A.
(1995)
J. Biol. Chem.
270,
23780-23784 |
20. |
Aguirre, V.,
Werner, E. D.,
Giraud, J.,
Lee, Y. H.,
Shoelson, S. E.,
and White, M. F.
(2002)
J. Biol. Chem.
277,
1531-1537 |
21. |
De Fea, K.,
and Roth, R. A.
(1997)
J. Biol. Chem.
272,
31400-31406 |
22. |
Yuan, M.,
Konstantopoulos, N.,
Lee, J.,
Hansen, L., Li, Z. W.,
Karin, M.,
and Shoelson, S. E.
(2001)
Science
293,
1673-1677 |
23. |
Jakobsen, S. N.,
Hardie, D. G.,
Morrice, N.,
and Tornqvist, H. E.
(2001)
J. Biol. Chem.
276,
46912-46916 |
24. |
Rui, L.,
Aguirre, V.,
Kim, J. K.,
Shulman, G. I.,
Lee, A.,
Corbould, A.,
Dunaif, A.,
and White, M. F.
(2001)
J. Clin. Invest.
107,
181-189 |
25. |
Weston, C. R.,
Lambright, D. G.,
and Davis, R. J.
(2002)
Science
296,
2345-2347 |
26. | Davis, R. J. (1999) Biochem. Soc. Symp. 64, 1-12[Medline] [Order article via Infotrieve] |
27. |
Kyriakis, J. M.,
and Avruch, J.
(2001)
Physiol. Rev.
81,
807-869 |
28. | Baud, V., and Karin, M. (2001) Trends Cell Biol. 11, 372-377[CrossRef][Medline] [Order article via Infotrieve] |
29. | Ip, Y. T., and Davis, R. J. (1998) Curr. Opin. Cell Biol. 10, 205-219[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Dong, C.,
Yang, D. D.,
Wysk, M.,
Whitmarsh, A. J.,
Davis, R. J.,
and Flavell, R. A.
(1998)
Science
282,
2092-2095 |
31. | Kuan, C. Y., Yang, D. D., Samanta Roy, D. R., Davis, R. J., Rakic, P., and Flavell, R. A. (1999) Neuron 22, 667-676[Medline] [Order article via Infotrieve] |
32. |
Sabapathy, K.,
Kallunki, T.,
David, J. P.,
Graef, I.,
Karin, M.,
and Wagner, E. F.
(2001)
J. Exp. Med.
193,
317-328 |
33. | Yang, D. D., Kuan, C. Y., Whitmarsh, A. J., Rincon, M., Zheng, T. S., Davis, R. J., Rakic, P., and Flavell, R. A. (1997) Nature 389, 865-870[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Coso, O. A.,
Teramoto, H.,
Simonds, W. F.,
and Gutkind, J. S.
(1996)
J. Biol. Chem.
271,
3963-3966 |
35. |
Shapiro, P. S.,
Evans, J. N.,
Davis, R. J.,
and Posada, J. A.
(1996)
J. Biol. Chem.
271,
5750-5754 |
36. | Lopez-Ilasaca, M., Li, W., Uren, A., Yu, J. C., Kazlauskas, A., Gutkind, J. S., and Heidaran, M. A. (1997) Biochem. Biophys. Res. Commun. 232, 273-277[CrossRef][Medline] [Order article via Infotrieve] |
37. | Minden, A., Lin, A., Claret, F. X., Abo, A., and Karin, M. (1995) Cell 81, 1147-1157[Medline] [Order article via Infotrieve] |
38. | Minden, A., Lin, A., McMahon, M., Lange-Carter, C., Derijard, B., Davis, R. J., Johnson, G. L., and Karin, M. (1994) Science 266, 1719-1723[Medline] [Order article via Infotrieve] |
39. |
Schwertfeger, K. L.,
Hunter, S.,
Heasley, L. E.,
Levresse, V.,
Leon, R. P.,
DeGregori, J.,
and Anderson, S. M.
(2000)
Mol. Endocrinol.
14,
1592-1602 |
40. |
Krause, D.,
Lyons, A.,
Fennelly, C.,
and O'Connor, R.
(2001)
J. Biol. Chem.
276,
19244-19252 |
41. | Miller, B. S., Shankavaram, U. T., Horney, M. J., Gore, A. C., Kurtz, D. T., and Rosenzweig, S. A. (1996) Biochemistry 35, 8769-8775[CrossRef][Medline] [Order article via Infotrieve] |
42. | Desbois-Mouthon, C., Blivet-Van Eggelpoel, M. J., Auclair, M., Cherqui, G., Capeau, J., and Caron, M. (1998) Biochem. Biophys. Res. Commun. 243, 765-770[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Standaert, M. L.,
Bandyopadhyay, G.,
Antwi, E. K.,
and Farese, R. V.
(1999)
Endocrinology
140,
2145-2151 |
44. |
Uchida, T.,
Myers, M. G., Jr.,
and White, M. F.
(2000)
Mol. Cell. Biol.
20,
126-138 |
45. |
Tournier, C.,
Hess, P.,
Yang, D. D., Xu, J.,
Turner, T. K.,
Nimnual, A.,
Bar-Sagi, D.,
Jones, S. N.,
Flavell, R. A.,
and Davis, R. J.
(2000)
Science
288,
870-874 |
46. |
Student, A. K.,
Hsu, R. Y.,
and Lane, M. D.
(1980)
J. Biol. Chem.
255,
4745-4750 |
47. |
Kohanski, R. A.,
Frost, S. C.,
and Lane, M. D.
(1986)
J. Biol. Chem.
261,
12272-12281 |
48. | Myers, M. G., Jr., Zhang, Y., Aldaz, G. A. I., Grammer, T. C., Glasheen, E. M., Yenush, L., Wang, L. M., Sun, X. J., Blenis, J., Pierce, J. H., and White, M. F. (1996) Mol. Cell. Biol. 16, 4147-4155[Abstract] |
49. |
Yasuda, J.,
Whitmarsh, A. J.,
Cavanagh, J.,
Sharma, M.,
and Davis, R. J.
(1999)
Mol. Cell. Biol.
19,
7245-7254 |
50. |
Bonny, C.,
Oberson, A.,
Negri, S.,
Sauser, C.,
and Schorderet, D. F.
(2001)
Diabetes
50,
77-82 |
51. |
Schwarze, S. R., Ho, A.,
Vocero-Akbani, A.,
and Dowdy, S. F.
(1999)
Science
285,
1569-1572 |
52. |
Asano, T.,
Kanda, A.,
Katagiri, H.,
Nawano, M.,
Ogihara, T.,
Inukai, K.,
Anai, M.,
Fukushima, Y.,
Yazaki, Y.,
Kikuchi, M.,
Hooshmand-Rad, R.,
Heldin, C. H.,
Oka, Y.,
and Funaki, M.
(2000)
J. Biol. Chem.
275,
17671-17676 |
53. | Skolnik, E. Y., Lee, C. H., Batzer, A. G., Vicentini, L. M., Zhou, M., Daly, R. J., Myers, M. G., Jr., Backer, J. M., Ullrich, A., White, M. F., and Schlessinger, J. (1993) EMBO J. 12, 1929-1936[Abstract] |
54. | Myers, M. G., Jr., Wang, L. M., Sun, X. J., Zhang, Y., Yenush, L., Schlessinger, J., Pierce, J. H., and White, M. F. (1994) Mol. Cell. Biol. 14, 3577-3587[Abstract] |
55. |
Whitmarsh, A. J.,
Cavanagh, J.,
Tournier, C.,
Yasuda, J.,
and Davis, R. J.
(1998)
Science
281,
1671-1674 |
56. |
Fukunaga, K.,
Noguchi, T.,
Takeda, H.,
Matozaki, T.,
Hayashi, Y.,
Itoh, H.,
and Kasuga, M.
(2000)
J. Biol. Chem.
275,
5208-5213 |
57. |
Gao, Z.,
Hwang, D.,
Bataille, F.,
Lefevre, M.,
York, D.,
Quon, M.,
and Ye, J.
(2002)
J. Biol. Chem.
277,
48115-48121 |
58. | DeFronzo, R. A. (1997) Diabetes Rev. 5, 177-269 |
59. | Facchini, F. S., Hua, N. W., Reaven, G. M., and Stoohs, R. A. (2000) Free Radic. Biol. Med. 29, 1302-1306[CrossRef][Medline] [Order article via Infotrieve] |
60. | Baron, S. H. (1982) Diabetes Care 5, 64-71[Abstract] |
61. |
Kim, J. K.,
Kim, Y. J.,
Fillmore, J. J.,
Chen, Y.,
Moore, I.,
Lee, J.,
Yuan, M., Li, Z. W.,
Karin, M.,
Perret, P.,
Shoelson, S. E.,
and Shulman, G. I.
(2001)
J. Clin. Invest.
108,
437-446 |
62. | White, M. F., and Myers, M. G. (2001) in Endocrinology (DeGroot, L. J. , and Jameson, J. L., eds), Vol. 3 , pp. 712-727, W. B. Saunders Co., Philadelphia |
63. | Hotamisligil, G. S., Shargill, N. S., and Spiegelman, B. M. (1993) Science 259, 87-91[Medline] [Order article via Infotrieve] |
64. |
Peraldi, P.,
Hotamisligil, G. S.,
Buurman, W. A.,
White, M. F.,
and Spiegelman, B. M.
(1996)
J. Biol. Chem.
271,
13018-13022 |
65. |
Uysal, K. T.,
Wiesbrock, S. M.,
and Hotamisligil, G. S.
(1998)
Endocrinology
139,
4832-4838 |
66. | Uysal, K. T., Wiesbrock, S. M., Marino, M. W., and Hotamisligil, G. S. (1997) Nature 389, 610-614[CrossRef][Medline] [Order article via Infotrieve] |