MOLECULAR AND CELLULAR BIOLOGY, Sept. 2003, p. 6255–6266
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 23, No. 17
Morphine Induces Desensitization of Insulin Receptor Signaling
Yu Li,1Shoshana Eitan,2Jiong Wu,1Christopher J. Evans,2Brigitte Kieffer,3
Xiaojian Sun,4and Roberto D. Polakiewicz1*
Cell Signaling Technology, Inc., Beverly, Massachusetts1; Department of Psychiatry and Biobehavioral Sciences,
Neuropsychiatric Institute, University of California, Los Angeles, Los Angeles, California2; IGBMC UMR 7104,
Parc d’Innovation, 67404 Illkirch Cedex, France3; and Endocrinology Division, University of
Vermont College of Medicine, Burlington, Vermont4
Received 14 April 2003/Returned for modification 21 May 2003/Accepted 5 June 2003
Morphine analgesia is mediated principally by the ?-opioid receptor (MOR). Since morphine and other
opiates have been shown to influence glucose homeostasis, we investigated the hypothesis of direct cross talk
between the MOR and the insulin receptor (IR) signaling cascades. We show that prolonged morphine
exposure of cell lines expressing endogenous or transfected MOR, IR, and the insulin substrate 1 (IRS-1)
protein specifically desensitizes IR signaling to Akt and ERK cascades. Morphine caused serine phosphory-
lation of the IR and impaired the formation of the signaling complex among the IR, Shc, and Grb2. Morphine
also resulted in IRS-1 phosphorylation at serine 612 and reduced tyrosine phosphorylation at the YMXM
p85-binding motifs, weakening the association of the IRS-1/p85 phosphatidylinositol 3-kinase complex. How-
ever, the IRS-1/Grb2 complex was unaffected by chronic morphine treatment. These results suggest that
morphine attenuates IR signaling to Akt by disrupting the IRS-1–p85 interaction but inhibits signaling to ERK
by disruption of the complex among the IR, Shc, and Grb2. Finally, we show that systemic morphine induced
IRS-1 phosphorylation at Ser612 in the hypothalamus and hippocampus of wild type, but not MOR knockout,
mice. Our results demonstrate that opiates can inhibit insulin signaling through direct cross talk between the
downstream signaling pathways of the MOR and the IR.
The clinically useful properties of morphine are often over-
shadowed by the development of tolerance and dependence
following chronic use. The mechanisms of morphine’s acute
and chronic actions have therefore been the focus of intense
research. Mouse gene-targeting studies have confirmed that
morphine-induced analgesia and dependence are mediated by
?-opioid receptors (MORs) (40). MOR signaling can be reg-
ulated at several levels, namely, receptor homo- and hetero-
dimerization (14), MOR desensitization and trafficking (8, 20,
70), or the downstream signaling pathways (36). It is not fully
understood how these different mechanisms regulating recep-
tor signaling are coordinated. The MOR is typically coupled to
Gi/o proteins, which inhibit adenylyl cyclase and modulate
both inwardly rectifying K?and voltage-dependent calcium
channels (36). Evidence has recently emerged that binding of
MOR by agonists, including morphine, leads to the activation
of G??- and phosphatidylinositol 3-kinase (PI3K)-dependent
signaling cascades (22, 36, 48, 49). These include the stimula-
tion of serine/threonine kinases such as ERK, Akt, and p70S6
kinase (22, 48, 49). However, the functional consequences of
MOR activation of ERK or Akt signaling pathways in vivo are
Activation of the Akt and ERK pathways is typically ob-
served upon mitogenic stimulation of receptor tyrosine kinases
(RTKs), including the insulin receptor (IR). Binding of insulin
to its receptor leads to activation of IR tyrosine kinase activity
and consequent tyrosine phosphorylation of several IR sub-
strates (IRS), including IRS-1 and IRS-2, and the adaptor
protein Shc (58, 66, 71). IRS proteins interact with Src homol-
ogy domain (SH2)-containing proteins such as the p85 subunit
of PI3K, Grb2, SHP2, Nck, and others (71). Activation of PI3K
leads to stimulation of Akt, which contributes to the stimula-
tion of glucose uptake, glycogen synthesis, and protein synthe-
sis (58, 66, 71). Association of IR with Shc and/or association
of IRS with Grb2 and consequent recruitment of SOS and Ras
lead to activation of the ERK pathway, resulting in mitogenic
effects and changes in gene expression (58, 66, 71). Sustained
activation of the IR or stress-activated pathways can result in
serine phosphorylation of IRS-1 and consequent attenuation
of insulin signaling (77). This mechanism is thought to contrib-
ute to acute and chronic insulin resistance.
There is evidence that signaling pathways activated by G-
protein-coupled receptors (GPCRs) and RTKs can be highly
coordinated (25). One of the most extensively studied cases of
such cross-regulation is the transactivation of the epidermal
growth factor receptor by different GPCRs, including MOR (5,
25). Cross talk between the IR and GPCRs appears to be
bidirectional and complex. For example, insulin attenuates cat-
echolamine actions through tyrosine phosphorylation of the
?2-adrenergic receptor (?2AR), diminishing its ability to in-
duce Gs-mediated accumulation of cyclic AMP (32). Insulin
also induces Akt-mediated serine phosphorylation of ?2AR,
and both tyrosine and serine phosphorylations of ?2AR con-
tribute to its rapid sequestration (15, 16). Other evidence sug-
gests that GPCR activation can attenuate insulin signaling.
Stimulation of ?3AR in adipocytes inhibits IR and IRS-1 sig-
naling to PI3K and glucose uptake (35), and in the heart and
vascular tissues, two distinct GPCR agonists, angiotensin II
and endothelin, inhibit insulin signaling (29, 68).
It is well established that opioids influence glucose ho-
* Corresponding author. Mailing address: Cell Signaling Technol-
ogy, Inc., 166B Cummings Center, Beverly, MA 01915. Phone: (978)
867-2369. Fax: (978) 867-2402. E-mail: email@example.com.
meostasis (18, 19). Early studies documented the hyperglyce-
mic effects of morphine and methadone when administered
centrally (18, 23, 30, 56) and suggested that heroin addiction
produces a metabolic state similar to that of non-insulin-de-
pendent diabetes mellitus (24). Although morphine and other
opiates may act indirectly via the sympathetic nervous system
to cause hyperglycemia, the possibility of a direct interaction
between opioid and insulin signaling pathways has not been
well explored. The present study addressed the question of
direct signaling interactions between the MOR and the IR-
signaling pathways. Our results demonstrate that morphine
stimulates serine phosphorylation of IRS-1 and the IR, result-
ing in disruption of functional signaling complexes that couple
the insulin response to the ERK and Akt pathways.
MATERIALS AND METHODS
Reagents. DAMGO, morphine, naloxone, and insulin were purchased from
Sigma. Puromycin was from Calbiochem. Protein A-agarose was purchased from
Roche. Glutathione-Sepharose was from Amersham-Pharmacia. A PolyFect
transfection kit was obtained from Qiagen. All of the antibodies used in this
study were from Cell Signaling Technology Inc. (Beverly, Mass.), except for the
anti-IR, anti-Grb2, and anti-Shc antibodies from Transduction Laboratories
(Lexington, Ky.). The protein kinase C (PKC) substrate antibody was raised and
characterized as previously described (75). The p85-binding motif antibody was
raised and characterized by following essentially the same procedures and assays
as for the PKC substrate antibody except for the sequences in the peptide
libraries used as antigens. Wild-type IRS-1–hemagglutinin (HA) and F6–IRS-
1–HA constructs were kindly provided by Michael J. Quon.
Cell culture. A stable Chinese hamster ovary cell line expressing the IR and
IRS-1 (CHO–IR–IRS-1) was transfected with a DNA construct expressing the
HA-tagged ??R cDNA. This cDNA was originally amplified by PCR from an
embryonic mouse brain cDNA library and cloned into a PEAK-10 vector (Edge
Biosystems). Transfected cells were maintained in Dulbecco modified Eagle
medium supplemented with 10% fetal bovine serum and puromycin (1.5 ?g/ml)
and were shown to be responsive to insulin and morphine (measured by Akt and
ERK activation), respectively. SK-N-SH human neuroblastoma cells were grown
in Dulbecco modified Eagle medium and 10% fetal bovine serum. In a typical
experiment, cells were grown in six-well plates for 24 h prior to treatment,
washed, and then incubated in serum-free medium overnight prior to ligand
stimulation as indicated in the figure legends. Incubation with inhibitors was
initiated 30 min (U0126, PD98059) and 5 min (naloxone) prior to ligand stim-
ulation. For desensitization experiments, cells were incubated overnight in se-
rum-free medium before morphine, insulin, and different inhibitors were added
to the medium for an additional 1.5 h. The cells were then washed three times in
phosphate-buffered saline and subjected to a second ligand stimulus for 5 min.
Extracts were then prepared and analyzed by immunoblotting as described be-
Animal experiments. Male MOR knockout (40) and littermate control mice 10
to 18 weeks old were used for all experiments and housed four per cage with food
and water provided ad libitum. All procedures were conducted in accordance
with the National Institutes of Health Guide for the Care and Use of Laboratory
Animals and were approved by the Institutional Animal Care and Use Commit-
tee. Morphine sulfate and naloxone were obtained from the National Institute on
Drug Abuse (Bethesda, Md.) drug supply program. Mice were habituated to the
injection procedure by daily (subcutaneous [s.c.]) injection for 6 days with saline.
On day 7, mice were injected s.c. with either saline or 2 mg of naloxone per kg
and then injected s.c. with either saline or 10 mg of morphine per kg 15 min later.
Thirty minutes following the morphine injection, mice were sacrificed and their
brains were removed. Brains were dissected on ice and immediately frozen at
Immunoprecipitation. Cells were grown in 10-cm-diameter plates for 24 h,
serum starved overnight, and treated as indicated in the figure legends. After
treatment, cells were scraped into 0.5 ml of lysis buffer (20 mM Tris-HCl [pH
7.4], 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, protease and
phosphatase inhibitors). Brain tissue extracts were prepared in the same buffer
with a Polytron homogenizer. Extracts were incubated on ice for 10 min and
centrifuged at 14,000 ? g for 10 min at 4°C. The supernatant was incubated with
the indicated antibodies at 4°C for 2 h. Protein A-agarose was then added, and
incubation under the same conditions was continued for 1 h more. Immune
complexes were washed three times with lysis buffer, and the pellets were resus-
pended in 3? sodium dodecyl sulfate (SDS) sample buffer (187.5 mM Tris-HCl
[pH 6.8], 6% SDS, 30% glycerol, 150 mM dithiothreitol, 0.03% bromophenol
blue) and boiled for 5 min. These samples were then subjected to immunoblot-
ting with the indicated antibodies.
Extract preparation and immunoblotting. For Western blotting experiments,
cell extracts were prepared by lysing the cells immediately after treatment in
Laemmli sample buffer and subjected to SDS–10% polyacrylamide gel electro-
phoresis and immunoblotted as previously described (48, 49). Peptide competi-
tion experiments were done with p85-binding motif antibody and PY-100 anti-
body alone or together with the following peptides at 1 ?g/ml: Y608-P (CLHT
DDGY[PO3]MPMS), Y608 (CLHTDDGY[PO3]MPMS), and Y891-P (CPKSP
GST-Grb2 pulldown assay. Cells grown on 10-cm-diameter plates were lysed
in 0.5 ml of lysis buffer. Extracts were incubated on ice for 10 min and centrifuged
at 14,000 ? g for 10 min at 4°C. Pulldown assays were conducted by adding 2 ?g
of glutathione S-transferase (GST) fusion protein-Sepharose slurry to 0.5 ml of
cell lysate. After 1 h of incubation at 4°C on a rotator, beads were washed three
times with lysis buffer, resolved by SDS-polyacrylamide gel electrophoresis, and
subjected to immunoblotting.
Morphine and insulin activate similar signaling cascades.
We have shown in previous studies that, similar to the effects
of insulin, MOR agonists such as DAMGO and morphine lead
to rapid activation of PI3K and ERK signaling cascades (48,
49). This was determined by measuring the enzymatic activi-
ties, as well as the phosphorylation status, of Akt, p70S6K,
MEK, and ERK (48, 49). In order to study the possible sig-
naling cross talk between the IR and MOR signaling pathways,
a construct expressing murine HA-tagged MOR cDNA was
stably transfected into a CHO cell line overexpressing the IR
and IRS-1 (67). This cell line expressed a number of MORs
similar to that of the CHO-MOR cell line we used in earlier
studies (data not shown) (48). With this CHO–MOR/IR/IRS-1
cell line, we first confirmed that DAMGO and morphine in-
duced the phosphorylation of ERK and Akt, as well as phos-
phorylation of their downstream in vivo substrates p90RSK (at
T573) and GSK-3? (at S9), respectively (Fig. 1a). Exposure of
the CHO–MOR/IR/IRS-1 cells to insulin provoked robust
phosphorylation of Akt, ERK, p90RSK, and GSK3? (Fig. 1a).
Insulin also activated these cascades in a CHO-MOR cell line
not overexpressing the IR and IRS-1 but with less efficacy,
presumably because of the lower number of IRs (data not
shown). The rapid induction of the Akt and ERK cascades by
either morphine (Fig. 1b) or insulin (Fig. 1c) followed similar
kinetics, except for the increased duration of phosphorylation
of Akt at S473 following insulin (Fig. 1c).
Morphine desensitizes IR signaling. Multiple lines of evi-
dence suggest a functional interaction between MOR and IR
signaling cascades: the existence of signaling cross talk between
the IR and other GPCRs (29, 31, 35, 68), a recent report
indicating that insulin pretreatment of Xenopus oocytes ex-
pressing the rat MOR increases the intrinsic efficacy of MOR
agonists (41), and the reported effects of opiates on glucose
metabolism (18, 19, 23, 24, 30, 56). To examine possible cross
talk between MOR and IR signaling, we exposed the CHO–
MOR/IR/IRS-1 cells to either morphine or insulin for 1.5 h,
washed them three times in serum-free medium, and then
exposed them again to either morphine or insulin for 5 min.
Figure 2a shows that preincubation of the cells with insulin had
little, if any, effect on MOR-induced phosphorylation of Akt
6256LI ET AL.MOL. CELL. BIOL.
and ERK. Compared to cells stimulated with morphine alone,
a slight enhancement of the phosphorylated Akt and ERK
signal was observed in cells preexposed to insulin. This en-
hancement is consistent with the results showing that insulin
pretreatment enhances MOR activation in Xenopus oocytes
(41). Alternatively, this enhancement could be explained by
prolonged residual insulin-induced phosphorylation of Akt
and ERK (Fig. 1c). In contrast to the weak effects of insulin on
MOR signaling, preexposure of the cells to morphine resulted
in dramatic modulation of insulin signaling. Incubation of
CHO–MOR/IR/IRS-1 cells with 1 ?M morphine for 1.5 h
completely abolished the ability of insulin to induce the phos-
phorylation of both Akt and ERK (Fig. 2a). This attenuation of
IR signaling was observed as soon as 15 min following mor-
phine exposure (Fig. 2c). In the same cells, ERK activation by
bFGF was unaffected by morphine pretreatment (Fig. 2b),
indicating that this receptor cross talk does not represent a
general effect of morphine exposure on the signaling of all
RTKs. Like morphine, ERK phosphorylation can also be in-
duced in CHO–MOR/IR/IRS-1 cells by another Gi-coupled
receptor ligand, lysophosphatidic acid (LPA). However, pre-
exposure of the cells to LPA for 1.5 h did not cause desensi-
tization of the insulin response (Fig. 2b). This suggests that
MOR modulation of IR signaling cannot be mediated by all
GPCRs that couple to inhibitory G proteins. The desensitizing
effect of morphine on the insulin response is blocked by the
opioid antagonist naloxone, as well as the MEK inhibitors
U0126 (Fig. 3a) and PD98059 (data not shown). Similar mod-
ulation of IR signaling was observed with other MOR selective
agonists, such as DAMGO, as well as in other cell lines ex-
pressing endogenous MOR and IR, such as the neuroblastoma
cell line SK-N-SH (4, 46) (Fig. 3b).
Morphine induces serine phosphorylation of the IR and
IRS-1. Insulin resistance can be caused by multiple mecha-
nisms, including elevated activity of protein or lipid phospha-
tases like PTEN, PTP1B, and SHIP2 (9, 58). Insulin resistance
can also result from serine/threonine phosphorylation of the
IR itself (6, 37, 65) and/or its main substrates, such as the IRS
docking proteins (77). Work by several groups has documented
the negative effects of serine phosphorylation on IRS-1 func-
tion (77), and serine phosphorylation of IRS proteins has been
implicated in insulin resistance associated with obesity and
trauma (2, 3, 52, 55). In one example, tumor necrosis factor
alpha signaling leads to JNK-dependent phosphorylation of rat
IRS-1 at S307 and consequent uncoupling of insulin signaling
(2, 3, 26, 55). Endothelin, a GPCR ligand, causes similar effects
by stimulating the PKC/ERK-mediated IRS-1 phosphorylation
at S612 (29, 42). IRS-1 phosphorylation at serines 632, 662, and
731 is induced by platelet-derived growth factor and negatively
regulates the induction of an IRS-1–PI3K complex in a mech-
anism involving a PI3K/Akt/mTOR cascade (38, 54, 64). Other
studies have implicated a negative feedback signal involving
IRS-1 phosphorylation by PKC? (53, 57), as well as by other
PI3K-dependent kinases leading to insulin-dependent proteo-
some degradation of IRS-1 (47, 74). We thus investigated the
effects of acute and prolonged exposure to morphine on IR
and IRS-1 tyrosine and serine phosphorylation. CHO–MOR/
IR/IRS-1 cells were either treated with morphine or insulin for
5 min or treated with morphine for 1.5 h prior to stimulation
with insulin for 5 min. Cell extracts were prepared, the IR was
immunoprecipitated with an IR antibody, and tyrosine and
serine phosphorylation status was analyzed by Western blot-
ting (Fig. 4). Stimulation with morphine had no detectable
effect on the general tyrosine phosphorylation status of the IR,
FIG. 1. Induction of Akt and ERK phosphorylation by morphine and insulin. (a) Cells expressing the IR, IRS-1, and the MOR were left
untreated (lane C) or treated with DAMGO (lane D; 1 ?M), morphine (M; 1 ?M), or insulin (I; 200 nM) for 5 min, extracted, and assayed by
Western blotting for levels of phospho-Akt (S473), phospho-p90RSK (T573), phospho-ERK (T202/Y204), and phospho-GSK3? (S9), as well as
total Akt and ERK. The time courses of Akt and ERK induction by morphine (1 ?M; b) and insulin (200 nM; c) are also shown.
VOL. 23, 2003 MORPHINE DESENSITIZES INSULIN SIGNALING6257
as measured by a general phosphotyrosine monoclonal anti-
body (PY-100) or more specifically measured by an antibody
directed against the phosphorylated Y1146 residue located in
the activation loop of the IR kinase domain (Fig. 4a). This
result indicates that MOR stimulation cannot transactivate the
IR. Furthermore, prolonged stimulation with morphine did
not attenuate the tyrosine phosphorylation of the IR induced
by insulin (Fig. 4a). These results and the fact that general
tyrosine phosphorylation of IRS-1 is also not diminished fol-
lowing morphine treatment (see Fig. 6) suggest that the IR
intrinsic kinase activity stimulated by insulin is not significantly
affected by MOR activation. However, since a general phos-
photyrosine antibody was used, we cannot rule out possible
alteration of the phosphorylation state of individual tyrosine
residues in the IR that are involved in the binding of specific
adaptor proteins. Reduced phosphorylation of some sites
FIG. 2. Desensitization of insulin signaling to Akt and ERK by
morphine. (a) Cells were treated with morphine (M; 1 ?M) or insulin
(I; 200 nM) for 1.5 h, washed, subjected to either morphine or insulin
for 5 min, and then assayed for levels of phospho-Akt, phospho-ERK,
and total Akt and ERK. (b) Cells were treated with morphine (1 ?M)
or LPA (10 ?M) for 1.5 h; washed; exposed to morphine (1 ?M), LPA
(10 ?M), insulin (200 nM), or bFGF (100 ng/ml) for 5 min; and then
analyzed for kinase activation. (c) Time course of MOR-mediated
desensitization of IR signaling via Akt and ERK. Cells were incubated
for various periods of time with 1 ?M morphine, washed, subjected to
200 nM insulin for 5 min, and then assayed for phospho-Akt, total Akt,
phospho-ERK, and total ERK.
FIG. 3. Inhibition of the ERK pathway blocks desensitization of
insulin signaling by morphine. CHO–MOR/IR/IRS-1 (a) or SK-N-SH
(b) cells were serum starved overnight and then pretreated with 1 ?M
morphine (a) or 200 nM DAMGO (b). Morphine and DAMGO pre-
treatments were performed in the absence or presence of 10 ?M
naloxone (a and b) or 10 ?M U0126 (a). After 1.5 h, the cells were
washed three times with serum-free medium and exposed to 200 nM
insulin for 5 min. Extracts were then assayed for levels of phospho-Akt
(S473), total Akt, phospho-ERK(T202/Y204), and total ERK.
6258LI ET AL.MOL. CELL. BIOL.
could escape detection in the background of the phosphoryla-
tion of other tyrosine residues in the IR when detected with a
general phosphotyrosine antibody such as PY-100 (see text
below and Fig. 6). Interestingly, both insulin and morphine
markedly induced serine phosphorylation of the IR as probed
with an antibody designed to detect serine phosphorylation
within a motif defined as phospho-S-X-R/K (Fig. 4b) (75).
Serine phosphorylation of the IR was blocked by preincubation
with either the MOR antagonist naloxone (10 ?M) or the
MEK inhibitors PD98059 (10 ?M) and U0126 (10 ?M). This is
consistent with the notion that MOR agonists can induce IR
phosphorylation at serine residues by kinases that phosphory-
late serine in the context of such a motif. Earlier studies sug-
gested that serine phosphorylation of the IR can be mediated
by PKCs (6, 37, 65). Our results indicate that a kinase, capable
of phosphorylating a motif defined as pS-X-R/K and activated
downstream of ERK, is responsible for the serine phosphory-
lation of the IR induced by morphine. The precise identity of
such a kinase remains to be determined.
We then asked whether acute or prolonged treatment with
morphine would result in serine phosphorylation of IRS-1.
CHO–MOR/IR/IRS-1 cells were treated with either morphine
or insulin for 5 min. Following treatment, extracts were pre-
pared and IRS-1 was immunoprecipitated with IRS-1-specific
antibodies, followed by Western blotting to assess the tyrosine
phosphorylation state of IRS-1 and phosphorylation at specific
serine residues known to affect IRS-1 function. Consistent with
the results shown in Fig. 4, morphine did not induce tyrosine
phosphorylation of IRS-1 or binding of IRS-1 to the p85 sub-
unit of PI3K in CHO–MOR/IR/IRS-1 cells (Fig. 5a, middle
and bottom). However, Fig. 5a shows that, like insulin treat-
ment (11, 12), acute treatment with morphine induces IRS-1
phosphorylation at S612. Similarly, prolonged stimulation with
morphine (1.5 h) led to IRS-1 phosphorylation at S612 (Fig.
5b). This phosphorylation could be fully reversed by naloxone
(10 ?M) (Fig. 5b) and the MEK inhibitors U0126 (10 ?M; Fig.
5b) and PD98059 (10 ?M; data not shown). Also, in SK-N-SH
cells, acute treatment with insulin or prolonged treatment (1.5
h) with the MOR selective agonist DAMGO induced IRS-1
phosphorylation at S612 (Fig. 5c). These results indicate that
the ERK cascade likely mediates MOR-induced IRS-1 phos-
phorylation at S612. IR-induced tyrosine phosphorylation of
IRS-1 creates active binding sites for various SH2 domain-
containing proteins, including p85, the regulatory subunit of
PI3K. Studies on the effects of IRS-1 phosphorylation at S612
have shown that this modification contributes to the dimin-
ished activation of PI3K (12, 38), which ultimately results in
diminished activation of Akt. Morphine-induced serine phos-
phorylation of IRS-1 could therefore have a role in the atten-
uation of the insulin response, as shown in Fig. 2 and 3.
Prolonged morphine exposure disrupts the complex between
IRS-1 and p85 PI3K but not that between IRS-1 and Grb2. We
next investigated whether prolonged exposure to morphine
affects insulin-induced tyrosine phosphorylation of IRS-1 and
the integrity of the IR–IRS-1 complex. IRS-1 couples the in-
sulin response to different pathways, including the Akt and
ERK pathways. Upon IR-mediated phosphorylation, specific
tyrosine residues in IRS-1 become docking sites for other
adaptors and effector molecules, such as p85, the regulatory
subunit of PI3K, and Grb2. To directly probe the tyrosine
phosphorylation of IRS-1 at the p85-binding site, we raised a
novel phosphospecific antibody that recognizes phosphoty-
rosine only in the context of the SH2 domain p85-binding
motif, phospho-YMXM (where X indicates any amino acid).
We prepared this antibody with a peptide library containing
this motif by following principles we have described recently
(75). The specificity of this antibody was assessed with an
HA-tagged IRS-1 mutant construct, IRS-1–F6, with six phe-
nylalanine residues substituted for Y465, Y612, Y632, Y662,
Y989, and Y941 in YMXM motifs of human IRS-1 (corre-
sponding to Y460, Y608, Y628, Y658, Y935, and Y983 in
mouse IRS-1) (17). NIH 3T3 cells were transfected with either
the IRS-1–F6 construct or a wild-type HA–IRS-1 construct
and then stimulated with insulin for 15 min. Epitope-tagged
IRS-1 proteins were immunoprecipitated with an anti-HA an-
tibody and analyzed by Western blotting with antibodies di-
rected against the p85-binding motif (pYMXM), phosphoty-
FIG. 4. Effect of morphine on IR phosphorylation. (a) CHO–
MOR/IR/IRS-1 cells were serum starved overnight, exposed to mor-
phine (M; 1 ?M) or insulin (I; 200 nM) for 5 min or exposed to 1 ?M
morphine for 1.5 h, washed, and then stimulated with 200 nM insulin
for 5 min. Cell lysates were subjected to immunoprecipitation with an
anti-IR antibody and then assayed for levels of phospho-Tyr (PY-100)
(top), phospho-IR (Y1146) (middle), and total IR (bottom). (b) Cells
were serum starved overnight and then treated with 200 nM insulin for
5 min or 1 ?M morphine for 1.5 h alone or together with naloxone (Nx;
10 ?M) or U0126 (10 ?M). Cell lysates were subjected to immuno-
precipitation (IP) with anti-IR antibody and then analyzed by Western
blotting (WB) with the phospho-PKC substrate (sub) and anti-IR
antibodies. C, control; PD, PD98059.
VOL. 23, 2003 MORPHINE DESENSITIZES INSULIN SIGNALING6259
rosine (PY-100), or the HA epitope. Figure 6a shows that
following insulin treatment, the p85-binding motif antibody
and the general phosphotyrosine antibody, PY-100, detect
wild-type IRS-1. In contrast, the IRS-1–F6 protein was not
detected by the p85-binding motif antibody following insulin
treatment although residual tyrosine phosphorylation was still
detected by PY-100, presumably because of phosphorylated
tyrosines not embedded in the YMXM motif (Fig. 6a, middle).
To further establish the specificity of the antibody, competition
experiments were performed with a phosphorylated peptide
containing the sequence corresponding to the p85-binding site
Y608 in mouse IRS-1. As controls, the unphosphorylated pep-
tide with the same Y608 sequence or a phosphorylated peptide
containing Grb2-binding site Y891 were used (data not shown).
These experiments demonstrated that the p85-binding motif
antibody had no general phosphotyrosine immunoreactivity
but reacted specifically with phosphorylated tyrosine in the
context of a YMXM sequence.
To determine the regulation of the p85-binding motif,
CHO–MOR/IR/IRS-1 cells were treated with morphine or in-
sulin acutely or with morphine for 1.5 h and then stimulated
with insulin. Cell extracts were then immunoprecipitated with
IRS-1 antibodies and analyzed by Western blotting as de-
scribed in the legend to Fig. 6b. Receptor activation was con-
firmed by induction of Akt phosphorylation (Fig. 6c). Although
morphine pretreatment did not reduce the general phospho-
tyrosine content of IRS-1 (Fig. 6b, middle), morphine pretreat-
ment caused a significant reduction in the tyrosine phosphor-
ylation of IRS-1 at the p85-binding motif pYMXM (Fig. 6b,
top). Similar results were obtained with SK-N-SH cells (Fig.
7c). These results underscore the importance of assessing the
phosphorylation state of specific tyrosine residues and their
respective docking proteins to obtain more precise data on
pathway activation. Consistent with the reduction in phosphor-
ylation at the pYMXM motif, prolonged exposure to morphine
significantly reduced the amounts of the IR and p85 pulled
down with an IRS-1 antibody in either CHO–MOR/IR/IRS-1
cells or SK-N-SH neuroblastoma cells (Fig. 7a and c, respec-
tively). This effect was blocked by naloxone (10 ?M) or the
MEK inhibitor U0126 (10 ?M). Morphine pretreatment, how-
ever, had no effect on the association between IRS-1 and Grb2
(Fig. 7b), the SH2 domain-containing adaptor protein that
typically connects IRS-1 to the Ras/ERK cascade (43, 62, 63).
This was consistent with our peptide competition results
indicating that IRS-1 phosphorylation at Grb2-binding sites
remained intact upon morphine exposure (data not shown).
Disruption of the IR/IRS-1/p85 complex could explain a mech-
anism for morphine desensitization of the insulin-mediated
activation of Akt (by disruption of the IRS-1–p85 complex) but
cannot entirely account for morphine attenuation of insulin
signaling via ERK (Fig. 2 and 3).
Prolonged morphine exposure disrupts the complex among
IR, Shc, and Grb2. IR activation leads to tyrosine phosphory-
lation of the phosphotyrosine-binding (PTB) domains in both
IRS-1 and Shc, and Grb2 subsequently binds to both docking
proteins (33, 71, 72). The Shc-Grb2-Sos complex formed upon
Shc phosphorylation may constitute the dominant pathway
coupling the IR to the Ras/ERK–mediated mitogenic effects of
insulin (59). Because morphine treatment did not disrupt the
complex between IRS-1 and Grb2 (Fig. 7b), we asked whether
FIG. 5. Serine phosphorylation of IRS-1 by morphine. CHO–
MOR/IR/IRS-1 (a and b) or SK-N-SH (c) cells were serum starved
overnight and then stimulated with morphine (M; 1 ?M) or insulin (I;
200 nM) for 5 min (a), with morphine alone or in the absence or
presence of naloxone (10 ?M) or U0126 (10 ?M) for 1.5 h; or with
insulin for 5 min, with or without preincubation with U0126 (10 ?M)
(b); and with insulin for 5 min or with 200 nM DAMGO (D) for 1.5 h
(c). Cell lysates then were immunoprecipitated (IP) with IRS-1 anti-
bodies and analyzed with phospho-IRS1 (S612), phosphotyrosine (PY-
100), PI3-kinase p85, and IRS-1 antibodies. WB, Western blot.
6260 LI ET AL.MOL. CELL. BIOL.
morphine interferes with the tyrosine phosphorylation of Shc
and with the interaction among Shc, the IR, and Grb2. CHO–
MOR/IR/IRS-1 cells were exposed to morphine alone or in the
presence of naloxone or U0126 for 1.5 h and subsequently
stimulated with insulin for 5 min. To assess tyrosine phosphor-
ylation of Shc, we prepared cell extracts, immunoprecipitated
them with either PY-100 or Shc antibodies, and then subjected
them to Western blotting with Shc or IR antibodies. Figure 8a
shows that preincubation with morphine for 1.5 h attenuated
the tyrosine phosphorylation of Shc by insulin, an effect that
could be blocked by naloxone and U0126. In contrast, tyrosine
phosphorylation of the IR was not reduced by naloxone and
U0126. We then tested the levels of the IR and Grb2 coim-
munoprecipitating with Shc. As anticipated, insulin induced
the formation of a complex containing the IR, Shc, and Grb2,
and morphine preincubation reduced the levels of the IR and
Grb2 coimmunoprecipitated by the Shc-specific antibody (Fig.
8b). The effect of morphine was blocked by naloxone (10 ?M)
or U0126 (10 ?M). To confirm this result, we tested the ability
of a GST-Grb2 fusion protein to pull down the IR, Shc, or
IRS-1 from extracts of cells stimulated with insulin alone or
exposed to morphine prior to insulin stimulation. GST-Grb2
was able to efficiently pull down the IR, Shc, and IRS-1 from
extracts of cells stimulated with insulin alone. However, pre-
incubation with morphine reduced the levels of the IR and Shc,
but not that of IRS-1, pulled down by GST-Grb2 (data not
shown). Together, these results support the hypothesis that
morphine pretreatment prevents the formation of an active
complex among the IR, Shc, and Grb2 but does not affect the
interaction between IRS-1 and Grb2 (Fig. 7). Thus, the effect
of morphine on the IR-Shc interaction is not due to the im-
pairment of the IR intrinsic kinase activity (Fig. 4) but instead
is likely a result of morphine-stimulated phosphorylation of IR
serine residues (Fig. 4b) that reduce the docking and phos-
phorylation of Shc (Fig. 8). Morphine-stimulated phosphory-
lation of IR-specific serine residues may also explain the at-
tenuated tyrosine phosphorylation of IRS-1 at YMXM motifs,
thus reducing IRS-1 interaction with p85 but not with Grb2
(Fig. 6 and 7).
Morphine induces IRS-1 phosphorylation at S612 in dis-
crete brain regions. To assess the physiological relevance of
our findings, we explored whether acute administration of
morphine to mice induces serine phosphorylation of IRS-1 at
the S612 site as determined in cell culture. It has been docu-
mented that the MOR mediates most of the analgesic effects of
morphine (40). However, since morphine can activate the ?-
and ?-opioid receptors, we compared the effects of morphine
administration in wild-type and MOR knockout mice. Mice
were habituated to the injection procedure by being injected
s.c. daily for 6 days with saline. On day 7, mice were injected
s.c. with either saline or naloxone and then injected s.c. with
either saline or morphine at 10 mg/kg 15 min later. Thirty
minutes later, mice were sacrificed and their brains were re-
moved. The brains were dissected on ice and immediately
frozen at ?70°C. The whole brains, hypothalamus, hippocam-
pus, and striatum were homogenized in lysis buffer. These
brain regions were selected because of the relative abundance
of MOR and IRS-1 expression (21). Tissue extracts were used
to immunoprecipitate IRS-1 as described previously, followed
by Western blotting with total and phospho-S612 IRS-1 anti-
FIG. 6. Effect of morphine on the tyrosine phosphorylation of
IRS-1. (a) Specificity of p85-binding motif antibody. NIH 3T3 cells
were transfected with wild-type and mutant F6–HA–IRS-1 constructs,
starved overnight, and treated with insulin (I; 200 nM) for 5 min.
Transfected IRS-1 proteins were immunoprecipitated with anti-HA
antibodies and analyzed with p85-binding motif (pYMXM; top), gen-
eral phosphotyrosine (PY-100; middle), or HA (bottom) antibodies.
(b) Cell lysates were prepared as described in the legend to Fig. 4a and
then subjected to immunoprecipitation (IP) with IRS-1 antibodies,
followed by analysis with phospho-p85-binding motif (pYMXM),
phosphotyrosine (pY-100), and IRS-1 antibodies. (c) Cell lysates from
panel b were analyzed for levels of phospho-Akt (S473) and total Akt.
WB, Western blotting.
VOL. 23, 2003 MORPHINE DESENSITIZES INSULIN SIGNALING6261
bodies. Increased IRS-1 phosphorylation at S612 in whole
brain extracts was observed following acute morphine admin-
istration, and this effect could be blocked by naloxone (Fig. 9a).
Furthermore, morphine-induced IRS-1 phosphorylation at
S612 was observed in the hypothalamus and hippocampus of
wild-type but not MOR knockout mice (Fig. 9b). No induction
of IRS-1 phosphorylation could be detected in the striatum
despite a high abundance of IRS-1 (Fig. 9b, bottom) and the
MOR. This demonstrates MOR modulation of IRS-1 serine
phosphorylation in specific brain areas.
This study demonstrates unidirectional cross talk between
MOR and IR signaling whereby opiate agonists like morphine
activate MOR signaling cascades that lead to an insulin-resis-
tant state. Attenuation of insulin signaling was measured by
the lack of insulin-induced activation of both Akt and ERK
cascades in transfected CHO (CHO–MOR/IR/IRS-1) cells
and in neuroblastoma cells endogenously expressing MOR and
IR (SK-N-SH cells). The specificity of the cross talk between
the MOR and the IR was established by a lack of MOR
modulation of another RTK signaling pathway (bFGF) and by
the fact that activation of a different Gi-coupled receptor (the
LPA receptor) did not desensitize IR signaling. Unlike the
reported transactivation of the epidermal growth factor recep-
tor by opioid receptor ligands (5), morphine did not modulate
IR tyrosine phosphorylation and activity. The desensitizing
effects of morphine on insulin signaling were mediated by the
ERK signaling cascade, as they were effectively abolished by
FIG. 7. Effect of morphine on IRS-1, IR, PI3K, and Grb2 interactions. CHO–MOR/IR/IRS-1 (a) or SK-N-SH (c) cells were treated with
morphine or DAMGO, respectively. Cell lysates were then prepared as described in the legend to Fig. 4a, and IRS-1 was immunoprecipitated with
IRS-1 antibodies, followed by analysis with IR, p85 PI3-kinase, p85-binding motif, and IRS-1 antibodies. (b) Cell lysates were prepared as described
in the legend to Fig. 4a, and IRS-1 was immunoprecipitated (IP) with IRS-1 antibodies and analyzed with anti-Grb2 and IRS-1 antibodies. The
same cell lysates were analyzed for levels of phospho-ERK and total ERK (bottom). WB, Western blotting; M, morphine; I, insulin.
6262 LI ET AL.MOL. CELL. BIOL.
both MEK1/2 inhibitors in a fashion similar to the homologous
desensitization of the MOR (48, 60). Our results also suggest
that the mechanism underlying the desensitization of insulin
signaling involves increased serine phosphorylation of both the
IR and IRS-1, resulting in uncoupling of the IR from its major
adaptor signaling complexes. In the case of IRS-1, we demon-
strate that morphine induced phosphorylation at Ser612 via
an ERK-dependent pathway. Accordingly, morphine pretreat-
ment had a profound effect on IRS-1 function: it diminished
tyrosine phosphorylation at the p85-binding motif YMXM
sites and the binding of p85 and the IR to IRS-1 without
detectable alteration of phosphorylation at other tyrosine res-
idues. Our preliminary results indicate that morphine stimu-
lates the phosphorylation of other serine residues in IRS-1, but
the effect of these phosphorylation events on insulin signaling
is not clear. Morphine-dependent IRS-1 phosphorylation at
S612 is reminiscent of endothelin-induced IRS-1 phosphoryla-
tion at S612 via a PKC/ERK-dependent pathway (38). Because
of their close proximity, phosphorylation of S612 may hinder
the binding of p85 to Y608 (12), one of the major p85-binding
FIG. 8. Effect of morphine on Shc, IR, and Grb2 interactions. (a)
Cell lysates were prepared as described in the legend to Fig. 4b. The
lysates were immunoprecipitated with phosphotyrosine antibodies
(PY-100) and then analyzed with anti-Shc and anti-IR antibodies. (b)
The same cell lysates as in panel a were immunoprecipitated (IP) with
anti-Shc antibody and then analyzed with Grb2, IR, and Shc antibod-
ies. WB, Western blotting.
FIG. 9. Morphine induces IRS-1 phosphorylation at S612 in the
mouse brain. Mice were injected s.c. with either saline or 2 mg of
naloxone (Nx) per kg, followed by s.c. injection of either saline or 10
mg of morphine (M) per kg 15 min later as described in Materials and
Methods. Extracts of whole brains from wild-type mice (a) or hypo-
thalamus, hippocampus, and striatum tissues from wild type (wt) or
MOR knockout (KO) mice (b) were prepared, and IRS-1 was immu-
noprecipitated (IP) with an IRS-1 antibody, followed by analysis with
anti-IRS-1 or phospho-IRS1 S612 antibodies. Histograms represent
the average percent induction of IRS-1 S612 phosphorylation of two
independent experiments normalized for total immunoprecipitated
IRS-1. WB, Western blotting; C, control.
VOL. 23, 2003MORPHINE DESENSITIZES INSULIN SIGNALING6263
sites in mouse IRS-1. A different study demonstrated the role
of the phosphorylation of serine residues adjacent to YMXM
motifs in the negative regulation of PI3K and Akt (13). MOR-
induced reduction of YMXM motif phosphorylation and the
reduced binding of p85 to IRS-1 are consistent with an atten-
uated Akt response. Our results support the premise that phos-
phorylation at S612 leads to the dissociation of IRS-1 from IR
and p85, the regulatory subunit of PI3K. MOR activation most
likely affects the binding of only a subset of IRS-1 binding
partners containing an SH2 domain, as evidenced by the lack
of modulation of the interaction between IRS-1 and Grb2 by
morphine. Whether morphine stimulation also disrupts the
association of IRS-1 with other binding partners, such as Nck,
Crk, Fyn, and SHP-2, remains unanswered.
How does morphine desensitize insulin signaling to ERK?
Following insulin stimulation, the IRS-1 and Shc adaptor pro-
teins bind the IR through their PTB domains. IRS-1 and Shc
have been shown to bind Grb2, relaying the insulin signal to
the Ras/ERK cascade (43, 62, 63). However, the relative con-
tribution of either Shc or IRS-1 to ERK activation has not
been fully characterized and may differ from one cell type to
another. A recent report suggested that the Shc/Grb2 complex,
rather than IRS-1/Grb2, may actually be the adaptor system
predominantly transmitting insulin signaling to the ERK path-
way (59). In our system, the interaction between IRS-1 and
Grb2 remained intact following MOR activation. This ob-
servation and the fact that morphine induced serine phos-
phorylation of the IR prompted us to test the integrity of the
complex among the IR, Shc, and Grb2. Our analysis of IR-
Shc-Grb2 complexes demonstrated that morphine pretreat-
ment effectively prevented the association of Shc with the IR
and that of Shc with Grb2. This result indicates that disruption
of the IR/Shc/Grb2 complexes plays a major role in the desen-
sitization of insulin signaling to the ERK pathway following
Several studies have reported a role for PKC phosphoryla-
tion in the inhibition of IR kinase activity (6, 37, 65). One of
these studies postulated S994 as a possible phosphate acceptor
site for PKCs (65). Ser994 in the human IR is in close prox-
imity to Y999, a putative Shc PTB site, although it is not
contained within a prototypical PKC site. Morphine had little
effect on IR kinase activity; at least as measured by autophos-
phorylation of the IR and tyrosine phosphorylation of IRS-1.
However, morphine induced marked serine phosphorylation of
the IR, as demonstrated by an antibody that detects phosphor-
ylation of consensus “PKC motifs” (75). These motifs, namely,
phospho-S-X-R/K, are found within the IR sequence in close
proximity to serine residues 727 and 1064. Serine phosphory-
lation of these IR residues may hinder the binding and phos-
phorylation of Shc, thus preventing the formation of an active
complex with Grb2. The reduced binding of Shc to the IR and
the diminished tyrosine phosphorylation of Shc induced by
morphine support this hypothesis. In future studies, it will be
important to determine the precise IR serine phosphorylation
site(s) following morphine or insulin stimulation and identify
the relevant kinase(s) downstream from ERK involved in this
event. Another layer of regulation that fits well with our ob-
servations involves potential modulation of Shc interaction
with CEACAM1. CEACAM1 is tyrosine phosphorylated by
the IR, and upon binding to Shc, this results in sequestration
and uncoupling of the IR from the Ras/ERK pathway (50).
Phosphorylation of CEACAM1 at S503 enhances this inhibi-
tory activity (50). It is tempting to speculate that a mechanism
by which morphine, or perhaps other stimuli, could enhance
insulin resistance may involve CEACAM1 phosphorylation of
This report demonstrates that MOR agonists can dramati-
cally modulate insulin signaling, at least at the level of the Akt
and ERK pathways. Further studies aimed at exploring the
direct interaction between opioid and insulin signaling systems
are critical to determining whether the hyperglycemic effects of
opiates can be explained by receptor cross talk. Where may a
physiologically relevant interaction between insulin and opioid
receptor signaling occur in vivo? IRs are expressed in most
tissues of the body, including “insulin-insensitive” tissues
such as the brain (21), and the IR, IRS-1, and the MOR are
abundantly expressed in many brain areas. Following systemic
morphine administration, we observed increased IRS-1 phos-
phorylation at S612 in the hypothalamus and hippocampus of
wild-type but not MOR knockout mice. IRS-1 phosphorylation
at this site was blocked by naloxone. These results indicate that
the effect of morphine on IRS-1 phosphorylation in vivo was
mediated by the MOR and not by other opioid receptors.
These results also support the physiological relevance of our
results obtained with cell culture systems. It is noteworthy that
morphine-induced IRS-1 phosphorylation occurred in discrete
brain regions such as the hypothalamus and hippocampus but
not in the striatum, where there is also abundant MOR and
IRS-1 expression. It remains to be determined whether the
MOR and the IR or IRS-1 have the same cellular localization
in different brain regions. It has recently been proposed that
the IR, together with leptin signaling, activates IRS-1and PI3K
cascades in the arcuate nucleus of the hypothalamus, thereby
contributing to the control of energy homeostasis (45). Inhibi-
tion of PI3K activity in the hypothalamus reverses the food
intake-lowering effects of insulin (44). An elegant study in
which the gene for the IR was specifically disrupted in neurons
showed that mutant mice displayed diet-sensitive obesity, with
increases in body fat and plasma leptin levels, mild insulin
resistance, elevated insulin levels and hypertriglyceridemia (7).
Interestingly, early studies documented that opiate antagonists
like naloxone and naltrexone significantly reduced food con-
sumption in both genetically and dietarily obese animals, lead-
ing to the concept that endogenous opioid peptides have a role
in regulating food intake (27, 39, 61). A more detailed analysis
of the effects of morphine on hypothalamic insulin signaling is
required to better understand how these two systems interact
and how this interaction affects energy metabolism.
Opiates may also modulate insulin-like growth factorI re-
ceptor signaling through serine phosphorylation of IRS-1, thus
neutralizing the neuroprotective and neurogenic effects of in-
sulin-like growth factor I on hippocampal neurons (1, 76). This
could have important implications when opiate drugs are used
chronically. Finally, the presence of opioid peptides and opioid
receptors in the pancreas, as well as the influence of ?-endor-
phin on endocrine pancreas function, has been reported (10,
28, 34). Whether morphine has any effect on pancreatic islets
through the MOR remains controversial (28, 69). Nonethe-
less, these observations raise overall the possibility that en-
dogenous opioids modulate insulin gene expression and secre-
6264 LI ET AL.MOL. CELL. BIOL.
tion through inhibition of IR signaling in islet ? cells (73). The
use of MOR and IR knockout mice may help confirm our
hypothesis that signaling triggered by activation of these re-
ceptors and their physiological functions are regulated in a
We thank Sandra Schieferl for technical assistance. HA-tagged
IRS-1 wild-type and mutant constructs and the SK-N-SH neuronal cell
line were provided by Michael J. Quon and Lakshmi Devi, respectively.
We also thank Morris Birnbaum and Al Moritz for critical reading of
the manuscript. We specially thank Michael Comb for encouragement
and helpful discussions.
1. Aberg, M. A., N. D. Aberg, H. Hedbacker, J. Oscarsson, and P. S. Eriksson.
2000. Peripheral infusion of IGF-I selectively induces neurogenesis in the
adult rat hippocampus. J. Neurosci. 20:2896–2903.
2. Aguirre, V., T. Uchida, L. Yenush, R. Davis, and M. F. White. 2000. The
c-Jun NH2-terminal kinase promotes insulin resistance during association
with insulin receptor substrate-1 and phosphorylation of Ser307. J. Biol.
3. Aguirre, V., E. D. Werner, J. Giraud, Y. H. Lee, S. E. Shoelson, and M. F.
White. 2002. Phosphorylation of Ser307 in insulin receptor substrate-1 blocks
interactions with the insulin receptor and inhibits insulin action. J. Biol.
4. Baumhaker, Y., M. Gafni, O. Keren, and Y. Sarne. 1993. Selective and
interactive down-regulation of ?- and ?-opioid receptors in human neuro-
blastoma SK-N-SH cells. Mol. Pharmacol. 44:461–467.
5. Belcheva, M. M., M. Szucs, D. Wang, W. Sadee, and C. J. Coscia. 2001.
?-Opioid receptor-mediated ERK activation involves calmodulin-dependent
epidermal growth factor receptor transactivation. J. Biol. Chem. 276:33847–
6. Bollag, G. E., R. A. Roth, J. Beaudoin, D. Mochly-Rosen, and D. E. Kosh-
land, Jr. 1986. Protein kinase C directly phosphorylates the insulin receptor
in vitro and reduces its protein-tyrosine kinase activity. Proc. Natl. Acad. Sci.
7. Bruning, J. C., D. Gautam, D. J. Burks, J. Gillette, M. Schubert, P. C.
Orban, R. Klein, W. Krone, D. Muller-Wieland, and C. R. Kahn. 2000. Role
of brain insulin receptor in control of body weight and reproduction. Science
8. Chavkin, C., J. P. McLaughlin, and J. P. Celver. 2001. Regulation of opioid
receptor function by chronic agonist exposure: constitutive activity and de-
sensitization. Mol. Pharmacol. 60:20–25.
9. Clement, S., U. Krause, F. Desmedt, J. F. Tanti, J. Behrends, X. Pesesse, T.
Sasaki, J. Penninger, M. Doherty, W. Malaisse, J. E. Dumont, Y. Marchand-
Brustel, C. Erneux, L. Hue, and S. Schurmans. 2001. The lipid phosphatase
SHIP2 controls insulin sensitivity. Nature 409:92–97.
10. Curry, D. L., L. L. Bennett, and C. H. Li. 1987. Stimulation of insulin
secretion by ?-endorphins (1–27 & 1–31). Life Sci. 40:2053–2058.
11. De Fea, K., and R. A. Roth. 1997. Modulation of insulin receptor substrate-1
tyrosine phosphorylation and function by mitogen-activated protein kinase.
J. Biol. Chem. 272:31400–31406.
12. De Fea, K., and R. A. Roth. 1997. Protein kinase C modulation of insulin
receptor substrate-1 tyrosine phosphorylation requires serine 612. Biochem-
13. Delahaye, L., I. Mothe-Satney, M. G. Myers, M. F. White, and E. Van
Obberghen. 1998. Interaction of insulin receptor substrate-1 (IRS-1) with
phosphatidylinositol 3-kinase: effect of substitution of serine for alanine in
potential IRS-1 serine phosphorylation sites. Endocrinology 139:4911–4919.
14. Devi, L. A. 2001. Heterodimerization of G-protein-coupled receptors: phar-
macology, signaling and trafficking. Trends Pharmacol. Sci. 22:532–537.
15. Doronin, S., E. Shumay, H. H. Wang, and C. C. Malbon. 2002. Akt mediates
sequestration of the b2-adrenergic receptor in response to insulin. J. Biol.
16. Doronin, S., H. H. Wang, and C. C. Malbon. 2002. Insulin stimulates phos-
phorylation of the b2-adrenergic receptor by the insulin receptor, creating a
potent feedback inhibitor of its tyrosine kinase. J. Biol. Chem. 277:10698–
17. Esposito, D. L., Y. Li, A. Cama, and M. J. Quon. 2001. Tyr612 and Tyr632 in
human insulin receptor substrate-1 are important for full activation of insu-
lin-stimulated phosphatidylinositol 3-kinase activity and translocation of
GLUT4 in adipose cells. Endocrinology 142:2833–2840.
18. Feldberg, W., and K. P. Gupta. 1974. Morphine hyperglycaemia. J. Physiol.
19. Feldman, M., R. S. Kiser, R. H. Unger, and C. H. Li. 1983. Beta-endorphin
and the endocrine pancreas. Studies in healthy and diabetic human beings.
N. Engl. J. Med. 308:349–353.
20. Finn, A. K., and J. L. Whistler. 2001. Endocytosis of the mu opioid receptor
reduces tolerance and a cellular hallmark of opiate withdrawal. Neuron
21. Folli, F., L. Bonfanti, E. Renard, C. R. Kahn, and A. Merighi. 1994. Insulin
receptor substrate-1 (IRS-1) distribution in the rat central nervous system.
J. Neurosci. 14:6412–6422.
22. Fukuda, K., S. Kato, H. Morikawa, T. Shoda, and K. Mori. 1996. Functional
coupling of the delta-, mu-, and kappa-opioid receptors to mitogen-activated
protein kinase and arachidonate release in Chinese hamster ovary cells.
J. Neurochem. 67:1309–1316.
23. Giugliano, D. 1984. Morphine, opioid peptides, and pancreatic islet function.
Diabetes Care 7:92–98.
24. Giugliano, D., A. Ceriello, A. Quatraro, and F. D’Onofrio. 1985. Endogenous
opiates, heroin addiction, and non-insulin-dependent diabetes.Lancet ii:769–
25. Gschwind, A., E. Zwick, N. Prenzel, M. Leserer, and A. Ullrich. 2001. Cell
communication networks: epidermal growth factor receptor transactivation
as the paradigm for interreceptor signal transmission. Oncogene 20:1594–
26. Hirosumi, J., G. Tuncman, L. Chang, C. Z. Gorgun, K. T. Uysal, K. Maeda,
M. Karin, and G. S. Hotamisligil. 2002. A central role for JNK in obesity and
insulin resistance. Nature 420:333–336.
27. Holtzman, S. G. 1975. Effects of narcotic antagonists on fluid intake in the
rat. Life Sci. 16:1465–1470.
28. Ipp, E., R. Dobbs, and R. H. Unger. 1978. Morphine and beta-endorphin
influence the secretion of the endocrine pancreas. Nature 276:190–191.
29. Jiang, Z. Y., Q. L. Zhou, A. Chatterjee, E. P. Feener, M. G. Myers, Jr., M. F.
White, and G. L. King. 1999. Endothelin-1 modulates insulin signaling
through phosphatidylinositol 3-kinase pathway in vascular smooth muscle
cells. Diabetes 48:1120–1130.
30. Johansen, O., T. Tonnesen, T. Jensen, R. Jorde, P. G. Burhol, and O.
Reikeras. 1992. Increments in glucose, glucagon and insulin after morphine
in rats, and naloxone blocking of this effect. Life Sci. 51:1237–1242.
31. Karoor, V., and C. C. Malbon. 1998. G-protein-linked receptors as substrates
for tyrosine kinases: cross-talk in signaling. Adv. Pharmacol. 42:425–428.
32. Karoor, V., L. Wang, H. Y. Wang, and C. C. Malbon. 1998. Insulin stimulates
sequestration of beta-adrenergic receptors and enhanced association of beta-
adrenergic receptors with Grb2 via tyrosine 350. J. Biol. Chem. 273:33035–
33. Kasus-Jacobi, A., D. Perdereau, S. Tartare-Deckert, E. Van Obberghen, J.
Girard, and A. F. Burnol. 1997. Evidence for a direct interaction between
insulin receptor substrate-1 and Shc. J. Biol. Chem. 272:17166–17170.
34. Khawaja, X. Z., I. C. Green, J. R. Thorpe, and M. A. Titheradge. 1990. The
occurrence and receptor specificity of endogenous opioid peptides within the
pancreas and liver of the rat. Comparison with brain. Biochem. J. 267:233–
35. Klein, J., M. Fasshauer, M. Ito, B. B. Lowell, M. Benito, and C. R. Kahn.
1999. ?3-Adrenergic stimulation differentially inhibits insulin signaling and
decreases insulin-induced glucose uptake in brown adipocytes. J. Biol. Chem.
36. Law, P. Y., Y. H. Wong, and H. H. Loh. 2000. Molecular mechanisms and
regulation of opioid receptor signaling. Annu. Rev. Pharmacol. Toxicol. 40:
37. Lewis, R. E., D. J. Volle, and S. D. Sanderson. 1994. Phorbol ester stimulates
phosphorylation on serine 1327 of the human insulin receptor. J. Biol. Chem.
38. Li, J., K. DeFea, and R. A. Roth. 1999. Modulation of insulin receptor
substrate-1 tyrosine phosphorylation by an Akt/phosphatidylinositol 3-kinase
pathway. J. Biol. Chem. 274:9351–9356.
39. Marin-Bivens, C. L., and D. H. Olster. 1999. Opioid receptor blockade
promotes weight loss and improves the display of sexual behaviors in obese
Zucker female rats. Pharmacol. Biochem. Behav. 63:515–520.
40. Matthes, H. W., R. Maldonado, F. Simonin, O. Valverde, S. Slowe, I.
Kitchen, K. Befort, A. Dierich, M. Le Meur, P. Dolle, E. Tzavara, J. Ha-
noune, B. P. Roques, and B. L. Kieffer. 1996. Loss of morphine-induced
analgesia, reward effect and withdrawal symptoms in mice lacking the mu-
opioid-receptor gene. Nature 383:819–823.
41. McLaughlin, J. P., and C. Chavkin. 2001. Tyrosine phosphorylation of the
mu-opioid receptor regulates agonist intrinsic efficacy. Mol. Pharmacol. 59:
42. Mothe, I., and E. Van Obberghen. 1996. Phosphorylation of insulin receptor
substrate-1 on multiple serine residues, 612, 632, 662, and 731, modulates
insulin action. J. Biol. Chem. 271:11222–11227.
43. Myers, M. G., Jr., L. M. Wang, X. J. Sun, Y. Zhang, L. Yenush, J. Schless-
inger, J. H. Pierce, and M. F. White. 1994. Role of IRS-1-GRB-2 complexes
in insulin signaling. Mol. Cell. Biol. 14:3577–3587.
44. Niswender, K. D., C. D. Morrison, D. J. Clegg, R. Olson, D. G. Baskin, M. G.
Myers, Jr., R. J. Seeley, and M. W. Schwartz. 2003. Insulin activation of
phosphatidylinositol 3-kinase in the hypothalamic arcuate nucleus: a key
mediator of insulin-induced anorexia. Diabetes 52:227–231.
45. Niswender, K. D., and M. W. Schwartz. 2003. Insulin and leptin revisited:
VOL. 23, 2003MORPHINE DESENSITIZES INSULIN SIGNALING6265
adiposity signals with overlapping physiological and intracellular signaling
capabilities. Front. Neuroendocrinol. 24:1–10.
46. Ota, A., J. Shemer, R. M. Pruss, W. L. Lowe, Jr., and D. LeRoith. 1988.
Characterization of the altered oligosaccharide composition of the insulin
receptor on neural-derived cells. Brain Res. 443:1–11.
47. Pederson, T. M., D. L. Kramer, and C. M. Rondinone. 2001. Serine/threo-
nine phosphorylation of IRS-1 triggers its degradation: possible regulation
by tyrosine phosphorylation. Diabetes 50:24–31.
48. Polakiewicz, R. D., S. M. Schieferl, L. F. Dorner, V. Kansra, and M. J. Comb.
1998. A mitogen-activated protein kinase pathway is required for mu-opioid
receptor desensitization. J. Biol. Chem. 273:12402–12406.
49. Polakiewicz, R. D., S. M. Schieferl, A. C. Gingras, N. Sonenberg, and M. J.
Comb. 1998. ?-Opioid receptor activates signaling pathways implicated in
cell survival and translational control. J. Biol. Chem. 273:23534–23541.
50. Poy, M. N., R. J. Ruch, M. A. Fernstrom, Y. Okabayashi, and S. M. Najjar.
2002. Shc and CEACAM1 interact to regulate the mitogenic action of insu-
lin. J. Biol. Chem. 277:1076–1084.
51. Poy, M. N., Y. Yang, K. Rezaei, M. A. Fernstrom, A. D. Lee, Y. Kido, S. K.
Erickson, and S. M. Najjar. 2002. CEACAM1 regulates insulin clearance in
liver. Nat. Genet. 30:270–276.
52. Qiao, L. Y., J. L. Goldberg, J. C. Russell, and X. J. Sun. 1999. Identification
of enhanced serine kinase activity in insulin resistance. J. Biol. Chem. 274:
53. Ravichandran, L. V., D. L. Esposito, J. Chen, and M. J. Quon. 2001. Protein
kinase C-? phosphorylates insulin receptor substrate-1 and impairs its abil-
ity to activate phosphatidylinositol 3-kinase in response to insulin. J. Biol.
54. Ricort, J. M., J. F. Tanti, E. Van Obberghen, and Y. Marchand-Brustel.
1997. Cross-talk between the platelet-derived growth factor and the insulin
signaling pathways in 3T3-L1 adipocytes. J. Biol. Chem. 272:19814–19818.
55. Rui, L., V. Aguirre, J. K. Kim, G. I. Shulman, A. Lee, A. Corbould, A. Dunaif,
and M. F. White. 2001. Insulin/IGF-1 and TNF-? stimulate phosphorylation
of IRS-1 at inhibitory Ser307 via distinct pathways. J. Clin. Investig. 107:
56. Sadava, D., D. Alonso, H. Hong, and D. P. Pettit-Barrett. 1997. Effect of
methadone addiction on glucose metabolism in rats. Gen. Pharmacol. 28:
57. Sajan, M. P., M. L. Standaert, G. Bandyopadhyay, M. J. Quon, T. R. Burke,
Jr., and R. V. Farese. 1999. Protein kinase C-? and phosphoinositide-depen-
dent protein kinase-1 are required for insulin-induced activation of ERK in
rat adipocytes. J. Biol. Chem. 274:30495–30500.
58. Saltiel, A. R., and C. R. Kahn. 2001. Insulin signalling and the regulation of
glucose and lipid metabolism. Nature 414:799–806.
59. Sasaoka, T., and M. Kobayashi. 2000. The functional significance of Shc in
insulin signaling as a substrate of the insulin receptor. Endocr. J. 47:373–381.
60. Schmidt, H., S. Schulz, M. Klutzny, T. Koch, M. Handel, and V. Hollt. 2000.
Involvement of mitogen-activated protein kinase in agonist-induced phos-
phorylation of the mu-opioid receptor in HEK 293 cells. J. Neurochem.
61. Shimomura, Y., J. Oku, Z. Glick, and G. A. Bray. 1982. Opiate receptors,
food intake and obesity. Physiol. Behav. 28:441–445.
62. Skolnik, E. Y., A. Batzer, N. Li, C. H. Lee, E. Lowenstein, M. Mohammadi,
B. Margolis, and J. Schlessinger. 1993. The function of GRB2 in linking the
insulin receptor to Ras signaling pathways. Science 260:1953–1955.
63. Skolnik, E. Y., C. H. Lee, A. Batzer, L. M. Vicentini, M. Zhou, R. Daly, M. J.
Myers, Jr., J. M. Backer, A. Ullrich, and M. F. White. 1993. The SH2/SH3
domain-containing protein GRB2 interacts with tyrosine-phosphorylated
IRS1 and Shc: implications for insulin control of ras signalling. EMBO J.
64. Staubs, P. A., J. G. Nelson, D. R. Reichart, and J. M. Olefsky. 1998. Platelet-
derived growth factor inhibits insulin stimulation of insulin receptor sub-
strate-1-associated phosphatidylinositol 3-kinase in 3T3-L1 adipocytes with-
out affecting glucose transport. J. Biol. Chem. 273:25139–25147.
65. Strack, V., A. M. Hennige, J. Krutzfeldt, B. Bossenmaier, H. H. Klein, M.
Kellerer, R. Lammers, and H. U. Haring. 2000. Serine residues 994 and
1023/25 are important for insulin receptor kinase inhibition by protein kinase
C isoforms ?2 and ?. Diabetologia 43:443–449.
66. Summers, S. A., V. P. Yin, E. L. Whiteman, L. A. Garza, H. Cho, R. L. Tuttle,
and M. J. Birnbaum. 1999. Signaling pathways mediating insulin-stimulated
glucose transport. Ann. N. Y. Acad. Sci. 892:169–186.
67. Sun, X. J., M. Miralpeix, M. G. Myers, Jr., E. M. Glasheen, J. M. Backer,
C. R. Kahn, and M. F. White. 1992. Expression and function of IRS-1 in
insulin signal transmission. J. Biol. Chem. 267:22662–22672.
68. Velloso, L. A., F. Folli, X. J. Sun, M. F. White, M. J. Saad, and C. R. Kahn.
1996. Cross-talk between the insulin and angiotensin signaling systems. Proc.
Natl. Acad. Sci. USA 93:12490–12495.
69. Verspohl, E. J., U. Berger, and H. P. Ammon. 1986. The significance of mu-
and delta-receptors in rat pancreatic islets for the opioid-mediated insulin
release. Biochim. Biophys. Acta. 888:217–224.
70. Whistler, J. L., H. H. Chuang, P. Chu, L. Y. Jan, and M. von Zastrow. 1999.
Functional dissociation of mu opioid receptor signaling and endocytosis:
implications for the biology of opiate tolerance and addiction. Neuron 23:
71. White, M. F. 1998. The IRS-signalling system: a network of docking proteins
that mediate insulin action. Mol. Cell. Biochem. 182:3–11.
72. Wolf, G., T. Trub, E. Ottinger, L. Groninga, A. Lynch, M. F. White, M.
Miyazaki, J. Lee, and S. E. Shoelson. 1995. PTB domains of IRS-1 and Shc
have distinct but overlapping binding specificities. J. Biol. Chem. 270:27407–
73. Xu, G. G., and P. L. Rothenberg. 1998. Insulin receptor signaling in the
beta-cell influences insulin gene expression and insulin content: evidence for
autocrine beta-cell regulation. Diabetes 47:1243–1252.
74. Zhande, R., J. J. Mitchell, J. Wu, and X. J. Sun. 2002. Molecular mechanism
of insulin-induced degradation of insulin receptor substrate 1. Mol. Cell.
75. Zhang, H., X. Zha, Y. Tan, P. V. Hornbeck, A. J. Mastrangelo, D. R. Alessi,
R. D. Polakiewicz, and M. J. Comb. 2002. Phosphoprotein analysis using
antibodies broadly reactive against phosphorylated motifs. J. Biol. Chem.
76. Zheng, W. H., S. Kar, and R. Quirion. 2002. Insulin-like growth factor-1-
induced phosphorylation of transcription factor FKHRL1 is mediated by
phosphatidylinositol 3-kinase/Akt kinase and role of this pathway in insulin-
like growth factor-1-induced survival of cultured hippocampal neurons. Mol.
77. Zick, Y. 2001. Insulin resistance: a phosphorylation-based uncoupling of
insulin signaling. Trends Cell Biol. 11:437–441.
6266 LI ET AL.MOL. CELL. BIOL.