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Endocrinology 144(4):1211–1218
Copyright © 2003 by The Endocrine Society
doi: 10.1210/en.2002-220723
Insulin Receptor Substrate-4 Is Expressed in Muscle
Tissue without Acting as a Substrate for the
Insulin Receptor
SYLVIA SCHREYER, DANIELA LEDWIG, IRINI RAKATZI, INGRID KLÖTING,
AND
JÜRGEN ECKEL
Molecular Cardiology (S.S., D.L., I.R., J.E.), Department of Clinical Biochemistry and Pathobiochemistry, German Diabetes
Research Institute, D-40225 Düsseldorf, Germany; and Institute of Pathophysiology (I.K.), University of Greifswald, D-17495
Karlsburg, Germany
Insulin receptor substrate (IRS) proteins represent key elements of the insulin-signaling cascade. IRS-4 is the most recently characterized member of the IRS family with an undefined in vivo function. In contrast to IRS-1 and IRS-2, IRS-4
exhibits a limited tissue expression, and IRS-4 protein has not
been detected in any mouse or primary human tissue so far.
The purpose of the present study was to analyze the expression of IRS-4 in rat muscle and human skeletal muscle cells
and assess involvement of IRS-4 in initial insulin signaling.
Using immunoblotting and immunoprecipitation, the specific
expression of IRS-4 protein could be demonstrated in rat soleus and cardiac muscle and human skeletal muscle cells, but
it was not significantly detectable in quadriceps and gastrocnemius. A prominent down-regulation of IRS-4 was observed
I
NSULIN RECEPTOR SUBSTRATE (IRS) proteins exert
key functions as signaling intermediates for the insulin
and the IGF-1 receptor (1–2). This involves the tyrosine phosphorylation of IRS proteins within short motifs that bind to
Src homology 2 domains of intracellular signaling proteins,
including phosphatidylinositol 3-kinase (3), growth factor
receptor-binding protein 2 (4), and the protein tyrosine phosphatase SHP-2/Syp (5), finally activating specific signaling
cascades. So far, four members of the IRS family (IRS-1, -2,
-3, and -4) have been identified with a similar general architecture and domain structure (6). Despite an apparent
functional redundancy in the IRS family, these proteins are
thought to play distinct roles in mediating insulin action.
This is based on their specific tissue and cellular expression
(7) and the unique structural features of each IRS protein,
which affect the interaction with upstream and downstream
proteins (8, 9).
IRS-4 is the most recently characterized member of the IRS
family that was initially detected in human embryonic kidney (HEK) 293 cells (10, 11). In vitro studies have shown that
IRS-4 binds to phosphatidylinositol 3-kinase and growth factor receptor-binding protein 2 (11) and that overexpression
of IRS-4 in rat adipocytes leads to the translocation of GLUT4
to the cell surface (12). However, IRS-4 has a different signaling capacity, compared with the other IRS proteins. Thus,
IRS-4 does not interact with SHP2 (11) and overexpression in
Abbreviations: ECL, Enhanced chemiluminescence; HEK, human
embryonic kidney; HRP, horseradish peroxidase; IRS, insulin receptor
substrate; RIPA, radioimmunoprecipitation assay; WOKW, Wistar Ottawa Karlsburg.
in heart and soleus muscle of WOKW rats, an animal model of
the metabolic syndrome. In human skeletal muscle cells, both
IRS-1 and IRS-2 are rapidly phosphorylated on tyrosine in
response to insulin, whereas essentially no tyrosine phosphorylation of IRS-4 was observed in response to both insulin
and IGF-I. Instead, a 2-fold increase in IRS-4 tyrosine phosphorylation was observed in myocytes subjected to osmotic
stress. In conclusion, IRS-4 protein is expressed in heart and
skeletal muscle in a fiber type specific fashion. Our data suggest that IRS-4 does not function as a substrate of the insulin
and the IGF-I receptor in primary muscle cells but may be
involved in nonreceptor tyrosine kinase signaling. (Endocrinology 144: 1211–1218, 2003)
32D cells failed to promote cell survival, in sharp contrast to
IRS-1 and IRS-2 (13). Furthermore, Tsuruzoe et al. (14) recently suggested that IRS-3 and IRS-4 may even act as negative regulators of the IGF-1 signaling pathway by suppressing the function of other IRS proteins.
In contrast to the in vitro studies, the in vivo function of
IRS-4 has remained elusive. Interestingly, IRS-4 exhibits a
more limited tissue expression, and it was not possible to
detect IRS-4 protein in any mouse tissue so far (15). This may
explain the absence of a discernible phenotype in knockout
mice lacking IRS-4 (16). However, mRNA expression of IRS-4
was recently demonstrated in different human and rodent
tissues including heart and skeletal muscle (17). In the
present study, we assessed the protein expression of IRS-4 in
rat muscle of different fiber type composition and in human
skeletal muscle cells. The data show specific expression of
IRS-4 in soleus and cardiac muscle with a prominent downregulation of IRS-4 expression in a rat model of the metabolic
syndrome. IRS-4 is not phosphorylated in response to insulin
and IGF-I in primary muscle cells but appears to be involved
in stress-induced cellular signaling.
Materials and Methods
Chemicals
Reagents for SDS-PAGE were supplied by Amersham Pharmacia
Biotech (Braunschweig, Germany) and Sigma (München, Germany).
BSA (fraction V, fatty acid free) was obtained from Boehringer (Mannheim, Germany), and protein A trisacryl beads were a product from
Pierce Chemical Co. (Rockford, IL). Polyclonal anti-IRS-1 and anti-IRS-2
antiserum were gifts from Dr. J. A. Maassen (Leiden, The Netherlands).
The polyclonal rabbit anti-IRS-4 antibody and the immunizing peptide
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Endocrinology, April 2003, 144(4):1211–1218
(residues 1240 –1257 of human IRS-4) were provided by Upstate Biotechnology (Lake Placid, NY). The antiphosphotyrosine antibody (RC20) coupled to horseradish peroxidase (HRP) was from Transduction
Laboratories, Inc. (Lexington, KY). HRP-conjugated goat-antirabbit IgG
antibody as secondary antibody for enhanced chemiluminescence (ECL)
detection was from Promega Corp. (Mannheim, Germany). Primary
human skeletal muscle cells and supplement pack for growth medium
were obtained from PromoCell (Heidelberg, Germany). Culture media
were purchased from Life Technologies, Inc. (Berlin, Germany). All
other chemicals were of the highest analytical grade commercially available and were purchased from Sigma.
Cell culture
HEK 293 cells were grown on 10-cm plates in DMEM supplemented
with 10% fetal calf serum and streptomycin/penicillin. Cells were used
upon reaching 80% confluence and were preincubated in serum-free
medium for 2 h before stimulation with insulin.
Cardiomyocytes from adult rat heart of normal Wistar rats were
isolated by perfusion of the heart with collagenase, as described in our
earlier reports (18, 19). The final cell suspension was centrifuged and
stored in liquid nitrogen until further use.
Primary human skeletal muscle cells obtained from satellite cells
isolated from M. rectus abdominis of healthy Caucasian donors were
supplied as proliferating myoblasts. Cells were cultured in ␣-modified
Eagle’s/Ham’s F-12 medium containing skeletal muscle cell growth
medium supplement pack up to near confluence as recently described
by us (20). For stimulation studies, 106 cells/dish were plated in growth
medium and were cultured for 5– 6 d. Myoblasts were then washed with
PBS and incubated for 24 h in the absence of serum before stimulation
with insulin.
Preparation of tissues
Male Wistar rats weighing 280 –320 g were used throughout the
experiments. All animals had free access to food and drinking water, and
all animal experimentation was conducted in accord with accepted
standards of humane animal care. Animals were killed by decapitation
Schreyer et al. • IRS-4 in Skeletal and Cardiac Muscle
and the heart and the gastrocnemius, quadriceps, and soleus muscle
were removed (21). In parallel, the brain of the animal was rapidly
excised and the hypothalamus was dissected, as detailed recently by
us (22).
In some experiments, male obese Wistar Ottawa Karlsburg (WOKW)
rats (23), at an age of 24 –28 wk and weighing 400 – 450 g, and agematched Wistar controls were used. Cardiac and soleus muscle of these
animals was removed as outlined above.
Immunoprecipitation
Cells were washed twice with ice-cold PBS and lysis was performed
by incubation in radioimmunoprecipitation assay (RIPA) lysis buffer (50
mm Tris-HCL, pH 7.4; 1% Nonidet P-40; 0.25% sodium-deoxycholate;
150 mm NaCl; 1 mm EDTA; 1 mm Na3Vo4; 1 mm NaF; and protease
inhibitor cocktail) for 2 h at 4 C with gentle agitation. The suspension
was centrifuged at 10,000 ⫻ g for 20 min and the supernatant (800 ␮l at
1 ␮g protein per microliter, if not otherwise indicated) was then incubated with antibodies (5 ␮l) against IRS-1, IRS-2, or IRS-4 at 4 C and
gently rocked overnight. The immunocomplexes were adsorbed to protein A-Sepharose beads for 2 h at 4 C during gentle agitation and
subsequently collected by centrifugation at 14,000 rpm for 30 sec at 4 C.
Beads were then washed three times with ice-cold PBS, incubated for 10
min at 95 C with 20 ␮l electrophoresis buffer, and the complete supernatant was used for Western blot analysis.
Western blotting
Tissues were lysed in RIPA buffer except for brain, which was lysed
in a buffer consisting of 50 mm Tris/HCl (pH 7.5), 150 mm NaCl, 1%
Triton X-100, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 2 mm
sodium orthovanadate, 100 mm NaF, 1 mm EDTA, and protease inhibitor cocktail. The tissues (200 mg) were homogenized in 2 ml lysis buffer
(10% wt/vol) using Ultra-Turrax tissue homogenizer (Jahnke Kunkel,
Staufen, Germany). Lysates were cleared by centrifugation at 10,000 rpm
for 20 min at 4 C. Protein determination of the supernatant was performed by the Bradford method using a protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Immunoprecipitates or total lysates were
FIG. 1. Western blot analysis of IRS-4 expression in HEK 293 cells and rat skeletal muscle. A, HEK 293 cells were cultured as described in
Materials and Methods and were lysed in RIPA buffer. One microgram cell lysate from three different cell batches (a– c) were resolved by
SDS-PAGE and immunoblotted for IRS-4. IRS-4 antibody blocked with the immunizing peptide was used as a specificity control. IRS-4 was
immunoprecipitated (IP) as described in Materials and Methods, and immunopellets were subjected to immunoblotting (ID) for IRS-4. Bands
were detected using an ECL system and LUMI imager analyzer. Representative experiments of five replicate experiments are shown. B–D,
Skeletal muscle samples from four individual rats (a– d) was lysed and subjected to immunoblotting for IRS-4 as outlined above, except using
20 ␮g tissue lysate per lane.
Schreyer et al. • IRS-4 in Skeletal and Cardiac Muscle
Endocrinology, April 2003, 144(4):1211–1218 1213
separated by SDS-PAGE using 7.5% horizontal gels and transferred to
polyvinylidene fluoride filters in a semidry blotting apparatus (24). For
phosphotyrosine detection, filters were blocked 60 min in Tris-buffered
saline containing 0.05% Tween 20 and 1% BSA. Thereafter filters were
incubated overnight with antiphosphotyrosine antibody (RC-20) coupled to HRP and subsequently processed for ECL detection using SuperSignal substrate (Pierce Chemical Co.). For detection of IRS-4, filters
were blocked with Tris-buffered saline containing 0.05% Tween 20 and
10% nonfat dry milk and incubated overnight with 1 ␮g/ml antibody.
Blocking of the IRS-4 antibody was achieved by preincubation of
1 ␮g/ml antibody with 10 ␮m immunizing peptide for 30 min at 4 C,
followed by immediate use for immunodetection. After extensive washing, filters were incubated with goat-antirabbit HRP-coupled antibody
and processed for ECL detection. Signals were visualized and evaluated
on a LUMI Imager workstation using image analysis software (Roche
Molecular Biochemicals, Mannheim, Germany).
Statistical analysis
All data analysis was performed using Prism (GraphPad Software,
Inc., San Diego, CA) or t-ease (ISI, Philadelphia, PA) statistical software.
Significance of reported differences was evaluated by using the null
hypothesis and t statistics for paired data. A P value less than 0.05 was
considered to be statistically significant.
Results
IRS-4 is expressed in rat skeletal and cardiac muscle
In the present study, we used an antiserum raised against
the carboxyl-terminal 18 amino acids of human IRS-4, somewhat different from the antisera used in earlier studies (11,
25). As shown in Fig. 1, the antiserum detects a prominent
160-kDa protein band in HEK 293 cell lysates that is completely absent when probing the blots with IRS-4 antiserum
blocked with the immunizing peptide. Furthermore, immunoprecipitation of IRS-4 from HEK cell lysates revealed the
presence of one protein band at 160 kDa (Fig. 1), most probably representing IRS-4. So far, rat IRS-4 has not been cloned;
however, the peptide used for immunization has 44% homology with murine IRS-4 (15). Lysates of rat skeletal muscle
with different fiber-type composition were probed with the
IRS-4 antiserum using the blocked antiserum as a specificity
control. As presented in Fig. 1, IRS-4 protein was specifically
detected in red soleus muscle at a molecular mass of 150 kDa.
In quadriceps and gastrocnemius, two muscles with mixed
fiber-type composition, a faint band at 150 kDa was observed, potentially representing IRS-4. It should be noted
that 20 ␮g skeletal muscle lysates were used for immunoblotting vs. only 1 ␮g HEK cell lysates.
Specific expression of IRS-4 protein was also observed in
cardiac muscle and isolated cardiomyocytes (Fig. 2A). Again,
the protein appeared as a 150-kDa protein band. Direct comparison between heart and soleus muscle showed a significantly higher (2- to 3-fold) abundance of IRS-4 in the heart
(Fig. 2B). IRS-4 protein was also detected in a preparation of
hypothalamic tissue that contains the leptin receptor and
exhibits leptin-stimulated signal transducer and activator of
transcription 3 phosphorylation (22), but it was absent from
the cerebellum (Fig. 3, lane c). These data confirm the specificity of our IRS-4 protein detection assay and are in excellent
agreement with a recent report by Numan and Russell (26)
showing that IRS-4 expression in the rat brain is nearly completely restricted to the hypothalamus.
FIG. 2. Protein expression of IRS-4 in cardiac and soleus muscle. A,
Hearts were removed from individual rats (a– d), and tissue lysates
(20 ␮g per lane) were subjected to immunoblotting for IRS-4, as
outlined in Fig. 1. Cardiomyocytes were prepared from adult rat as
described in Materials and Methods. Lysates were then immunoblotted for IRS-4. B, Heart and soleus muscle lysates were resolved by
SDS-PAGE within the same gel and were immunoblotted for IRS-4.
Signal intensities were quantified by LUMI imager software and are
expressed as arbitrary units. Data are mean values of four different
animals ⫾ SD. *, Significantly different at P ⬍ 0.005.
FIG. 3. Protein expression of IRS-4 in rat brain. Dissection of the
hypothalamus was performed as described in Materials and Methods.
Twenty micrograms hypothalamic lysates (a, b, d, e) or cerebellum (c)
were then immunoblotted for IRS-4, as outlined in Fig. 1. A representative experiment of three is shown.
IRS-4 expression is reduced in heart and soleus muscle of
WOKW rats
Recent studies have shown that WOKW rats develop a
nearly complete metabolic syndrome with obesity, hypertension, dyslipidemia, hyperinsulinemia, and impaired glucose tolerance (23, 27). Furthermore, development of the
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Endocrinology, April 2003, 144(4):1211–1218
Schreyer et al. • IRS-4 in Skeletal and Cardiac Muscle
FIG. 4. Expression of IRS proteins in heart and soleus muscle of WOKW rats. Heart and soleus muscle of WOKW and Wistar rats was removed,
tissues were lysed, and immunoblotted (20 ␮g per lane) for IRS-4 (A), IRS-1 (B), and IRS-2 (C). Tissues from individual animals are marked
a– d. Signal intensities were quantified by LUMI imager software and are expressed as arbitrary units. Data are mean values of four different
animals ⫾ SD. *, Significantly different at P ⬍ 0.05; **, significantly different at P ⬍ 0.005.
Schreyer et al. • IRS-4 in Skeletal and Cardiac Muscle
Endocrinology, April 2003, 144(4):1211–1218 1215
syndrome in the WOKW rat is under polygenic control (28).
We have now assessed the expression of IRS-4 in heart and
skeletal muscle of these animals and compared it with normal Wistar rats. As shown for Wistar rats (see Figs. 1 and 2),
IRS-4 was present only in soleus and cardiac muscle but was
not specifically detectable in quadriceps and gastrocnemius
(data not shown). The direct comparison of IRS-4 expression
in Wistar and WOKW rats is presented in Fig. 4A. Cardiac
IRS-4 abundance was moderately (about 50%) but significantly reduced in WOKW rats. A very prominent (80%)
reduction of IRS-4 expression was observed in soleus muscle
(Fig. 4A). For comparison we also assessed expression of
IRS-1 (Fig. 4B) and IRS-2 (Fig. 4C) in heart and soleus muscle
of WOKW rats. In contrast to IRS-4, IRS-1 expression remained unaltered in the heart, whereas IRS-2 expression was
not affected in both tissues.
IRS-4 is expressed in human skeletal muscle cells but does
not act as a substrate for insulin and IGF-I receptors
Attempts were then made to study the phosphorylation of
IRS-4 in response to insulin in the cardiomyocytes. Unfortunately, we were unable to immunoprecipitate IRS-4 from
rat cardiomyocytes and cardiac muscle using the antiserum
generated against the carboxy-terminal peptide of human
IRS-4. Furthermore, immunoprecipitates with an antiphosphotyrosine antibody after insulin stimulation did not contain IRS-4 (data not shown). We therefore decided to use
primary human skeletal muscle cells to test involvement of
IRS-4 in insulin signaling. As can be seen from Fig. 5, IRS-4
was immunoprecipitated from the myoblasts; however, we
were unable to detect any tyrosine phosphorylation of IRS-4
after different times of insulin exposure. In contrast, tyrosine
phosphorylation of IRS-4 in response to insulin was clearly
detectable in HEK 293 cells (Fig. 5).
To provide additional evidence for the inability of IRS-4 to
function as a substrate for the insulin receptor in human
skeletal muscle cells, we immunoprecipitated IRS-1, -2, and
-4 from basal and insulin-stimulated cells followed by immunoblotting for phosphotyrosine (Fig. 6). Both IRS-1 and
IRS-2 show a prominent tyrosine phosphorylation in response to insulin. However, the tyrosine phosphorylation of
IRS-4 remained undetectable under these conditions, despite
being expressed in the human skeletal muscle cells (Fig. 6).
Instead of the insulin receptor, IGF-I receptor signaling
may involve tyrosine phosphorylation of IRS-4 (25). Therefore, IRS-4 was immunoprecipitated from human myocytes
and HEK 293 cells treated with IGF-I or insulin followed by
immunoblotting for phosphotyrosine (Fig. 7). In HEK cells
IGF-I was equipotent to insulin resulting in a 4-fold increase
in IRS-4 tyrosine phosphorylation, in agreement with the
results of Fantin et al. (11). In the myocytes IGF-I even produced a dephosphorylation of IRS-4 (Fig. 7). By subjecting
the cells to osmotic shock, we then tested the possibility that
tyrosine kinases different from the insulin and IGF-I receptor
might use IRS-4 as a substrate for downstream signaling. As
presented in Fig. 7, osmotic stress produced a 2-fold increase
in IRS-4 tyrosine phosphorylation in the human skeletal muscle cells without being effective in HEK 293 cells.
FIG. 5. Effect of insulin on the tyrosine phosphorylation of IRS-4. A,
Human skeletal muscle cells were cultured as detailed in Methods.
After preincubation under serum-free conditions, the cells were stimulated with insulin (100 nM) for the indicated times followed by cell
lysis and immunoprecipitation (IP) (800 ␮g protein) of IRS-4. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted (ID)
with anti-IRS-4 and antiphosphotyrosine antibodies, respectively.
Signals were visualized using ECL detection. B, HEK cells were
stimulated with the indicated concentrations of insulin for 10 min
followed by cell lysis, immunoprecipitation (of 100 ␮g protein), and
immunoblotting as outlined above. Representative blots of four replicate experiments are shown.
Discussion
IRS-4 was initially identified in the HEK 293 cell line in
which it is expressed at high excess over IRS-1 and IRS-2 (11).
To date, IRS-4 protein has been detected only in these cells
and some human breast cancer cell lines (15). However, IRS-4
mRNA is expressed in a variety of human and rodent tissues
including pituitary, thyroid, ovary, prostate, hypothalamus,
liver, heart, and skeletal muscle (29). We now report the
detection of IRS-4 protein in rat skeletal and cardiac muscle
and primary human skeletal muscle cells using an antiserum
against the carboxyl-terminal 18 amino acids of human IRS-4.
Three lines of evidence support the specificity of IRS-4 detection using this antiserum: cross-reactivity with IRS-1/2
was excluded (Fig. 6); a single protein band at 160 kDa was
readily detected in HEK cells (Fig. 1); and blocking the antiserum with the immunizing peptide completely abolished
IRS-4 detection in soleus and cardiac muscle (Figs. 1 and 2).
IRS-4 could not be significantly detected in quadriceps and
gastrocnemius, two muscles with a mixed fiber-type composition. This finding agrees with the data of Fantin et al. (15),
who were unable to detect IRS-4 protein in mouse quadri-
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Endocrinology, April 2003, 144(4):1211–1218
Schreyer et al. • IRS-4 in Skeletal and Cardiac Muscle
FIG. 6. Tyrosine phosphorylation of IRS family members in human skeletal muscle cells in response to insulin. Human skeletal muscle cells
were stimulated with insulin (100 nM) for 2.5 min followed by cell lysis and immunoprecipitation (of 800 ␮g protein) of IRS-1, IRS-2, and IRS-4.
Immunoprecipitates were immunoblotted with antiphosphotyrosine and the corresponding IRS-antibodies, respectively. Representative blots
of three replicate experiments are shown.
FIG. 7. Tyrosine phosphorylation of IRS-4 in response to IGF-I and osmotic shock. Human skeletal muscle cells (left panel) or HEK 293 cells
(right panel) were stimulated for 10 min with insulin (100 nM), IGF-I (70 nM), or osmotic shock (500 mM mannitol) followed by cell lysis and
immunoprecipitation (800 ␮g protein) of IRS-4. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antiphosphotyrosine
and anti-IRS-4 antibodies, respectively. Representative blots of three replicate experiments are shown. Signal intensities were quantified by
LUMI imager software and are expressed as arbitrary units. Data are mean values of three different experiments ⫾ SD. *, Significantly different
at P ⬍ 0.05; **, significantly different at P ⬍ 0.005.
ceps. However, in contrast to our work, these authors were
also unable to detect IRS-4 in the heart (15). The reason for
this discrepancy remains unclear but may be related to the
different antibodies used and/or the difference between the
rat and the mouse.
IRS-1 and IRS-2 exhibit a widespread tissue distribution
(17) and are expressed at the protein level in both red and
white muscle (30, 31). We report here that IRS-4 protein is
preferably detectable in oxidative muscle with the highest
level in the heart. Oxidative red muscles are rich in type I
Schreyer et al. • IRS-4 in Skeletal and Cardiac Muscle
fibers and have a higher content of GLUT4 (32) and a higher
insulin sensitivity (33). Specific expression of IRS-4 in red
soleus and cardiac muscle may suggest the potential involvement of IRS-4 in the regulation of GLUT4 translocation in
these tissues, in agreement with the data by Zhou et al. (12)
showing that overexpression of IRS-4 induces GLUT4 translocation. Unfortunately, we were not able to assess insulin
signaling to IRS-4 in rat tissues. However, our results obtained in primary human skeletal muscle cells show that
IRS-4 does not function as a substrate for the insulin and
IGF-I receptor kinase. Interestingly, a prominent downregulation of IRS-4 protein in cardiac and skeletal muscle was
observed in WOKW rats, an animal model of obesity and
insulin resistance (23, 27). On the other hand, IRS-1 was
affected only in skeletal muscle in these animals with IRS-2
remaining unaltered, demonstrating a highly specific metabolic regulation of different IRS proteins. It must be kept in
mind, however, that IRS-4-null mice have only mild defects
in growth, reproduction, and glucose homeostasis (16). Future work will be needed to define the functional implications of normal and perturbed IRS-4 expression in heart and
skeletal muscle.
A key finding of the present study consists of the observation that IRS-4 protein is expressed in primary human
skeletal muscle cells at a considerable level without acting as
a substrate for the insulin receptor kinase. This observation
is consistent with the absence of insulin resistance in IRS-4null mice (16). A potential explanation may be related to the
relative abundance of IRS-1/2 vs. IRS-4 in muscle tissue.
Thus, the insulin receptor may be saturated with IRS-1
and/or IRS-2 excluding IRS-4 from phosphorylation by the
insulin receptor kinase. The inverse situation was reported
for HEK 293 cells, in which IRS-4 is expressed at a 20- to
30-fold excess over IRS-1/2 (11). In these cells, as confirmed
in the present study, IRS-4 is phosphorylated in response to
insulin, whereas IRS-1 and IRS-2 do not participate in insulin
signaling (11). We therefore concluded that IRS-4 is not a
substrate for the insulin receptor kinase in muscle tissue
under physiological conditions. Instead of the insulin receptor, the IGF-I receptor may represent an interesting candidate
for using IRS-4 for downstream signaling. Qu et al. (25) reported that IRS-4 is implicated in IGF-I receptor mitogenic
signaling in cell lines overexpressing IRS-4 and the IGF-I
receptor. Furthermore, IRS-4-null mice exhibit a mild defect
in growth (16). However, our data obtained with human
myoblasts do not support a role for IRS-4 in IGF-I receptor
signaling in primary muscle tissue.
In contrast to insulin and IGF-I, hyperosmotic shock was
found to induce a prominent tyrosine phosphorylation of
IRS-4 in human skeletal muscle cells. It is well established
that protein tyrosine kinases play a pivotal role in the
signaling of hyperosmotic stress (34 –36), involving both
members and nonmembers of the Src family of tyrosine
kinases (36). Our results suggest that IRS-4 may be activated by nonreceptor tyrosine kinases and may be involved in stress-induced signaling in human skeletal muscle. Further work will be needed to identify the protein
kinase mediating this effect. Interestingly, insulin-mimetic
signaling to glucose transport involving tyrosine phosphorylation of IRS-1/2 by nonreceptor tyrosine kinases
Endocrinology, April 2003, 144(4):1211–1218 1217
like pp59 (Lyn) has recently been reported (37), and this
pathway may also include IRS-4.
In summary, we show here that IRS-4 protein is expressed
in rat cardiac and soleus muscle and primary human skeletal
muscle cells. However, IRS-4 does not function as a substrate
for the insulin and IGF-I receptor, most likely because of
competition with IRS-1 and/or IRS-2. It is suggested that
IRS-4 may exert a physiological role in heart and skeletal
muscle, potentially involving tyrosine kinases different from
the insulin receptor.
Acknowledgments
The secretarial assistance of Birgit Hurow is gratefully acknowledged.
Received July 17, 2002. Accepted December 16, 2002.
Address all correspondence and requests for reprints to: Professor Dr.
Jürgen Eckel, German Diabetes Research Institute, Auf’m Hennekamp
65, D-40225 Düsseldorf, Germany. E-mail: [email protected].
This work was supported by the Ministerium für Wissenschaft und
Forschung des Landes Nordrhein-Westfalen, the Bundesministerium
für Gesundheit, EU COST Action B17, and the Jühling Foundation.
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