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INTRODUCTION |
The IRSs1 play a key
role in signal transduction from the insulin receptor (reviewed in Ref.
1). They are the major intracellular targets for phosphorylation by the
activated insulin receptor tyrosine kinase. In addition, they are also
substrates for the insulin-like growth factor I receptor and for
tyrosine kinases associated with the receptors for growth hormone and
some interleukins and interferons. Four members of the IRS family
(IRS-1, -2, -3, and -4) are now known. Each IRS contains at its N
terminus a pleckstrin homology domain and a phosphotyrosine binding
domain, both of which are required for efficient phosphorylation by the
insulin receptor tyrosine kinase. The large C-terminal portion of each IRS contains many tyrosine phosphorylation sites in motifs that can
associate with SH2 domain-containing proteins, notably PI 3-kinase, the
adaptor protein Grb-2 (complexed with Sos, the guanine nucleotide
exchange factor for Ras), and the tyrosine phosphatase SHP-2. The
interaction of the tyrosine-phosphorylated IRSs with PI 3-kinase and
Grb-2/Sos stimulates their activities, leading to the elevation of PI
3,4,5-trisphosphate and the GTP form of Ras. These in turn activate
protein kinase cascades, resulting in the well established cellular
effects of insulin, including the stimulation of glucose transport,
glycogen synthesis, and protein synthesis and alterations in gene
transcription (reviewed in Refs. 1 and 2).
The occurrence of four members of the IRS family raises the question of
the physiological roles of each. Targeted disruption of the
IRS genes provides an approach to address this question. To
date mice lacking IRS-1 and IRS-2 have been described. Mice deficient
in IRS-1 are growth-retarded and moderately insulin-resistant (3, 4).
Mice lacking IRS-2 develop diabetes early in life due to severe insulin
resistance combined with a failure of the pancreatic
cells to
proliferate (5). From these phenotypes it can be concluded that IRS-1
and 2 play important roles in the regulation of growth and glucose homeostasis.
We recently purified and cloned IRS-3 (6). To determine the
physiological role of IRS-3, we have now generated mice with targeted
disruption of the IRS-3 gene. We report here that
IRS-3-null mice do not exhibit abnormalities in growth or
glucose homeostasis. In adipocytes IRS-3 is a prominent
insulin-elicited Tyr(P) protein that associates with PI 3-kinase and
SHP-2, and weakly with Grb-2 (7). Consequently, we examined insulin
effects in adipocytes from the IRS-3-null mice. Insulin
stimulation of glucose transport was not impaired. Moreover, the
absence of IRS-3 did not lead to detectable increases in the tyrosine
phosphorylation of IRS-1/2 or their association with PI 3-kinase or
SHP-2.
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EXPERIMENTAL PROCEDURES |
Generation of IRS-3-null Mice--
Mouse P1 genomic clones
containing the IRS-3 gene were isolated for us by Genome
Systems Inc, by PCR screening of a P1 library of mouse ES cell genomic
DNA (mouse strain 129) with two primers derived from the rat IRS-3
cDNA. The presence of the IRS-3 gene was confirmed by
Southern blotting of restriction digests with a probe from the rat
cDNA. Subsequently, we subcloned several restriction fragments of
the mouse gene encoding IRS-3 into pBluescript and sequenced the coding
region. In addition, we sequenced the insert of a plasmid that contains
the complete coding cDNA for mouse IRS-3 (expressed sequence tag
number 557145 from Genome Systems, Inc). Comparison of the two
sequences revealed that the coding region of the mouse IRS-3
gene contains only a single small intron of 344 bp near the amino
terminus. The identity of the two sequences in the coding region
definitely established that the P1 clones contained IRS-3.
As we were preparing this information for publication, another group
published the same information (8), and so the details are not
presented here.
In order to construct the targeting vector for disruption of the
IRS-3 gene, two overlapping fragments (a 6.0-kb
SpeI-SpeI and a 9.3-kb
EcoRI-EcoRI fragment; see Fig. 1A), as
well as a larger fragment encompassing these two (16.5-kb
EagI-EagI fragment), were subcloned into
pBluescriptSK+, and mapped for restriction sites. For the targeting
vector, which was assembled in pBluescriptSK+, a 2.9-kb
SpeI-EcoRI fragment containing the entire coding
region of IRS-3 was replaced in opposite orientation by a
1.8-kb fragment containing the neo gene driven by the
phosphoglycerate kinase promoter (PGKneo) and linked to
phosphoglycerate kinase polyadenylation sequences (Fig. 1A).
The neo gene was flanked by a 3.5-kb
SpeI-SpeI fragment and a 2.5-kb
EcoRI-EcoRI fragment derived from the
IRS-3 regions 5' and 3' to the coding region, respectively.
A 2.6-kb thymidine kinase gene under the regulation of the
phosphoglycerate kinase promoter was placed at the 3' end of the
targeting vector.
The targeting vector was linearized with NotI and
electroporated into J1 ES cells. Colonies that were resistant to G418
(Life Technologies, Inc.) and gancyclovir (Syntex) were picked and
expanded as described (9) and screened for homologous recombination by
Southern blotting of EcoRI-digested genomic DNA. Seventeen ES clones out of 177 showed the 8.2-kb fragment characteristic for
disruption of IRS-3 as well as the 9.3-kb fragment from the wild-type allele (see Fig. 1B). Four of these ES clones were
injected into blastocysts derived from C57BL/6 and BALB/c mice in the
facility directed by Dr. Arlene Sharpe (Department of Pathology,
Brigham and Women's Hospital, Boston, MA). One clone yielded highly
chimeric male mice, and these were bred with female wild-type C57BL/6
and BALB/c mice to obtain IRS-3+/
mice. The
mice heterozygous for IRS-3 were mated to obtain
IRS-3-null mice. These and their wild-type littermates were
used in the experiments described in Table I and Figs. 2 and 3. For the
other experiments, wild-type and IRS-3-null mice were
obtained by breeding the IRS-3+/+ and
IRS-3
/
offspring of the heterozygous
breeding pairs, respectively. Unless otherwise stated, experiments were
carried out on mice of the 129 × C57BL/6 background. The mice
were housed under a constant light (6 a.m. to 6 p.m.) and dark
cycle and fed Teklad LM-485 mouse/rat diet (Harlan Teklad).
Mice were genotyped by Southern blotting and also by PCR analysis of
genomic DNA obtained from tail snips. Southern blotting was performed
on EcoRI-digested DNA using a digoxigenin-labeled probe that
was prepared according to the manufacturer's (Roche Molecular
Biochemicals) instructions by PCR amplification of a 0.9-kb
SacI-XbaI fragment located upstream of the 5'
homologous sequence of the targeting vector (Fig. 1A). Wild
type and IRS-3-null mice showed the expected 9.3- and 8.2-kb
fragments, respectively (Fig. 1B). For PCR analysis, the
sense and antisense primers for the IRS-3 gene corresponded
to nucleotides 1559-1581 and 2017-2039, respectively, in the coding
region of IRS-3 (Fig. 1 of Ref. 8), and were expected to
yield a product of 481 bp. The sense and antisense primers for the
neo gene were 5'-AGG ATC TCG TCG TGA CCC ATG-3' and 5'-AAG
GCG ATA GAA GGC GAT GC-3', respectively, and were expected to yield a
233-bp product. As shown in Fig. 1C, PCR
analysis yielded the fragments of the predicted sizes. Results from
both methods of genotyping agreed.

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Fig. 1.
Targeted disruption of the IRS-3
gene. A, maps of the mouse IRS-3
locus, the targeting vector, and the mutant IRS-3 locus.
Restriction enzymes are abbreviated as: E, EcoRI;
S, SpeI. The coding region of IRS-3 is
shown as a hatched box. B, genotyping
of wild-type (+/+), heterozygote (+/ ), and IRS-3-null
( / ) mice by Southern blot analysis. Genomic DNA digested with
EcoRI was hybridized with a probe derived from a sequence
upstream of the 5' IRS-3 homologous recombination region
(indicated in A). The EcoRI fragments of the
wild-type and recombinant alleles, which were at approximately 9.3 and
8.2 kb, respectively, based upon comparison with size markers, are
indicated. C, genotyping of wild-type (+/+), heterozygote
(+/ ), and IRS-3-null mice ( / ) by PCR analysis. Primers
from the IRS-3 and neo genes yielded products of
approximately 480 and 230 bp, respectively, based upon comparison with
size markers.
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Analysis of Blood Glucose and Plasma Insulin Concentrations and
of Glucose Tolerance--
Blood samples were collected from the tails
cut at the tip of either randomly fed or fasted mice. For the former,
samples were taken between 7 and 8 a.m.; for the latter, mice were
fasted from 5 p.m. to 9 a.m., and then samples taken. Blood
glucose was measured with the Precision·G blood glucose testing
system (Medisense). For measurement of plasma insulin, heparinized (10 units/ml) blood samples were spun at 8000 rpm in a microcentrifuge for
10 min at 4 °C, and the supernatant plasma was used in an insulin
radioimmune assay (Linco Research Inc., catalog numbers RI and SRI
13K). For the glucose tolerance test, mice were fasted from 5 p.m.
to 9 a.m., and then an oral glucose bolus of 1 g of
D-glucose/kg of body weight as a solution of 0.25 g/ml was
introduced with a feeding needle. Glucose levels were determined in
blood drops obtained from the tail vein immediately before and at 10, 20, 30, 60, 90, and 120 min after the administration of glucose.
Isolation of Adipocytes and Assay of Glucose
Transport--
Epididymal or parametrial fat pads were removed from
male or female 12-16-week-old mice sacrificed with CO2.
Adipocytes were isolated by collagenase digestion of fat pads (10), as
follows. Fat pads were added to collagenase (type I from Worthington
Biochemical Corp.) (1 mg/ml, 2 mg/g of tissue) that was dissolved in
KRH-BSA (120 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM
KH2PO4, 1.2 mM MgSO4,
20 mM HEPES, 2.5% BSA, 200 nM adenosine, pH
7.4). The fat pads in the collagenase solution were minced with
scissors and incubated in a 37 °C shaking water bath (100 rpm) for
1 h. The adipocytes were filtered from debris through 0.4-mm Nitex
nylon mesh (Tetko) and then washed by flotation with KRH-BSA.
Glucose transport was assayed by the uptake of
[U-14C]D-glucose after the method described
in (11), as follows. The loosely packed cells in KRH-BSA were diluted
10-fold to give an approximate 5% cell suspension. Aliquots of the
cell suspension (100 µl) were added to 350 µl of KRH-BSA containing
insulin (Humulin R, Lilly Corp.) at the stated concentrations and
incubated in a 37 °C shaking water bath (80 rpm) for 30 min. To each
tube 50 µl of 40 µM
[U-14C]D-glucose (ICN) in KRH-BSA (0.6 µCi/aliquot) was added, and the incubation continued for another 30 min. Glucose uptake was terminated by separating the medium from the
cells in a 200-µl aliquot from each assay tube by centrifugation
through 200 µl of dinonyl phthalate. The cell-associated
radioactivity was determined by scintillation counting. Nonspecific
association of radioactive glucose with cells was assessed by
performing the assay in the presence of 15 µM
cytochalasin B. For each condition the measurements were done in quadruplicate.
Measurements of adipocyte number, lipid content, and relative GLUT4
content were also made with aliquots of the adipocyte preparations used
for the transport assays. For determination of the cell number,
adipocytes were fixed with osmium tetroxide and counted in a Coulter
counter, as described in (12). For the determination of the lipid
content, total lipids were extracted and weighed, according to Ref. 13,
as follows. A 200-µl aliquot of the 5% cell suspension was mixed
with 2.7 ml of 40:10:1 isopropanol/heptane/1 N
H2SO4, followed by the addition of 1.8 ml of
heptane and 1.0 ml of water. The mixture was vortexed and centrifuged
briefly. A 1.0-ml aliquot of the organic layer was evaporated, and the lipid weighed. For the determination of the GLUT4 content, 200 µl of
the 5% cell suspension was spun through dinonyl phthalate to remove
the medium. The isolated adipocytes were solubilized in 100 µl of SDS
sample buffer and immunoblotted with antibodies against the C-terminal
peptide of GLUT4 (14) at 5 µg/ml, as described below. These three
analyses were performed in quadruplicate.
Preparation of Adipocyte Lysates--
Adipocytes were isolated
as described above, except that the buffer was KRBH-BSA (120 mM NaCl, 4 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2,
10 mM NaHCO3, 30 mM HEPES, 1% BSA,
3 mM D-glucose, and 200 nM
adenosine, pH 7.4). The cells were washed with KRBH to remove the
albumin, diluted in KRBH to give an approximately 10% cell suspension,
and then treated with or without 500 nM insulin (Humulin R)
for 5 min at 37 °C. Adipocytes were centrifuged at 80 × g for 1 min to obtain a packed cell suspension. The
infranatant was removed, and the cells were lysed in the following
buffers (approximately 2 ml/ml of packed cells) to yield a final
protein concentration of 1-2 mg/ml. When samples were used directly
for SDS-gel electrophoresis and immunoblotting, the lysis buffer
contained 1% SDS, 20 mM dithiothreitol, 2 mM
EDTA, 40 mM HEPES, 150 mM NaCl, pH 7.5, with
phosphatase inhibitors (10 mM sodium pyrophosphate, 10 mM NaF, 10 mM Na3VO4)
and protease inhibitors (10 µg/ml aprotinin, 10 µM
leupeptin, 10 µM EP475, 1 µM pepstatin A).
Samples were held at 100 °C for 5 min, and the DNA was sheared with
a 23-gauge needle. When samples were used for immunoprecipitation, the
SDS in the above lysis buffer was replaced with 1% nonaethylene glycol dodecyl ether (Thesit, Roche Molecular Biochemicals), and
dithiothreitol was omitted. The lysates were centrifuged at 14,000 × g for 10 min, and the supernatant was frozen in liquid
nitrogen and stored at
80 °C until use for immunoprecipitation as
described below. Protein concentration was measured by a precipitating
Lowry assay (15).
Antibodies--
Antibodies against the 85-kDa subunit of PI
3-kinase (catalog number 06-195), IRS-1 (06-248), IRS-2 (06-506), and
Tyr(P) (monoclonal 4G10) were purchased from Upstate Biotechnology,
Inc. Antibodies against SHP-2 (catalog number sc-280) were from Santa
Cruz Biotechnology, and one against Tyr(P) coupled to horseradish
peroxidase (RC20:HRPO) was from Transduction Laboratories.
Immunoprecipitation and Immunoblotting of Adipocyte
Proteins--
Adipocyte lysates (250 µg) at 1 mg/ml were incubated
on ice for 2 h with antibodies against PI 3-kinase (2 µl
antisera) or SHP-2 (5 µg) or as a control with irrelevant rabbit
immunoglobulin (5 µg). Immune complexes were collected onto 20 µl
of protein A-Sepharose for 2 h. Beads were washed twice with the
Thesit-containing lysis buffer. The immunoprecipitated proteins were
solubilized by holding the beads at 100 °C for 5 min in 100 µl SDS
sample buffer (4% SDS, 20 mM dithiothreitol, 10%
glycerol, 90 mM TrisCl, 1 mM EDTA, 0.004%
bromphenol blue, 10 mM Na3VO4, pH
6.8, with the same protease inhibitors as in the lysis buffer).
Proteins in the SDS samples of the adipocyte lysates or
immunoprecitates were separated by gel electrophoresis and transferred to Immobilon P membranes (Millipore) by electroblotting in 25 mM Tris, 192 mM glycine, 20% methanol, 0.01%
SDS. The membranes were blocked with 1% BSA in TBS (20 mM
TrisCl, 150 mM NaCl, pH 7.4) for 30 min and then incubated
in 0.2% BSA, 0.3% Tween 20, plus TBS for 90 min with antibodies
against Tyr(P) (0.5 µg/ml 4G10), IRS-1 (1 µg/ml), IRS-2 (1 µg/ml), SHP-2 (1 µg/ml), or PI 3-kinase (1:1000 dilution of serum).
Membranes were washed with 0.3% Tween 20/TBS and incubated with the
horseradish peroxidase conjugates of goat anti-mouse (Pierce) or goat
anti-rabbit immunoglobulin (Bio-Rad) for 30 min, and reactive proteins
were detected by enhanced chemiluminescence (SuperSignal, Pierce).
Immunoblotting for Tyr(P) with RC20:HRPO was performed according to the
instructions of the manufacturer (Transduction Laboratories).
Statistical Analysis--
For each experiment, unpaired
two-tailed t tests were used to compare the mean values for
the wild-type and the IRS-3-null mice. Differences between
means were taken to be not significant for p values greater
than 0.05. Data displayed in the text are expressed as mean ± S.E.
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RESULTS |
Growth and Development of IRS-3-null Mice--
Mice heterozygous
for targeted disruption of the IRS-3 gene were obtained by
crossing four chimeric male mice with wild-type female mice. From 10 IRS-3+/
breeding pairs, a total of 435 pups
with an average litter size of 8 was obtained. The respective ratios of
IRS-3+/+, IRS-3+/
, and
IRS-3
/
mice were 19.8:54.2:25.9 for the
males and 25.1:47.1:27.8 for the females. Subsequently six breeding
pairs of wild-type and of IRS-3-null mice were set up.
Within the same period, the former produced 252 mice in 35 litters,
while the latter yielded 197 mice in 32 litters; the average litter
sizes were 7 and 6, respectively. These breeding data indicate that the
lack of IRS-3 does not affect fertility or embryonic viability.
To assess the effects of the absence of IRS-3 on growth, groups of
wild-type and IRS-3-null mice were weighed at intervals between the ages of 3 and 30 weeks (Fig.
2). Within this time period, the growth
curves for the wild-type and IRS-3-null female mice
overlapped. For the males, the wild-type mice were slightly heavier
than their IRS-3-null counterparts at 10-30 weeks, but the
difference is not statistically significant. The weights of the major
organs of the mice (brain, lung, liver, heart, kidney, spleen,
pancreas, quadriceps muscle, thymus, interscapular brown fat,
epididymal fat), measured for male mice at 10 months of age, were the
same for the wild-type and IRS-3-null mice (data not shown).
Moreover, histological examination of these tissues did not reveal any
differences in morphology between the wild-type and null mice. Thus, we
conclude that IRS-3-null mice show normal growth and
development.

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Fig. 2.
Growth curves for wild-type and
IRS-3-null mice. Weights were determined for male
and female wild-type ( ) and IRS-3-null ( ) mice at
intervals. For all the groups, each time point presents the weights
from 7-8 mice expressed as mean ± S.E.
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Blood Glucose and Plasma Insulin Levels in IRS-3-null Mice--
To
determine whether glucose homeostasis was affected by IRS-3 deficiency,
the concentrations of blood glucose and plasma insulin in the fed and
fasted state were compared between wild-type and IRS-3-null
mice. For both male and female mice of 9-13 weeks of age, the
genotypes showed no significant difference in the fed and fasted blood
glucose levels or in the fed plasma insulin levels (Table
I). In the case of the fasted plasma
insulin level, the female wild-type and null mice showed no significant
difference, but the male IRS-3-null mice showed a level 60%
that of the wild-type (p = 0.009). These measurements
were repeated on the same groups of male and female mice at 6 months of
age, in order to determine whether any effects of IRS-3 deficiency
develop with age. For both sexes, there were no significant differences
between the wild-type and IRS-3-null mice in any of the four
parameters, including the fasted plasma insulin concentration in the
males. In order to test whether IRS-3 deficiency had an effect in a
different genetic background, the same measurements were carried out on mice with a 129 × BALB/c background. The mice were 6 months of age; for each sex, 6 wild-type and 6 IRS-3-null mice were
examined. For both males and females, no significant differences were
observed between genotypes for each of the four parameters (data not
shown). In conclusion, with the possible exception of insulin in fasted young male mice, levels of blood glucose and plasma insulin in mice
were not altered by IRS-3 deficiency.
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Table I
Blood glucose and plasma insulin levels for wild-type (+/+) and
IRS-3-null ( / ) mice in the fed and fasted state
Measurements were made at the ages of 9-13 weeks and 6 months for both
male and female mice. Values are displayed as mean ± S.E. The
number of mice in each group is indicated in parentheses.
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Glucose Tolerance Tests--
To characterize glucose homeostasis
in the absence of IRS-3 further, the blood glucose concentrations at
various times after an oral glucose bolus were determined for male and
female mice of 10-13 weeks of age (Fig.
3). For the males, there was no
difference in blood glucose levels between wild-type and
IRS-3-null mice. In the case of the females, the
IRS-3-null mice showed slightly greater blood glucose levels
than the wild-type mice at 10 and 20 min; however, the differences were
not statistically significant. In order to determine if IRS-3
deficiency affects glucose tolerance at an older age, glucose tolerance
tests were also performed on male and female mice at 6 months of age.
For each sex, 5 wild-type and 5 IRS-3-null mice were
examined. With both sexes, there was no difference in the response to
the oral glucose load (data not shown). Among the 5 older males of each
genotype, there was one that had developed obesity (weight greater than
34 g; see Fig. 2); these two mice exhibited higher and more
prolonged elevation of the blood glucose level compared with the
non-obese mice. The glucose tolerance test was also carried out with
129 × BALB/c mice of 6 months of age. Six wild-type and 6 IRS-3-null mice of each sex were examined. For both sexes,
no difference between the genotypes was observed (data not shown).
Among the 6 older 129 × BALB/c males of each genotype, there was
a single obese one. Unlike the obese males on the 129 × C57BL/6
background, these mice did not exhibit an impaired glucose tolerance
test.

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Fig. 3.
Glucose tolerance test with fasted wild-type
and IRS-3-null mice. The test was performed on
male and female wild-type ( ) and IRS-3-null mice ( ) at
the age of 10-13 weeks. Data are from 7-9 mice in each group and are
expressed as mean ± S.E.
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Glucose Transport in Isolated Adipocytes--
To determine whether
the lack of IRS-3 affected glucose transport, adipocytes from wild-type
and IRS-3-null mice were compared for their capacity to
transport D-glucose. For adipocytes derived from male
(epididymal) and female (parametrial) fat depots, the values for basal
and maximal insulin-stimulated glucose transport were not significantly
different between genotypes, although the average values for
insulin-stimulated transport in the IRS-3-null mice were
slightly lower (Fig. 4). The values for
the transport rate in Fig. 4 are expressed per milligram of lipid. The
lipid content per adipocyte was the same for the wild-type and
IRS-3-null mice of each sex (Fig. 4, legend). In addition,
the amount of the insulin-responsive glucose transporter GLUT4 per mg
of lipid, as detected by immunoblotting, was the same for the wild-type and IRS-3-null mice of each sex (data not shown). Finally,
the amount of epididymal or parametrial adipose tissue (grams dissected per mouse) was also the same (data not shown). Thus, the transport rates in the wild-type and IRS-3-null adipocytes are also
similar when expressed per cell, per GLUT4, or per total adipose
tissue.

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Fig. 4.
Glucose transport in isolated adipocytes from
wild-type and IRS-3-null mice. Uptake of
[U-14C]D-glucose by isolated adipocytes from
male or female mice is shown for the basal (open
bar) and maximal insulin-stimulated state (10 nM
insulin) (closed bar). Each bar
represents data from five to six independent experiments expressed as
mean ± S.E. The respective -fold stimulation by insulin was
3.4 ± 0.4, 2.7 ± 0.4, 5.4 ± 0.9, and 5.2 ± 0.4 for adipocytes from wild-type and IRS-3-null male, and
wild-type and IRS-3-null female mice. In three or four of
these experiments, the cell number and lipid content of adipocytes from
wild-type and null male and wild-type and null female mice were
measured, and the values were 0.207 ± 0.058, 0.216 ± 0.005, 0.120 ± 0.018, and 0.107 ± 0.010 µg/cell,
respectively.
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The insulin dose that elicited a half-maximal response
(ED50) was determined from three or four independent
experiments in which glucose uptake was measured at 0.03, 0.1, 0.3, and
10 nM insulin for epididymal adipocytes and 0.01, 0.03, 0.1, and 10 nM insulin for parametrial adipocytes. The
ED50 values were 0.064 ± 0.017, 0.032 ± 0.009, 0.034 ± 0.011, and 0.029 ± 0.011 nM for adipocytes from wild-type and IRS-3-null male, and wild-type
and IRS-3-null female mice, respectively. The small
differences between EC50 values are not significant. Thus,
the lack of IRS-3 in adipocytes had no significant effect on glucose
transport, either in terms of the values for basal and maximal
insulin-stimulated transport or the dose of insulin eliciting
half-maximal stimulation.
Insulin-elicited Tyrosine Phosphorylation in Adipocytes--
SDS
lysates of basal and insulin-stimulated adipocytes from wild-type and
IRS-3-null mice were immunoblotted with antibodies against
Tyr(P) (Fig. 5). In adipocytes from
wild-type mice insulin stimulated the tyrosine phosphorylation of
proteins at 160-190, 90, and 60 kDa. These correspond to IRS-1/2, the
subunit of the insulin receptor, and IRS-3, respectively.
Adipocytes from the IRS-3-null mice showed similar
insulin-elicited tyrosine phosphorylation, with the exception that the
60-kDa band was absent. The intensities of the Tyr(P) signals from
IRS-1/2 and the insulin receptor
subunit were the same for
adipocytes from the wild-type and null mice. These results thus confirm
the absence of IRS-3 protein in the IRS-3-null mice, and in
addition show that the absence of IRS-3 did not result in any
detectable compensatory increase in the tyrosine phosphorylation of
IRS-1/2 or the insulin receptor.

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Fig. 5.
Insulin-stimulated tyrosine phosphorylation
in adipocytes. Basal ( ) and insulin-stimulated (+) adipocytes
from wild-type and IRS-3-null female mice were lysed with
SDS-containing buffer and immunoblotted with RC20:HRPO to detect Tyr(P)
proteins. The amount of protein per lane for loads 1 and 1/2 are 120 and 60 µg, respectively. Bands corresponding to phosphorylated
IRS-1/2, insulin receptor subunit (IR), and IRS-3 are
indicated. Molecular size markers in kDa are shown on the
right. Repetition of this experiment with adipocytes from
male mice gave similar results.
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In other experiments we have immunoblotted SDS lysates of basal and
insulin-treated adipocytes from wild-type and IRS-3-null mice for Tyr(P) with a different antibody, the monoclonal 4G10, rather
than the monoclonal conjugate RC20:HRPO presented in Fig. 5. The
results were similar to those shown in Fig. 5, with the exception that
the signal from the phosphorylated
subunit of the insulin receptor
was relatively more intense and the background in the region of IRS-1/2
was stronger (data not shown). We have also measured the relative
amounts of the IRS-1 and IRS-2 proteins by immunoblotting the SDS
lysates for these; there was no difference in amounts between wild-type
and IRS-3-null adipocytes (data not shown). We attempted to
detect the IRS-3 protein in the SDS lysates of wild-type adipocytes by
immunoblotting with affinity-purified antibodies against the C-terminal
portion of mouse IRS-3, which are described in Ref. 7. Unfortunately,
IRS-3 was not detected by this method, probably because of its
relatively low abundance and/or the relative lack of sensitivity of the antibodies.
Association of the IRSs with PI 3-Kinase and SHP-2 in
Adipocytes--
It has previously been found that insulin treatment of
adipocytes causes the association of PI 3-kinase with IRS-1, -2, and -3 and the association of SHP-2 with IRS-1 and -3 (7, 16, 17). It was
therefore possible that an increased association of IRS-1 and/or -2 with these enzymes might occur as a compensation for IRS-3 deficiency.
To examine this possibility, PI 3-kinase and SHP-2 were
immunoprecipitated from lysates of basal and insulin-treated wild-type
and IRS-3-null adipocytes and immunoblotted for associated IRSs with antibodies against Tyr(P) (Fig.
6). In the case of the PI 3-kinase
immunoprecipitates, the intensities of the Tyr(P) signal from IRS-1/2
in the samples from insulin-treated wild-type and null adipocytes were
similar. This finding suggests that the absence of IRS-3 did not result
in significant enhanced association of IRS-1/2 with PI 3-kinase. As
expected, no Tyr(P) signal from IRS-3 was present in the PI 3-kinase
immunoprecipitate from insulin-treated IRS-3-null
adipocytes. In the case of the SHP-2 immunoprecipitates, in agreement
with our previous study (7), IRS-3 was the primary IRS associated with
SHP-2 in insulin-treated wild-type adipocytes. The absence of IRS-3 in
the null adipocytes did not lead to increased binding of other Tyr(P)
proteins to this phosphatase. The SHP-2 immunoprecipitates from basal
and insulin-treated cells contained a Tyr(P) protein of approximately
120 kDa, the amount of which was unaffected by insulin treatment or
IRS-3 absence. This is most likely the recently described SHPS-1, a
tyrosine-phosphorylated 120-kDa protein that binds to the SH2
domains of SHP-2 (18).

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Fig. 6.
Association of PI 3-kinase and SHP-2 with
IRSs in adipocytes. Basal ( ) and insulin-stimulated (+)
adipocytes from wild-type and IRS-3-null mice were
immunoprecipitated with antibodies against the 85-kDa subunit of PI
3-kinase (PI3K) or SHP-2 and immunoblotted for Tyr(P) with monoclonal
4G10. Loads 1, 1/2, and 1/4 were immunoprecipitates derived from 250, 125, and 62.5 µg of lysate protein, respectively. Molecular size
markers in kDa are shown on the right. Repetition of these
experiments with adipocytes from mice of the opposite sex gave similar
results. Control immunoprecipitations with irrelevant rabbit
immunoglobulin did not show any bands upon immunoblotting.
Immunoblotting of samples of the lysates taken before and after
immunoprecipitation with each immunoprecipitating antibody showed that
the efficiencies for immunoprecipitation of the 85-kDa subunit and
SHP-2 were approximately 90%.
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DISCUSSION |
Earlier studies presented data to suggest that the absence of
IRS-3 might affect growth and development or glucose transport in
adipocytes and glucose homeostasis. In the case of growth and development, it has been found that IRS-3 mRNA is expressed at day
7 of embryonic life, a time at which neither IRS-1 or IRS-4 mRNA
are expressed (8, 19). The expression of IRS-2 mRNA during
embryonic growth has not been determined. In the case of glucose
transport and glucose homeostasis, a previous study proposed that
approximately half of the insulin stimulation of glucose transport in
adipocytes was due to signaling through IRS-3 (20). This proposal was
based on the findings that for mouse adipocytes lacking IRS-1, insulin
stimulates glucose transport only approximately half as well as in
wild-type adipocytes, and more IRS-3 than IRS-2 immunoprecipitates with
PI 3-kinase, as assessed by Tyr(P) immunoblotting. Moreover, it has
recently been reported that reduction of the GLUT4 content specifically
in adipose tissue in transgenic mice results in impaired glucose
tolerance (21). Thus, reduction in insulin-stimulated glucose transport
in adipose tissue might be expected to have the same effect.
Despite these suggested roles for IRS-3, the IRS-3-null mice
were normal with regard to growth and development. Moreover, glucose
homeostasis, as measured by their fed and fasted blood glucose and
plasma insulin levels and by the oral glucose tolerance test, was not
impaired. Finally, insulin stimulation of glucose transport in
adipocytes was not significantly affected.
In contrast, transgenic mice deficient in IRS-1 or IRS-2 have unique
phenotypes (3-5). Mice lacking IRS-1 are 30-50% smaller than
wild-type mice, but show no other developmental abnormalities. They are
mildly insulin-resistant; the blood glucose concentration is normal,
but the insulin level is approximately twice the normal value. Also,
their blood glucose level does not fall as markedly upon an
intraperitoneal injection of insulin. Mice deficient in IRS-2 are 10%
smaller than normal. They progressively develop severe hyperglycemia at
an early age due to a combination of insulin resistance of the liver
and muscle and a failure of the
cells of the pancreas to
proliferate, which leads to insufficient compensatory insulin
secretion. These phenotypes show that IRS-1 and -2 are essential for
growth and glucose homeostasis and that each has unique functions,
which cannot be fully substituted by another IRS.
The lack of a phenotype for the IRS-3-null mice and the
occurrence of ones for the IRS-1 and -2 null mice may partially be explained in terms of the tissue distributions and relative amounts of
these three IRSs. As assessed by Northern blotting, IRS-1 and -2 are
expressed in all the major mouse tissues (liver, skeletal muscle,
heart, brain, kidney, lung, spleen, testis), whereas among these
tissues, IRS-3 mRNA is undetectable in skeletal muscle, a major
target for insulin action, as well as brain, spleen, and testis (8,
22). Moreover, even in those tissues where IRS-3 mRNA is expressed,
it is likely that the amount of IRS-3 protein is considerably less than
the amounts of IRS-1 and -2. For example, in liver, a major target for
insulin action, insulin-stimulated tyrosine phosphorylation of IRS-3 is
not detected by immunoprecipitation with antibodies against Tyr(P) or
PI 3-kinase followed by immunoblotting with antibodies against Tyr(P),
whereas IRS-1 and IRS-2 are prominent insulin-elicited Tyr(P) proteins
(23, 24). In fact, despite the presence of its mRNA in liver,
heart, kidney, and lung, the only tissue in which the IRS-3 protein has
been definitively detected to date is white adipose tissue (7).
We examined whether the absence of IRS-3 in adipocytes led to
compensatory changes in other signaling components. In addition to
IRS-3, adipocytes express IRS-1 and IRS-2, but not IRS-4 (17, 19, 24).
There was no change in the amounts of IRS-1 or -2 and no detectable
increase in the extent of their combined tyrosine phosphorylation in
response to insulin. Compensation for the absence of IRS-3 could also
occur by increased association of IRS-1 or 2 with PI 3-kinase and
SHP-2, the SH2 domain-containing proteins that normally associate with
the Tyr(P) form of IRS-3. However, the absence of any significant
increase in the association of the Tyr(P) forms of IRS-1/2 with either
SH2 domain protein suggests that this is not the case.
It is important to note that our present data do not provide
information about the effect of IRS-3 deficiency on the overall insulin-stimulated activation of PI 3-kinase and production of PI
3,4,5-trisphosphate. In future it will be of interest to compare insulin-stimulated production of PI 3,4,5-trisphosphate in wild-type and IRS-3-null adipocytes. A recent study examined the
distribution of IRS-1, -2, and -3 in subcellular fractions of rat
adipocytes, as well as their insulin-stimulated association with, and
activation of, PI 3-kinase (17). Unfortunately, the analysis did not
measure the relative amounts and activity of PI 3-kinase associated
with each IRS, and so the contribution of the complex between IRS-3 and
PI 3-kinase to the overall activation of PI 3-kinase in adipocytes is
not known. This study did show that IRS-3 and its complex with PI
3-kinase are primarily located in the plasma membrane, whereas IRS-1
and -2 and their complexes with PI 3-kinase are primarily in low
density microsomes. Thus, it remains possible that IRS-3, by virtue of
activation of PI 3-kinase in a unique location, has some unique, as yet
unidentified function in adipocytes. There is considerable evidence
that insulin stimulation of glucose transport in adipocytes requires
activation of PI 3-kinase through its association with the IRSs,
although there are also findings that conflict with this conclusion
(reviewed in Ref. 25). Our data show that association of PI 3-kinase
with IRS-3 is not essential for this insulin effect.
In adipocytes, IRS-3 appears to be the predominant IRS that associates
with the tyrosine phosphatase SHP-2 upon insulin treatment (Ref. 7 and
Fig. 6). The interaction of the SH2 domains of SHP-2 with Tyr(P) motifs
activates this phosphatase (26). Consequently, in the
IRS-3-null adipocytes, insulin-stimulated activation of SHP-2 is predicted to be substantially reduced. In the future it may be
possible to test this prediction by measurements of SHP-2 activity.
Results in earlier studies suggested that the association of SHP-2 with
IRSs might be required for full activation of the mitogen-activated
protein kinase cascade by insulin (27). However, a recent study has
shown conclusively that this is not the case for a cell line that
contains only IRS-1 (28). Thus, the role of the SHP-2/IRS association
in insulin signaling is currently unknown, and the consequences of its
reduction in the IRS-3-null adipocytes cannot be predicted.
However, these adipocytes may provide a system in which to examine this question.
In conclusion, at this point we have not found any abnormalities in
IRS-3-null mice, although insulin signaling through IRS-3 is
absent in their adipocytes. It is possible that in wild-type mice IRS-3
plays significant roles, and that in the null mice other IRSs function
in its place. Similarly, the phenotypes of the mice lacking IRS-1 and
IRS-2 may not be more severe because IRS-3 functions in part in their
place. Such functional redundancy has been found for other gene
families, such as the Src kinases and the retinoic acid receptors (29,
30). It should be possible to determine whether this is the case for
the IRSs by generating and characterizing mice lacking two IRSs. The
fact that each IRS gene is on a different chromosome (19) will
facilitate this approach.