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J Biol Chem, Vol. 273, Issue 45, 29686-29692, November 6, 1998
From The Third Department of Internal Medicine, Adipocytes contain three major substrate proteins
of the insulin receptor, termed IRS-1, IRS-2, and IRS-3. We
demonstrated that IRS-1 and IRS-2 are located mainly in the low density
microsome (LDM) fraction and are tyrosine phosphorylated in response to insulin stimulation, leading to phosphatidylinositol (PI) 3-kinase activation. In contrast, IRS-3 is located mainly in the plasma membrane
(PM) fraction and contributes to PI 3-kinase activation in the PM
fraction. The different cellular localizations of IRS proteins may
account for the mechanism of insulin resistance induced by a high fat
diet, considering that PI 3-kinase activation in the LDM fraction is
reportedly essential for the translocation of GLUT4 in adipocytes. High
fat feeding in rats increased both protein and mRNA levels of IRS-3
but decreased those of IRS-1 and IRS-2 in epididymal adipocytes. As a
result, selective impairment of insulin-induced PI 3-kinase activation
was observed in the LDM fraction, whereas PI 3-kinase activation was
conserved in the PM fraction. This is the first report showing that
different IRS proteins function in different subcellular compartments,
which may contribute to determining the insulin sensitivity in adipocytes.
Insulin induces numerous cell activities in adipose tissue, which
include cell proliferation and differentiation, stimulation of glucose
uptake, inhibition of lipolysis, translocation of various membrane
proteins such as transferrin receptor and insulin-like growth factor II
receptor, and synthesis and/or secretion of leptin (1-3). Although it
remains unclear which step in the insulin signaling pathway is related
to each of these individual activities, it seems certain that
insulin-stimulated phosphatidylinositol 3-kinase (PI
3-kinase)1 activation plays a
critical role in the translocation of GLUT4 from intracellular vesicles
to the cell surface (4-6). In addition, Yang et al. (7)
suggested that PI 3-kinase activation in the intracellular compartment,
but not on the plasma membrane, is necessary for the efficient
translocation of GLUT4 (7).
In adipocytes it has been reported that not only IRS-1 and IRS-2, but
also a protein of approximately 60 kDa referred to as pp60, are major
substrates that are tyrosine phosphorylated by the insulin receptor
(8-10). Recently, pp60 was cloned and termed IRS-3 in rat and mouse
adipocytes, and its structure was shown to have several similarities to
IRS-1 and IRS-2 (11, 12). Sequence alignment of IRS-3 with the other
members of the IRS family revealed that these IRS proteins contain
pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains that
are highly conserved (11, 12). In addition, there is conservation of many tyrosine phosphorylation motifs responsible for interactions with
downstream signaling molecules containing SH2 domains, including PI
3-kinase. However, IRS-3 is far smaller than IRS-1/2 and has some
regions that have no homology to IRS-1/2. Therefore, IRS-3 may have
some unique role in insulin signaling and glucose metabolism. IRS-1 and
IRS-2 have been shown to participate in the insulin signaling pathway
whereby insulin stimulates translocation of GLUT4 in adipose cells (13,
14).
The aim of this study was to clarify the unique role of IRS-3 compared
with IRS-1 and IRS-2. Our data suggest that different IRS proteins may
be important in the activation of SH2-containing proteins including PI
3-kinase in different subcellular compartments, which may induce
different cellular activities. In addition, we investigated how the
expression levels of IRS-3 as well as IRS-1 and IRS-2 are regulated in
high fat-fed rat adipocytes, in which insulin resistance is present.
The different regulation of IRS-3 from that of IRS-1 and IRS-2 would
induce a different degree of PI 3-kinase activation in the different
subcellular locations, which may be involved in the pathogenesis of
insulin resistance. This is the first paper showing the different role
of IRS-3 and also suggesting the possibility that the difference in
regulation of expression of IRS proteins may affect insulin sensitivity
in fat cells.
Materials--
Bovine serum albumin, fraction V, was purchased
from Intergen (Purchase, NY). Collagenase and PI were purchased from
Sigma (St. Louis, MO). Insulin was purchased from Novo Nordisk
(Denmark). [ Animals--
Male Sprague-Dawley rats, 5 weeks old and 110-140
g, were purchased from Tokyo Experimental Animals (Tokyo, Japan) and
housed under controlled light (12/12 h) and temperature conditions
with free access to food and water.
After a 2-3-day acclimatization period, the rats were divided into a
normal chow group and a high fat diet group and fed ad libitum for 2 weeks. The high fat diet consisted of 58% lard
(w/w), 30% fish powder, 10% skimmed milk, and a 2% vitamin and
mineral mixture (equivalent to 7.5% carbohydrate, 24.5% protein, and
60% fat). The normal chow proportions were 54% carbohydrate, 20%
protein, and 4.5% fat.
Antibodies--
Specific antibodies against IRS-1 and IRS-2 were
prepared as described previously (15). The antibody against IRS-3 was
prepared by immunizing rabbits with a synthetic peptide derived from
its specific amino acid sequence of the carboxyl terminus
(PPLEVPGAAPGNSPHSYASIKF). Anti-p85 Isolation of Adipocytes and Insulin Stimulation--
All of the
rats were killed by decapitation after 12-14 h of fasting. Isolated
adipocytes were prepared from epididymal fat pads by shaking at
37 °C for 20 min in Krebs-Ringer bicarbonate buffer containing
collagenase (3 mg/ml) and bovine serum albumin (10 mg/ml), according to
the methods of Robdell (16).
Isolated adipocytes were preincubated in 1% bovine serum albumin and
Krebs-Ringer bicarbonate buffer for 15 min and then stimulated with
10 Cell Fractionation--
Isolated adipocytes incubated with or
without insulin were homogenized and fractionated as described
previously by Simpson et al. (17) with several
modifications. 1 volume of HES buffer with 1 mM vanadate, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride was added to isolated adipocytes, and the
mixture was homogenized by 10 strokes in a Potter-Elvehjem Teflon
pestle homogenizer. The homogenates were centrifuged at 16,000 × g for 15 min at 4 °C, the solidified fat cake was removed carefully, and the supernatant was saved for preparation of the microsomal membrane fraction. The initial pellet was resuspended and
recentrifuged once before being resuspended in 5 ml of buffer, applied
to a 1.12 M sucrose cushion containing 20 mM
Tris-HCl and 1 mM EDTA, and centrifuged at 101,000 × gmax for 70 min. The mitochondria, nuclei, and
cell debris were collected as a pellet; however, in this experiment, we
did not analyze this fraction further. The plasma membranes (PM),
collected at the interface, were resuspended in 50 ml of buffer and
centrifuged at 48,000 × gmax for 45 min.
The pellet was resuspended at approximately 2 mg of protein/ml.
The initial supernatant was centrifuged at 48,000 × gmax for 20 min, yielding a pellet of high
density microsomal membranes (HDM). The supernatant was then
recentrifuged at 212,000 × gmax for 70 min, yielding a second pellet of low density microsomal membranes
(LDM), and the remaining supernatant was condensed by Centricon-30 and
used as cytosol. All pellets were resuspended in 1-10 ml of buffer and
repelleted before final resuspension at 1 mg of protein/ml.
PI 3-Kinase Assay--
Isolated adipocytes were prepared, and
some were stimulated with insulin as described above. Subcellular
fractions were separated as described above, and precipitated fractions
were resuspended in ice-cold buffer A (50 mM Hepes, pH 7.5, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 2 mM
Na3VO4, 10 mM sodium pyrophosphate,
10 mM NaF, 2 mM EDTA, 1% Nonidet P-40, 10%
glycerol, 2 µg/ml aprotinin, 5 µg/ml leupeptin, and 34 µg/ml
phenylmethylsulfonyl fluoride). Protein concentrations were determined
by BCA protein assay using bovine serum albumin as the standard. IRS-1,
IRS-2, IRS-3, and tyrosine-phosphorylated proteins were
immunoprecipitated from aliquots of the supernatant containing 1 mg of
protein with anti-IRS-1, anti-IRS-2, anti-IRS-3, or 4G10 antibodies,
respectively, followed by protein A-Sepharose 6MB. The assays of PI
3-kinase activities in the immunoprecipitates were performed as
described previously (15).
Western Blotting--
Isolated adipocytes were prepared, and
some were stimulated with insulin as described above. For total
cellular homogenates, isolated adipocytes were homogenized in ice-cold
buffer B (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 10% glycerol, 1.5 mM MgCl2, 1%
Triton X-100, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride) and kept on ice for 30 min. The
homogenates were subjected to centrifugation at 15,000 × g for 30 min at 4 °C, the solidified fat cake was removed
carefully, and the supernatants were used as samples. Subcellular
fractions were separated as described above, and precipitated fractions
were resuspended in ice-cold buffer B. The protein concentration was
determined by BCA protein assay using bovine serum albumin as the
standard, and the concentrations were adjusted to 1 mg/ml. IRS-1,
IRS-2, IRS-3, and tyrosine-phosphorylated proteins were
immunoprecipitated from each supernatant containing 1 mg of protein
with 2 µg/ml anti-IRS-1, anti-IRS-2, anti-IRS-3, or 4G10 antibodies,
respectively, followed by protein A-Sepharose 6MB, so that the
efficiency of immunoprecipitation was the same among the samples of
fractions. In some experiments, anti-IRS-3 antibody was chemically
cross-linked with protein A-Sepharose 4FF to decrease the amount of
immunoglobulin heavy chain in the immunoprecipitate and to detect the
amount of IRS-3 protein the in immunoprecipitates. Immunoprecipitates
were washed three times with buffer B and boiled in Laemmli sample
buffer containing 100 mM dithiothreitol. Aliquots of
samples were subjected to SDS-PAGE (7.5% or 10%). Electrotransfer of
proteins from the gel to PVDF membrane was performed for 3 h at 80 V (constant); immunoblotting with each of the aforementioned antibodies
was performed with Enhanced Chemiluminescence (ECL), and band
intensities were quantified with a PhosphorImager GS-525 using Imaging
Screen CH.
RNase Protection Assay--
In vitro transcription
for riboprobe and RNase protection assay was performed as described
previously (15). A rat IRS-3 cDNA corresponding to nucleotides
982-1526 was obtained by polymerase chain reaction based on the
reported sequence. The 544 base pairs of rat IRS-3 cDNA were
subcloned in pBluescript2 SK+ and used for in vitro
transcription. Pooled samples of 20 µg of total RNA from adipose
tissue were used and hybridized with the riboprobe of IRS-1, IRS-2, or
IRS-3. After treatment with RNase, the protected fragments were
resolved on 5% polyacrylamide-urea gel, subjected to autoradiography,
and the band intensities were determined by PhosphorImager GS-525 using
Imaging Screen BI.
Efficiency of Immunodepletion of Anti-IRS-3 Antibody and
Immunospecificity of Anti-IRS-1/2 Antibody--
To determine the
efficiency of immunoprecipitation with anti-IRS-3 antibody, the amount
of IRS-3 in the supernatant after immunoprecipitation was determined.
As shown in Fig. 1, IRS-3 in the second
immunoprecipitation was only 6% of that in the first. These results
indicate that the anti-IRS-3 antibody used in this study immunodepleted
IRS-3 from the cell lysate effectively.
The specificity of anti-IRS-1 and anti-IRS-2 antibody was demonstrated.
As shown in Fig. 1B, IRS-1 was detected in anti-IRS-1 immunoprecipitates, whereas IRS-1 was not detected in anti-IRS-2 immunoprecipitates by anti-IRS-1 immunoblotting. IRS-2 was also detected in anti-IRS-2 immunoprecipitates, although it was not detected
in anti-IRS-1 immunoprecipitates (data not shown). These results
indicate that the anti-IRS-1 and anti-IRS-2 antibodies used in this
study recognized specific proteins.
Subcellular Localization of IRS-3 Different from That of IRS-1 and
IRS-2 in Adipocytes--
The subcellular localization of IRS-1, IRS-2,
and IRS-3 protein in isolated adipocytes was investigated in the
presence or absence of insulin stimulation. After homogenization, the
cell lysate of fat cells was fractionated into fractions of PM, HDM, LDM, and cytosol. Indicated amounts of protein from each fraction were
immunoprecipitated with specific antibodies, electrophoresed, and
immunoblotting was performed using the specific antibody against IRS-1,
IRS-2, or IRS-3 (Figs. 2A,
3A, and
4, top panel, respectively). IRS-1 and IRS-2 were revealed to be most abundant in the LDM sample. In
contrast, IRS-3 was more abundant in the PM sample and was also present
in the LDM sample.
Treatment with insulin at 37 °C for 5 min did not significantly
affect the subcellular distributions of IRS-1, IRS-2, and IRS-3 but did
induce their marked tyrosine phosphorylation (Figs. 2B,
3B, and 4, second panel from top,
respectively). The tyrosine phosphorylation of IRS-1 and IRS-2 was
detected mainly in the LDM sample, whereas that of IRS-3 was detected
more strongly in the PM sample than in the LDM sample. These results of
phosphorylation level are in good accordance with the results of the
subcellular distribution of each IRS protein.
Similar results were obtained regarding the amount of p85 PI 3-Kinase Activity Associated with IRS-1, IRS-2, and IRS-3 in
Each Fraction of Isolated Adipocytes--
The subcellular localization
of PI 3-kinase activity in anti-IRS-1, anti-IRS-2, and anti-IRS-3
antibody immunoprecipitates from isolated adipocytes was investigated
(Figs. 2D, 3D, and 4, bottom panel,
respectively). Enhancement of the kinase activity by insulin
stimulation was observed irrespective of the fraction. In anti-IRS-1
and anti-IRS-2 antibody immunoprecipitates, the majority of PI 3-kinase
activity was detected in LDM, and the activity in the PM samples was
less than 10% of that in the LDM samples. In contrast, PI 3-kinase
activity associated with IRS-3 was detected mainly in the PM, and a
much lower activity was detected in the LDM samples.
Taking the different total amounts of protein obtained from each of the
subcellular fractions into consideration, the distribution of each IRS
protein, the tyrosine phosphorylation level, and PI 3-kinase protein
and activity associated with each IRS protein were calculated in whole
PM, LDM, HDM, and cytosol fractions and are summarized in Table
I. Briefly, 74% and 69% of the PI
3-kinase activity associated with IRS-1 and IRS-2, respectively, were
detected in the LDM fraction in the insulin-stimulated condition,
whereas 77% of that associated with IRS-3 was detected in the PM
fraction.
High Fat Diet Feeding Up-regulates IRS-3 but Down-regulates IRS-1
and IRS-2--
It is well known that a high fat diet induces the
enlargement of adipocytes and also insulin resistance in glucose
uptake. A diet high in fat or a normal diet was given to rats for 2 weeks, and epididymal fat cells were used to investigate the regulation of expression levels of IRS-1, IRS-2, and IRS-3. An RNase protection assay and immunoblotting were performed to measure the amounts of
mRNA and protein, respectively. The high fat diet decreased mRNA levels of IRS-1 and IRS-2 by 24 and 27% (Fig.
5, A and B), respectively, compared with the controls, whereas the mRNA level of
IRS-3 was up-regulated significantly by 49% (Fig. 5C).
Similar regulation was observed regarding the protein levels. IRS-1 and IRS-2 proteins in high fat-fed rat adipocytes were revealed to be
decreased by 27 and 52%, respectively, compared with the controls (Fig. 5, D and E), whereas IRS-3 protein level
was increased by 282% (Fig. 5F).
These results indicate that the expression level of IRS-3 is regulated
differently from that of IRS-1 and IRS-2 and that these different
regulations are likely to act at the transcriptional level.
Effects of High Fat Diet on IRS Protein Content in PM and LDM
Fractions in Adipocytes--
LDM and PM fractions were prepared from
adipocytes of high fat-fed rats and control rats. The amount of IRS-1
in the PM of normal diet rats was 11% of that in LDM (Fig.
6A). The amount of IRS-1 in
the adipocytes of high fat-fed rats in both PM and LDM was decreased by
31 and 74%, respectively, compared with the controls. Thus, the effect
of a high fat diet on IRS-1 protein amount was more pronounced in
LDM.
IRS-2 expression was also dominant in the LDM in adipocytes of normal
diet rats. The high fat diet reduced the IRS-2 protein level in both PM
and LDM compared with normal diet by 24 and 59%, respectively. Taken
together, in high fat-fed rat adipocytes, the decreases in IRS-1 and
IRS-2 content were more marked in the LDM fraction than those in the PM
fraction. In contrast, the amount of IRS-3 protein was demonstrated to
be increased similarly in both PM (by 62%) and LDM fractions (by
26%).
Effect of High Fat Diet on PI 3-Kinase Activity Associated with
Anti-IRS-1, Anti-IRS-2, Anti-IRS-3, and Anti-phosphotyrosine
Immunoprecipitates in PM and LDM--
The PI 3-kinase activity
associated with anti-IRS-1, anti-IRS-2, and anti-IRS-3
immunoprecipitates was determined. The high fat diet decreased the PI
3-kinase activity associated with IRS-1 and IRS-2 after insulin
stimulation in the LDM by 46 and 43%, respectively. In contrast, the
PI 3-kinase activity associated with IRS-3 after insulin stimulation
was significantly increased in both LDM and PM fractions by high fat diet.
Panel C of Fig. 7
demonstrates the PI 3-kinase activity associated with anti-IRS-3
immunoprecipitates. The high fat diet increased PI 3-kinase activity
associated with anti-IRS-3 immunoprecipitates in the basal state (PM,
1.7-fold; LDM, 1.9-fold). Insulin-induced PI 3-kinase activation
associated with anti-IRS-3 immunoprecipitates was also increased by a
high fat diet in both PM and LDM (PM, 1.5-fold; LDM, 2.4-fold).
PI 3-kinase activity associated with anti-phosphotyrosine
immunoprecipitates may represent the increment of those (associated with) anti-IRS-1, anti-IRS-2, and anti-IRS-3 immunoprecipitates. Fig.
7D demonstrates that insulin-induced PI 3-kinase activation associated with anti-phosphotyrosine antibody was decreased in both PM
and LDM in high fat-fed rats by 10 and 40%, respectively.
Insulin exerts numerous cellular activities in various cells.
Insulin signaling is initiated by the binding of insulin to its
specific receptor on the cell surface. By the activated insulin receptor tyrosine kinase, several substrates reportedly are
phosphorylated on their tyrosine residues. In rat adipocytes, not only
IRS-1 and IRS-2, but also pp60, were found to be major substrates of the insulin receptor (1-3). Recently, pp60 was cloned from rat and
mouse adipocytes and termed IRS-3 (11, 12). IRS-3 was shown to contain
an amino-terminal PH domain, followed by a PTB domain, and these
domains are highly homologous (about 50% identical amino acids) to
those of IRS-1 and IRS-2 (11, 12). In addition, there is conservation
of many tyrosine phosphorylation motifs responsible for interactions
with downstream signaling molecules containing SH2 domains including PI
3-kinase, SHP2, and Grb-2 (12). Thus, to date no functional difference
of IRS-3 from IRS-1 and IRS-2 has been reported.
IRS-3 mRNA is expressed abundantly in adipocytes and hepatocytes
but is also highly expressed in the first part of embryonic life, when
IRS-1 mRNA is barely detected (11). These results suggest that
IRS-3 may be responsible not only for the regulation of metabolic
functions in adipocytes and hepatocytes but also for differentiation
and/or proliferation.
In this study we used specific antibodies for IRS-1, IRS-2, and IRS-3
and determined the subcellular localization of these proteins and PI
3-kinase activity mediated by each of these insulin receptor
substrates. Insulin-stimulated PI 3-kinase activity associated with
IRS-1 and IRS-2 was located mainly in the LDM (74 and 69% of total
activity in cells, respectively), whereas it was barely detected in
plasma membrane (19 and 15% of total activity in cells, respectively)
(Table I). In fact, IRS-1 and IRS-2 proteins were barely detected in
PM. Because increased amounts of IRS-1 and IRS-2 in the LDM were not
detected in our experimental condition, PI 3-kinase activation
associated with IRS-1 and IRS-2 in LDM resulted from an increase in the
PI 3-kinase associated with the YMXM motif in IRS-1 and
IRS-2 phosphorylated on tyrosine residues by insulin.
Heller-Harrison et al. (18) reported that insulin action on
3T3-L1 adipocytes progressively decreased the amount of IRS-1 protein
associated with the LDM fraction. Although we could not observe a
similar decrease in IRS-1 in the LDM of isolated rat adipocytes, we
speculate that this contradiction may be caused by differences of the
cell types and/or experimental conditions such as serum starvation.
In contrast to the case of IRS-1 and IRS-2, IRS-3 protein was detected
in both PM and LDM, irrespective of stimulation with insulin. As
reflected by the subcellular distribution of IRS-3, 77% of the PI
3-kinase activity associated with IRS-3 in the whole cell was detected
in the PM fraction, and only 12% was in the LDM fraction (Table I).
Thus, only IRS-3 efficiently contributes to the insulin-induced PI
3-kinase activation on the plasma membrane in adipocytes.
Although it remains unclear how the subcellular distributions of these
signaling molecules are determined, it seems that the PH domain plays
an important role in anchoring the protein to the membrane via its
association with phospholipid (19, 20). On the other hand, the PTB
domain of IRS proteins is reportedly essential for association with the
insulin receptor (21). Therefore, the NH2-terminal portion
containing the PH domain and PTB domain could have some role in
determining the location of IRS proteins. However, because these
portions are highly conserved between IRS-3 and IRS-1/2, it is quite
unlikely that these portions contribute to their different subcellular
distributions. Because there is no extended homology in the portion
outside the PH and PTB domains between IRS-3 and either IRS-1 or IRS-2,
it seems reasonable to consider that there is a region other than PH
and PTB domains responsible for the different subcellular
distributions. Further study is necessary to clarify this issue.
Insulin induces numerous cell activities in adipose tissue, which
include cell proliferation and differentiation, stimulation of glucose
and amino acid uptake, inhibition of lipolysis and translocation of
various membrane proteins such as transferrin receptor and insulin-like
growth factor II receptor, and synthesis and/or secretion of leptin
(1-3). Among them, insulin-stimulated PI 3-kinase activation plays a
critical role in the translocation of GLUT4 from intracellular vesicles
to the cell surface (4-6). In addition, Yang et al. (7)
suggested that PI 3-kinase activation in the intracellular compartment,
but not on the PM, is necessary for the translocation of GLUT4, because
treatment with epidermal growth factor or platelet-derived growth
factor, which stimulate PI 3-kinase activity in the whole cell as
strongly as insulin, failed to induce translocation of the glucose
transporter to the cell surface as fully as that with insulin (7).
Assuming that this is the case, PI 3-kinase activation on the plasma
membrane by IRS-3 may not contribute to insulin-induced GLUT4
translocation, but one in LDM by IRS-1 and IRS-2 may be much more
important to contribute to insulin-induced GLUT4 translocation.
A high fat diet is one of the major causes inducing insulin resistance
with respect to the insulin-induced translocation of GLUT4 to the cell
surface (22-26). We demonstrated that high fat diet up-regulates IRS-3
but down-regulates IRS-1 and IRS-2. As reflected by these altered
expression levels, PI 3-kinase activation associated with IRS-1 and
IRS-2 was decreased markedly, whereas that associated with IRS-3 was
increased. Because IRS-3 and IRS-1/2 are located mainly in the PM and
LDM, respectively, as a consequence of their altered expression levels,
insulin-induced PI 3-kinase activation is impaired markedly in the LDM,
whereas that on the PM is maintained. We speculate that this
abnormality in the portion in the cell where PI 3-kinase activation
occurs may be one of the mechanisms causing insulin resistance in high
fat diet-induced insulin resistance, in terms of glucose uptake.
Although the role of PI 3-kinase activation on the PM induced by IRS-3
in adipocytes remains unclear, several possibilities can be raised.
Treatment of adipocytes with platelet-derived growth factor, epidermal
growth factor, or fibroblast growth factor reportedly induces a
decrease in the number of developing fat cells and the activity of
glycerol-3-phosphate dehydrogenase, a marker of adipocyte differentiation (27). In addition, this inhibitory action regarding the
adipocyte differentiation was associated with markedly potent stimulation of cell proliferation. The inhibitory effect of
platelet-derived growth factor and epidermal growth factor on adipocyte
differentiation was suggested to be induced by the reduction of
peroxisome proliferator-activated receptor *
This work was supported by Grant-in-aid 09470214 for
Scientific Research (to T. A.) from the Ministry of Education,
Science, and Culture of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
81-3-3815-5411 (ext. 3133); Fax: 81-3-5803-1874; E-mail:
asano-tky{at}umin.ac.jp.
The abbreviations used are:
PI 3-kinase, phosphatidylinositol 3-kinase; IRS insulin receptor substrate, PH,
pleckstrin homology; PTB, phosphotyrosine binding; PM, plasma membrane; HDM, high density microsome; LDM, low density microsome; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis.
Different Subcellular Distribution and Regulation of Expression
of Insulin Receptor Substrate (IRS)-3 from Those of IRS-1 and
IRS-2*
,,,, Institute for Adult Diseases,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]ATP was purchased from ICN (Costa
Mesa, CA). Aluminum-backed silica gel thin layer chromatographic plates
were purchased from Merck (Darmstadt, Germany). Protein A-Sepharose 6MB
was purchased from Amersham Pharmacia Biotech (U. K). All other
chemicals were purchased from Wako (Osaka, Japan).
antibody and anti-phosphotyrosine
antibody (4G10) were purchased from Upstate Biochemistry (Lake Placid, NY).
7 M insulin for 5 min. After insulin
stimulation, adipocytes were washed three times with HES buffer (20 mM Hepes, pH 7.5, 1 mM EDTA, 255 mM
sucrose, 10 mM NaF, 1 mM sodium pyrophosphate)
and used for PI 3-kinase assay or Western blotting immediately or after
cell fractionation.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Efficiency of immunodepletion of anti-IRS-3
antibody and immunospecificity of anti-IRS-1/2 antibody.
Panel A, a cell lysate of isolated adipocytes was prepared
as described under "Experimental Procedures." 1 mg of cell lysate
was incubated for 2 h at 4 °C with anti-IRS-3 antibody that
chemically cross-linked with protein A-Sepharose 4FF. After
centrifugation, the supernatant was removed and used subsequently for
another two immunoprecipitations. Immunoprecipitates were boiled as
described under "Experimental Procedures" and subjected to
SDS-PAGE. Immunoblotting was performed with anti-IRS-3 antibody to
detect IRS-3 (upper panel). The bar graph
represents the amount of IRS-3 in the first (1), second
(2), and third (3) immunoprecipitates.
Panel B, a cell lysate of isolated adipocytes was prepared,
and 1 mg of cell lysate was immunoprecipitated with anti-IRS-1 and
anti-IRS-2 antibody. Immunoprecipitates were subjected to SDS-PAGE,
transferred to PVDF membrane, and immunoblotted with anti-IRS-1
antibody.
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Fig. 2.
Subcellular localization of IRS-1 in isolated
adipocytes. The epididymal fat pad was excised, and adipocytes
were isolated as described under "Experimental Procedures."
Isolated adipocytes were incubated with or without insulin for 5 min at
37 °C, and PM, LDM, HDM, and cytosol (Cyt) were prepared
as described. Aliquots of each fraction (1 mg) were incubated with
anti-IRS-1 antibody and subsequently precipitated by adding protein
A-Sepharose 4FF. Immunoprecipitates were subjected to SDS-PAGE and
transferred to PVDF membrane. Immunoblotting was performed with
anti-IRS-1, anti-phosphotyrosine antibody (4G10), and anti-p85 antibody
to measure IRS-1 protein (panel A), tyrosine phosphorylation
of IRS-1 (panel B), and p85 associated with IRS-1
(panel C), respectively. In addition, PI 3-kinase activity
in the anti-IRS-1 immunoprecipitate from each fraction was assayed as
described under "Experimental Procedures" (panel
D).
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Fig. 3.
Subcellular localization of IRS-2 in isolated
adipocytes. The epididymal fat pad was excised, and adipocytes
were isolated as described under "Experimental Procedures."
Isolated adipocytes were incubated with or without insulin for 5 min at
37 °C, and PM, LDM, HDM, and cytosol (Cyt) were prepared
as described. Aliquots of each fraction (1 mg) were incubated with
anti-IRS-2 antibody and subsequently precipitated by adding protein
A-Sepharose 4FF. Immunoprecipitates were subjected to SDS-PAGE and
transferred to PVDF membrane. Immunoblotting was performed with
anti-IRS-2, anti-phosphotyrosine antibody (4G10), and anti-p85 antibody
to measure IRS-2 protein (panel A), tyrosine phosphorylation
of IRS-2 (panel B), and p85 associated with IRS-2
(panel C), respectively. In addition, PI 3-kinase activity
in the anti-IRS-2 immunoprecipitate from each fraction was assayed as
described under "Experimental Procedures" (panel
D).
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Fig. 4.
Subcellular localization of IRS-3 in isolated
adipocytes. The epididymal fat pad was excised, and adipocytes
were isolated as described under "Experimental Procedures."
Isolated adipocytes were incubated with or without insulin for 5 min at
37 °C, and PM, LDM, HDM, and cytosol (Cyt) were prepared
as described. Aliquots of each fraction (1 mg) were incubated with
anti-IRS-3 antibody and subsequently precipitated by adding protein
A-Sepharose 4FF. Immunoprecipitates were subjected to SDS-PAGE and
transferred to PVDF membrane. Immunoblotting was performed with
anti-IRS-3, anti-phosphotyrosine antibody (4G10), and anti-p85 antibody
to measure IRS-3 protein (top panel), tyrosine
phosphorylation of IRS-3 (second panel from
top), and p85 associated with IRS-3 (third
panel from top). In addition, PI 3-kinase activity in
the anti-IRS-3 immunoprecipitate from each fraction was assayed as
described under "Experimental Procedures" (bottom
panel).
, a
regulatory subunit of PI 3-kinase, associated with each of the IRS
proteins, when stimulated with insulin. In the insulin-stimulated condition, p85 associated with IRS-1 and IRS-2 was detected
predominantly in the LDM sample (Figs. 2C and 3C,
respectively), whereas that associated with IRS-3 was detected mainly
in the PM, but a smaller amount was also detectable in the LDM sample
(Fig. 4, second panel from bottom).
Protein characteristics in IRS
View larger version (36K):
[in a new window]
Fig. 5.
Effect of high fat diet on expression of
IRS-1, IRS-2, and IRS-3 in isolated adipocytes. RNA in the
epididymal fat pad was isolated as described under "Experimental
Procedures." Then 20 µg of total RNA was used for RNase protection
assay with radiolabeled antisense riboprobes for IRS-1, IRS-2, and
IRS-3 mRNA. Protected fragments were resolved on 5% polyacrylamide
urea gel, subjected to autoradiography, and RNase-protected band
intensities were analyzed with a PhosphorImager. The images show
RNase-protected bands from three control and three high fat-fed rats.
The bar graph represents quantitation of the results of
three independent experiments. Results are indicated as percent of
control (panels A-C). Total cell homogenate was prepared as
described under "Experimental Procedures." IRS-1, IRS-2, and IRS-3
were immunoprecipitated with their specific antibodies. The amounts of
IRS proteins were determined by immunoblotting with the specific
antibodies against each of the IRS proteins. * indicates a significant
difference from the control at p < 0.05; ** indicates
a significant difference from the control at p < 0.01
View larger version (20K):
[in a new window]
Fig. 6.
Effect of high fat diet on protein levels of
IRS-1, IRS-2, and IRS-3 in plasma membrane and low density microsome of
isolated adipocytes. Aliquots of fractionated homogenate were
immunoprecipitated with anti-IRS-1, anti-IRS-2, and anti-IRS-3
antibodies and subsequently with protein A-Sepharose 4FF.
Immunoprecipitates were subjected to SDS-PAGE, transferred to PVDF
membrane, and immunoblotted with specific antibodies using an ECL plus
kit. The quantitation was performed with a Bio-Rad PhosphorImager with
Screen-CH. The bar graph represents quantitation of the representative
results of three independent experiments. Results are presented as % of each protein level in LDM of normal diet rats. Protein levels of
IRS-1, IRS-2, and IRS-3 are represented in panels A,
B, and C, respectively.
View larger version (22K):
[in a new window]
Fig. 7.
Effect of high fat diet on PI 3-kinase
activity associated with anti-IRS-1, anti-IRS-2, anti-IRS-3, and
anti-phosphotyrosine immunoprecipitates in plasma membrane and low
density microsome of isolated adipocytes. Aliquots of fractionated
homogenate were immunoprecipitated with anti-IRS-1, anti-IRS-2, and
anti-IRS-3 antibodies and subsequently with protein A-Sepharose 6MB. PI
3-kinase assay for each immunoprecipitate was performed as described
under "Experimental Procedures." The quantitation was performed
with a Bio-Rad PhosphorImager with Screen-BI. The bar graph
represents quantitation of the representative results of three
independent experiments. Results are presented as the percent of each
insulin-stimulated PI 3-kinase activity in LDM of normal diet rats. PI
3-kinase activity associated with anti-IRS-1, anti-IRS-2, anti-IRS-3,
and anti-phosphotyrosine antibody is represented in panels
A, B, C, and D,
respectively.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1 transcriptional
activity caused by phosphorylation of peroxisome proliferator-activated
receptor 1 by mitogen-activated protein kinase (28). Taking these
previous reports into consideration, it can be speculated that IRS-3
phosphorylated mainly on the plasma membrane, similarly to the
receptors of epidermal growth factor or platelet-derived growth factor,
may have a role promoting cell proliferation and inhibiting adipocyte
differentiation. In addition to the functions of adipocytes discussed
above, fat cells have more specific functions that are affected by
insulin stimulation. It would be of great interest to clarify which IRS
protein transduces the signal inducing the individual insulin-induced
cell activity. Further study of this issue is needed.
FOOTNOTES
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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