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Departamento de Bioquimica y Biologia Molecular II
(A.M.V., M.L., M.B.) Instituto de Bioquimica Centro Mixto
Consejo Superior de Investigaciones Cientificas/Universidad
Complutense Facultad de Farmacia Universidad Complutense
28240-Madrid, Spain
Joslin Diabetes Center (S.P., M.F.W.,
M.B.) Harvard Medical School Boston, Massachusetts 02215
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, and
IRS-1- and IRS-2-associated phosphatidylinositol (PI) 3-kinase
activation to the same extent, the effect of insulin being stronger
than the effect of IGF-I at the same physiological dose (10
nM). Furthermore, insulin/IGF-I stimulated
IRS-1-associated Grb-2 phosphorylation. However, IRS-2-associated Grb-2
phosphorylation was barely detected. Pull-down experiments with
glutathione-S-transferase-fusion proteins containing
SH2-domains of p85 revealed a strong association between IRS-1 and
IRS-2 with p85 in response to insulin/IGF-I, the insulin effect
being stronger than IGF-I. However, the Grb-2-SH2 domain showed
functional differences. While a strong association between IRS-1/Grb-2
was found, IRS-2/Grb-2 association was virtually absent in response to
insulin/IGF-I, as also demonstrated in competition studies with a
phosphopeptide containing the phosphotyrosine 895 residue within the
putative Grb-2-binding domain. Finally, insulin/IGF-I stimulated
tyrosine phosphorylation of the three SHC proteins (46, 52, and 66
kDa). Moreover, insulin/IGF-I markedly increased the amount of
Grb-2-associated SHC proteins by the same extent. Our results suggest
that both IRS-1 and IRS-2 are required for phosphatidylinositol
3-kinase activation that leads to adipogenic and thermogenic
differentiation of fetal brown adipose tissue; meanwhile, IRS-1 and
SHC, but not IRS-2, associate with Grb-2 leading to the
ras-mitogen-activated protein kinase-signaling pathway required for
fetal brown adipocyte proliferation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunits and two membrane-spanning ß-subunits that
possess tyrosine kinase activity (3, 4). These receptors are believed
to transduce signals, at least in part, by phosphorylation of cellular
proteins. It is well known that phosphorylation of the insulin receptor
substrate-1 (IRS-1) upon multiple tyrosine residues results in the
interaction with SH2 domain-containing proteins including the growth
factor receptor-bound-2 (Grb-2/Sem5), the protein tyrosine phosphatase
SHPTP2, and the p85 subunit of phosphatidylinositol 3-kinase (PI
3-kinase) that leads to activation of various signaling pathways,
including the stimulation of the p21.ras-MAPK cascade (5, 6, 7, 8). To elucidate the role of IRS-1 in insulin/IGF-I action, two laboratories (9, 10) created IRS-1-deficient mice by targeted gene mutation. These mice showed an alternative high molecular weight substrate of the insulin receptor named IRS-2, which has been recently purified and cloned (11, 12). IRS-2 has been found to be the major substrate of tyrosine kinases activated by interleukin (IL)-4 in murine lymphohemopoietic cells (13). Furthermore, the expression of IRS-2 was detected in a variety of cell types, including fibroblasts, liver, skeletal muscle, and brain (12). However, the exact role of both IRS proteins in mediating physiological functions in the different tissues has not yet been clarified.
Fetal brown adipocyte primary cultures offer a nonfibroblastic mesenchymal cell model that has proven to be an excellent system by which to study the role of IGF-I/insulin in the proliferation (14, 15, 16) and differentiation processes (15, 17, 18, 19, 20), as well as their balance in the insulin/IGF-I signal transduction pathways (18, 21, 22). These cells bear a large number of high-affinity insulin and IGF-I receptors per cell (15, 20), allowing a variety of studies to be performed with no overexpression of signaling molecules. We have recently shown that IRS-1 is tyrosine phosphorylated in fetal brown adipocytes upon insulin/IGF-I stimulation, resulting in the activation of IRS-1-associated PI 3-kinase (18, 21, 22). Importantly, this enzyme is a requirement for IGF-I-induced brown adipocyte adipogenic and thermogenic differentiation, but not for mitogenesis (18). However, the presence of IRS-2 in brown adipose tissue and its potential role in the insulin/IGF-I signaling cascade throughout late fetal development has not yet been established.
In the present study, we demonstrate that IRS-1 and IRS-2 proteins equally contribute to the association and subsequent activation of PI 3-kinase by insulin/IGF-I that lead to adipogenic and thermogenic differentiation of primary fetal brown adipocytes. However, the association of IRS-2 with the adapter protein Grb-2 is very poor, IRS-1 and SHC being the main docking proteins involved in the activation of the ras-MAPK pathway in brown adipose tissue before birth.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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shows the tyrosine
phosphorylation of the IRS proteins induced by insulin or IGF-I
treatment. As shown in Fig. 1, both insulin/IGF-I induce a significant
increase in tyrosine phosphorylation of IRS-1, as compared with control
cells, the insulin effect being stronger than the effect of IGF-I at
the same dose (10 nM). When the anti-IRS-2
immunoprecipitates were analyzed by anti-Tyr(P) Western blotting, we
found a strong phosphorylation band showing a slightly higher molecular
mass (approximately 190 kDa) in insulin/IGF-I-treated cells. However,
the level of tyrosine phosphorylation of this band was similar to the
level that we previously found in the IRS-1 immunoprecipitates, the
effect of insulin being also stronger than the effect of IGF-I at the
same dose. These results suggest that both IRS-1 and IRS-2 are involved
in the insulin/IGF-I signaling in fetal brown adipocytes.
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Subunit of
Phosphatidylinositol 3-Kinase by Insulin/IGF-I subunit of PI 3-kinase as
compared with IRS-1. Serum-deprived cells were incubated with 10
nM insulin or 10 nM IGF-I for 5 min at 37 C.
After incubation, cell lysates were prepared, and equal amounts of
protein were immunoprecipitated either with the anti-IRS-1 or
anti-IRS-2 GST-fusion protein antibodies. The immune complexes were
analyzed by Western blotting with the anti-p85 polyclonal antibody.
As shown in Fig. 2A, control cells did
not show any p85 bound to either IRS-1 or IRS-2. However, treatment
of brown adipocytes with 10 nM insulin or 10 nM
IGF-I resulted in a marked increase in the amount of p85 bound to
either IRS-1 or IRS-2, insulin stimulating more binding of p85 to
either IRS-1 or IRS-2 than IGF-I did (Fig. 2A).
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subunit of PI 3-kinase in fetal brown adipocytes, quiescent cells were
stimulated with 10 nM insulin or 10 nM IGF-I
for 5 min at 37 C. The cells were lysed, and 600 µg of protein were
immunoprecipitated with the anti-p85 polyclonal antibody as
described in Materials and Methods. The immune complexes
were subjected to Western blotting analysis with the anti-IRS-1 and
anti-IRS-2 polyclonal antibodies. As shown in Fig. 2B
, we found a huge
increase in the amount of both IRS-1 and IRS-2 in the anti-p85
immunoprecipitates upon treatment with insulin/IGF-I, insulin
stimulating more binding of either IRS-1 or IRS-2 to p85 than IGF-I
did.
Both IRS Proteins Induce PI 3-Kinase Activity in Fetal Brown
Adipocytes
We have recently demonstrated the stimulation of IRS-1-associated
PI 3-kinase enzymatic activity in fetal brown adipocyte primary
cultures treated with insulin or IGF-I (21, 22). Our next step was to
investigate whether PI 3-kinase activity was also associated with IRS-2
as it was to IRS-1, under the same experimental conditions. Upon
stimulation with 10 nM insulin or 10 nM IGF-I,
cell lysates were subjected to immunoprecipitation with the anti-IRS-1
or IRS-2 antibodies and assayed for PI 3-kinase activity as described
in Materials and Methods. As shown in Fig. 3, insulin/IGF-I markedly increased IRS-1
and also IRS-2-associated PI 3-kinase activity, the effect of insulin
on both activities being stronger than that induced by IGF-I. Similar
PI 3-kinase activity associated with IRS-1/IRS-2 induced by insulin was
found after two successive immunoprecipitations with anti-IRS-1 and
anti-IRS-2 antibodies of the same lysate (results not shown).
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, control cells showed
a considerable amount of IRS-1-associated Grb-2. However,
IRS-2-associated Grb-2 was barely detected. Insulin/IGF-I induced a
marked increase in the amount of IRS-1-associated Grb-2 but a very poor
increase in the amount of IRS-2-associated Grb-2. To confirm these
data, we analyzed the presence of IRS-1 and IRS-2 in anti-Grb-2
immunoprecipitates. Cells were stimulated with 10 nM
insulin or 10 nM IGF-I for 5 min at 37 C, and lysates were
immunoprecipitated with the anti-Grb-2 polyclonal antibody as described
in Materials and Methods. The immune complexes were
subjected to Western blotting analysis with the anti-IRS-1 and
anti-IRS-2 GST-fusion protein polyclonal antibodies. As shown in Fig. 4B, insulin/IGF-I induced a marked increase in the amount of
Grb-2-associated IRS-1. However, the amount of Grb-2-associated IRS-2
in response to insulin/IGF-I was virtually undetectable.
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As shown in Fig. 5, the
NH2-terminal SH2 domain of p85 bound strongly to both
IRS-1 and IRS-2 upon stimulation with 10 nM insulin or 10
nM IGF-I, insulin stimulating more binding than IGF-I did.
However, the SH2 domains of Grb-2 revealed functional differences.
Thus, control cells showed a basal amount of Grb-2-associated IRS-1,
but not Grb-2-associated IRS-2. Insulin/IGF-I markedly increased the
amount of Grb-2-associated IRS-1. However, the amount of
Grb-2-associated IRS-2 in response to insulin/IGF-I was virtually
undetectable (Fig. 5).
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, A and B. Dissociation of IRS-2 from
Grb-2 was much more sensitive (ED50 = 0.01
µM) (Fig. 6D) to the phosphopeptide than dissociation of
IRS-1 from Grb-2 (ED50 > 1 µM) (Fig. 6C),
suggesting that the Grb-2-binding site of IRS-1 displays a much higher
affinity for Grb-2 than that of IRS-2.
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, control cells showed basal
phosphorylation bands of the three SHC proteins. However, 10
nM insulin/IGF-I increased the 46- and 52-kDa SHC
phosphotyrosine bands by 2-fold and the 66-kDa SHC phosphotyrosine band
by 3-fold.
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, there is an
important association of SHC with Grb-2 before stimulation of brown
adipocytes with insulin/IGF-I. However, insulin/IGF-I markedly
increased by the same extent the amount of Grb-2-associated SHC
proteins. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Previous studies performed in IRS-1-deficient mice showed that the
residual insulin/IGF-I action of these animals correlated with the
appearance of IRS-2 bound to the p85 subunit of PI 3-kinase in a
tissue-dependent manner, providing evidence of an alternative and
independent signaling pathway in the insulin/IGF-I action (9, 10, 23, 24, 25). Furthermore, IRS-2 has been found to be the major high
molecular mass protein phosphorylated on tyrosine in hematopoietic
cells (13). However, in fetal brown adipocytes, IRS-1 and IRS-2
strongly associate with the p85 subunit, indicating that both
docking proteins are contributing to the PI 3-kinase activation
throughout late fetal development. In a recent work, we have shown that
PI 3-kinase is an essential requirement for the IGF-I-induced
adipogenic and thermogenic differentiation of fetal brown adipocytes
(18). These results, together with the data presented here, suggest
that IRS-2 signaling may be as necessary as IRS-1 for the complete
differentiation of brown adipose tissue before birth, in contrast to
other adipose cells such as primary white adipocytes and differentiated
3T3-L1 fibroblasts in which phosphorylated IRS-2 was not found in
response to insulin (12). The relative contribution of IRS-1/IRS-2 to
the adipogenic and thermogenic differentiation is difficult to
determine in primary cultures and deserves further experimental work in
these cells from IRS-1-deficient mice. Actually, PI 3-kinase activation
in response to insulin is severely impaired in IRS-1-deficient
fibroblasts, although a strong association between IRS-2/p85 was
found (26). The fact that insulin turned out to be a stronger signal in
activating IRS-1 and IRS-2-associated PI 3-kinase activity, as compared
with IGF-I, further supports our previous data showing that insulin was
a more potent signal than IGF-I in adipogenic and thermogenic
differentiation (20).
The adapter protein Grb-2 has been shown to link IRS-1 to the activation of ras and the MAPK cascade (6, 27). Activation of p21.ras to its GTP active form has been shown to occur in fetal brown adipocytes upon insulin or IGF-I stimulation (21, 22). Importantly, serum-starved brown adipocytes showed a considerable percentage of ras in its active GTP form (21), as a consequence of their intrinsic mitogenic competence (15). Results presented in this paper show that there is a significant IRS-1/Grb-2 association in control cells in the absence of external stimuli, which could explain the intrinsic ras.GTP activation that we found in fetal brown adipocyte primary cultures. Furthermore, there is a marked increase in the amount of Grb-2-associated IRS-1 in response to insulin/IGF-I. However, despite the fact that the amino acid sequence of IRS-2 predicts a tyrosine phosphorylation motif that could serve as a Grb-2 binding site (11, 12, 28), our data show that the association between IRS-2/Grb-2 in response to insulin/IGF-I is virtually absent. In addition, the phosphopeptide competition assay revealed that IRS-2 binds to Grb-2 with 2 orders of magnitude lower affinity than IRS-1. Therefore, a molecular mechanism that may account for the differential signaling between IRS-1 and IRS-2 underlies the differential affinities for Grb-2 of their conserved Grb-2 binding domains, probably due to the different environment surrounding this domain in both molecules. These data are consistent with the severe inhibition of the DNA synthesis found in IRS-1-deficient fibroblasts, in which the presence of IRS-2 could not compensate for the absence of IRS-1 in maintaining the mitogenic response of IGF-I (26).
SHC has recently been established as another early phosphotyrosine substrate, which has been proposed as the predominant signaling molecule coupling insulin receptors to p21 ras.GTP formation in Rat-1 fibroblasts (29, 30). More recently, SHC signaling, rather than IRS-1, has been proposed to account for the proliferation of Rat-1 fibroblasts in response to insulin (31). In fetal brown adipocyte primary cultures, the three SHC proteins are tyrosine phosphorylated in response to insulin/IGF-I. Furthermore, we have found a marked increase in the amount of SH2Grb-2 fusion protein associated with SHC in response to insulin/IGF-I. The results presented here indicate that in fetal brown adipocytes IRS-1 and SHC, but not IRS-2, are the docking proteins that lead to the ras-MAPK cascade after insulin and IGF-I stimulation. The fact that IGF-I is as potent as insulin in inducing Grb-2-SHC association is in agreement with the role of IGF-I as a potent mitogen for fetal brown adipocytes, in a p21.ras-dependent manner (16).
In summary, the findings described in this paper strongly suggest that
IRS-1 and IRS-2 contribute equally to the association with the p85
that leads to the PI 3-kinase activation in response to insulin/IGF-I,
a signaling required for the onset of brown adipose tissue adipogenic
and thermogenic differentiation before birth. Moreover, IRS-1 and SHC,
but not IRS-2, associate with the adapter protein Grb-2 that leads to
the ras-MAPK cascade, a signaling required for fetal brown adipocyte
proliferation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-phosphatidylinositol, and
L--phosphatidyl-L-serine were purchased from
Sigma Chemical Co. (St. Louis, MO). Collagenase and Protein A-agarose
were purchased from Boehringer Mannheim (Mannheim, Germany).
Glutathione Sepharose beads were purchased from Pharmacia (Upsala,
Sweden). [
32P]ATP (3000 Ci/mmol) was purchased from
Amersham (Aylesbury, U.K.). All other reagents used were of the purest
grade available.
Cell Culture
Brown adipocytes were obtained from interscapular brown adipose
tissue of 20-day Wistar rat fetuses and isolated by collagenase
dispersion as previously described (15). Cells were plated at 5 x
106 cells/100 mm or 1–1.2 x 106
cells/60-mm tissue culture plates in MEM supplemented with 10% FCS to
allow cell attachment to the plastic surface of the plates. After 4–6
h of culture at 37 C, cells were rinsed twice with PBS, and 80% of the
initial cells were attached. Cells were maintained for 20 h in a
serum-free medium supplemented with 0.2% (wt/vol) BSA. At this time
cells were treated for 5 min with insulin or IGF-I at the doses
indicated in the text and in the figure legends. Control cells were
cultured in the absence of factors in the medium.
Immunoprecipitations
Quiescent fetal brown adipocytes (after 20 h of serum
deprivation) were treated with insulin or IGF-I for 5 min and
subsequently lysed at 4 C in 1 ml of a solution containing 10
mM Tris-HCl, 5 mM EDTA, 50 mM NaCl,
30 mM sodium pyrophosphate, 50 mM NaF, 100
µM Na3VO4, 1% Triton X-100, and
1 mM phenylmethylsulfonyl fluoride, pH 7.6 (lysis
buffer). Lysates were clarified by centrifugation at 15,000 x
g for 10 min, and the supernatants were transferred to a
fresh tube. After protein content determination, equal amounts of
protein were immunoprecipitated at 4 C with the corresponding
antibodies, and the immune complexes were collected on Protein
A-agarose or anti-mouse Ig-agarose beads. Immunoprecipitates were
washed three times with lysis buffer and extracted for 10 min at 95 C
in 2 x SDS-PAGE sample buffer (200 mM Tris-HCl, 6%
SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol, pH
6.8) and analyzed by SDS-PAGE and as described in Results
and in the figure legends. Anti-p85 and anti-Grb-2 polyclonal
antibodies were from Santa Cruz (Santa Cruz Biotechnology, Palo Alto,
CA). Anti-SHC polyclonal antibody was from Upstate Biotechnology (Lake
Placid, NY). For IRS-1/IRS-2 immunoprecipitations, polyclonal
antibodies were obtained as described (12). For anti-Tyr(P)
immunoprecipitations, the Py72 monoclonal antibody was the generous
gift of Dr. E. Rozengurt and J. Sinnett-Smith (Imperial Cancer Research
Foundation, London). The monoclonal anti-Tyr(P) antibody used for
Western blotting (Py20) was purchased from Santa Cruz.
Western Blotting
After SDS-PAGE, proteins were transferred to Immobilon membranes
and were blocked using 5% nonfat dried milk in 10 mM
Tris-HCl and 150 mM NaCl, pH 7.5, and incubated overnight
with several antibodies as indicated in 0.05% Tween-20, 1% nonfat
dried milk in 10 mM Tris-HCl and 150 mM NaCl,
pH 7.5. Immunoreactive bands were visualized using the enhanced
chemiluminescence Western blotting protocol (Amersham, Arlington
Heights, IL).
PI 3-Kinase Activity
PI 3-kinase activity was measured by in vitro
phosphorylation of phosphatidylinositol as described (32). Fetal brown
adipocytes were incubated with IGF-I in the absence or presence of PI
3-kinase inhibitors as indicated in the figure legends. After washing
with ice-cold PBS, cells were solubilized in lysis buffer containing
leupeptin (10 µg/ml), aprotinin (10 µg/ml), and 1 mM
phenylmethylsulfonyl fluoride. Lysates were clarified by centrifugation
at 15,000 x g for 10 min at 4 C, and proteins were
immunoprecipitated with the anti-IRS-1/IRS-2 polyclonal antibodies. The
immunoprecipitates were washed successively in PBS containing 1%
Triton X-100 and 100 µM Na3VO4
(twice), 100 mM Tris (pH 7.5) containing 0.5 M
LiCl, 1 mM EDTA and 100 µM
Na3VO4 (two times), and 25 mM Tris
(pH 7.5) containing 100 mM NaCl and 1 mM EDTA
(twice). To each pellet were added 25 µl of 1 mg/ml
L--phospha-tidylinositol/L--phosphatidyl-L-serine
sonicated in 25 mM HEPES (pH 7.5) and 1 mM
EDTA.
The PI 3-kinase reaction was started by the addition of 100
nM [32P]ATP (10 µCi) and 300
µM ATP in 25 µl of 25 mM HEPES, pH 7.4, 10
mM MgCl2, and 0.5 mM EGTA. After 15
min at room temperature, the reaction was stopped by the addition of
500 µl CHCl3-methanol (1:2) in a 1% concentration HCl
plus 125 µl chloroform and 125 µl HCl (10 mM). The
samples were centrifuged, and the lower organic phase was removed and
washed once with 480 µl methanol-100 mM HCl plus 2
mM EDTA (1:1). The organic phase was extracted, dried
in vacuo, and resuspended in chloroform. Samples were
applied to a silica gel TLC plate. TLC plates were developed in 2
N propanol-1-acetic acid (65:35, vol/vol), dried,
visualized by autoradiography, and quantitated by scanning laser
densitometry (Molecular Dynamics personal densitometer, Sunnyvale,
CA).
Differential Binding of SH2 Domains from p85 and Grb-2 with
the IRS-Proteins
GST-fusion proteins containing nSH2p85 and
SH2Grb-2 were prepared as described (7). Cell lysates were
prepared from unstimulated, insulin-stimulated, or IGF-I-stimulated
fetal brown adipocytes in lysis buffer. The extracts were clarified by
centrifugation at 12,000 x g for 15 min at 4 C. The
supernatants were incubated with 10 µg of the GST fusion proteins
containing SH2 domains as indicated at 4 C for 1 h and
precipitated with glutathione Sepharose at 4 C for 1 h, washed
twice in 50 mM Tris/HCl (pH 7.4) containing 100
mM NaCl, 250 µg/ml BSA, 0.2 mM
Na3VO4, and 0.4 mM
phenylmethylsulfonyl fluoride, and boiled for 5 min in Laemmli sample
buffer. Samples were separated on 7% SDS-PAGE and analyzed by
immunoblotting.
Phosphopeptide Competition Studies
Phosphopeptide binding competition assay was performed as
described (33). Lysates from insulin-stimulated cells (600–800 µg of
protein) prepared as described above were incubated with the anti-Grb-2
antibody for 30 min at 4 C, divided into 1-ml aliquots, and then
incubated at 4 C for 14 h without or with various doses
(0.001–100 µM) of a synthetic phosphopeptide
(PPEPKSPGEY(P)VNIEFG) corresponding to the IRS-1/IRS-2 consensus Grb-2
binding domain (11). The immune complexes were collected on 40 µl
protein A-agarose, resolved on a 7% gel, and analyzed by
immunoblotting with the anti-IRS-1 and anti-IRS-2 antibodies.
Protein Determination
Protein determination was performed by the Bradford dye method
(34), using the Bio-Rad (Richmond, CA) reagent and BSA as the
standard.
Experimental Animals
The animals used for the required experiments in this report
were treated in accord with the "Guidelines for Care and Use of
Experimental Animals."
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FOOTNOTES |
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This work was supported by Grant SAF96/0115 from the Comision Interministerial de Ciencia y Tecnologia, Spain.
Received for publication May 27, 1997. Revision received October 29, 1997. Accepted for publication January 27, 1998.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 L. Poretsky, N. A. Cataldo, Z. Rosenwaks, and L. C. Giudice The Insulin-Related Ovarian Regulatory System in Health and Disease Endocr. Rev., August 1, 1999; 20(4): 535 - 582. [Abstract] [Full Text] [PDF]
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