From the Developmental Endocrinology Branch, NICHD,
National Institutes of Health, Bethesda, Maryland 20892-1862, § Division of Endocrinology, University of North Carolina
Medical School, Chapel Hill, North Carolina 27514, and
¶ Dipartimento di Biologia e Patologia Cellulare and Centro di
Endocrinologia e Oncologia Sperimentale CNR, University of Naples
Medical School, Naples, Italy
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ABSTRACT |
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Insulin receptor substrates (IRSs) are tyrosine-phosphorylated following stimulation with insulin, insulin-like growth factors (IGFs), and interleukins. A key question is whether different IRSs play different roles to mediate insulin's metabolic and growth-promoting effects. In a novel system of insulin receptor-deficient hepatocytes, insulin fails to (i) stimulate glucose phosphorylation, (ii) enhance glycogen synthesis, (iii) suppress glucose production, and (iv) promote mitogenesis. However, insulin's ability to induce IRS-1 and gab-1 phosphorylation and binding to phosphatidylinositol (PI) 3-kinase is unaffected, by virtue of the compensatory actions of IGF-1 receptors. In contrast, phosphorylation of IRS-2 and generation of IRS-2/PI 3-kinase complexes are markedly reduced. Thus, absence of insulin receptors selectively reduces IRS-2, but not IRS-1 phosphorylation, and the impairment of IRS-2 activation is associated with lack of insulin effects. To address whether phosphorylation of additional IRSs is also affected, we analyzed phosphotyrosine-containing proteins in PI 3-kinase immunoprecipitates from insulin-treated cells. However, these experiments indicate that IRS-1 and IRS-2 are the main PI 3-kinase-bound proteins in hepatocytes. These data identify IRS-2 as the main effector of both the metabolic and growth-promoting actions of insulin through PI 3-kinase in hepatocytes, and IRS-1 as the main substrate mediating the mitogenic actions of IGF-1 receptors.
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INTRODUCTION |
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Insulin, IGF-1,1 and IGF-2, acting through insulin and IGF-1 receptors, promote a wide range of metabolic and growth-promoting functions in typical insulin target cells, such as liver, muscle, and fat, and to a lesser extent in other tissues. The mechanism by which insulin regulates energy metabolism and promotes cell growth has been extensively studied. In recent years, a consensus has emerged that phosphorylation of IRS molecules by the insulin receptor kinase is important for insulin action (1). IRS molecules engage in the formation of signaling complexes with numerous adapter molecules and enzymes via their pY-X-X-M motifs (2-6). Thus, the IRS signaling system provides an elegant explanation for the diversity of insulin signaling (7). Nevertheless, the role of different IRSs in insulin signaling, as well as the role of the numerous additional substrates of the insulin receptor kinase that are distinct from IRS has remained elusive.
Progress in this area has been hampered by the lack of suitable in vitro systems in which phosphorylation of individual molecules can be correlated with specific biologic functions. In fact, insulin-responsive cell lines such as 3T3-L1 adipocytes or L6 myoblasts possess an endogenous complement of signaling molecules, so that the effects of individual components can be addressed only by way of overexpression or inhibition experiments. It is significant that much progress in our understanding of the IRS system has derived from studies of the myeloid cell line 32D, which carries a functional knock-out of these molecules (8-10). However, 32D cells may not be representative of classic target tissues of insulin action.
Targeted mutagenesis of genes of the insulin and IGF signaling system in mice has provided clues as to the functional differences among related molecules (11, 12). We and others, for example, have shown that insulin receptors are indeed the master switch of the insulin signaling pathway (13, 14), and that IGF-1 receptors contribute little to metabolic regulation (15-17). Likewise, the phenotype of mice with a genetic ablation of IRS-1 has suggested that IRS-1 plays a more important role in mediating growth than metabolic responses (18, 19). Interestingly, however, combined heterozygosity for an insulin receptor and an IRS-1 null allele triggers synergistic interactions to impair insulin action and causes insulin-resistant diabetes in mice, suggesting that IRS-1 can also affect metabolism (20). On the other hand, mice lacking IRS-2 develop lethal diabetic ketoacidosis as a result of combined insulin resistance and insulin deficiency, indicating that IRS-2 plays a crucial role in the development of mechanisms regulating fuel homeostasis (21).
The derivation of cell lines from mice with targeted mutations provides an important tool to dissect the function of these molecules in vitro. For example, Bruning et al. (22) have been able to show that IRS-1 and IRS-2 are not functionally interchangeable in mediating various growth-promoting functions of IGF-1 in fibroblasts of IRS-1-deficient mice.
In this study, we have analyzed insulin action in permanent cultures of hepatocytes from mice lacking insulin receptors. We generated these cells using a well established procedure entailing transformation with a temperature-sensitive mutant SV40 virus (23). We asked whether, in the absence of insulin receptors, IGF-1 receptors could mediate the typical metabolic and growth-promoting responses of insulin in hepatocytes. We report that insulin, acting through the IGF-1 receptor, mediates IRS-1, but not IRS-2 phosphorylation, and that the failure to phosphorylate IRS-2 correlates with the inability of these cells to mediate insulin's characteristic actions. Phosphorylation of gab-1, and association of other phosphotyrosine-containing proteins with the p85 subunit of PI 3-kinase, were similar in normal and insulin receptor-deficient cells. These findings correlate IRS-2 phosphorylation with both the metabolic and growth-promoting actions of insulin, and suggest that individual IRS molecules play a more specific role in signal transduction than previously recognized.
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MATERIALS AND METHODS |
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Derivation of Cell Lines--
Livers of transgenic mice lacking
insulin receptors and normal controls were isolated between embryonic
day 18.5 and postnatal day 1. The livers were digested with collagenase
(Worthington), and the resulting cells were plated in 6-cm culture
dishes. Transformation with a temperature-sensitive mutant SV40 was
performed as described previously (23). Multiple clones were isolated
and characterized. To confirm the hepatocyte lineage, albumin secretion
in the medium was measured using an enzyme-linked immunosorbent assay.
The experiments described in this study were performed on two
independent clones of /
cells, chosen based on high levels of
albumin secretion. I-insulin and 125I-IGF-1
binding were performed according to standard techniques (13). Cells
were maintained in
-minimal essential medium supplemented with 1 mM L-glutamine, 200 nM
dexamethasone, and 4% fetal calf serum at 33 °C. Preliminary
experiments were carried out at both the permissive temperature for
viral replication (33 °C) and the nonpermissive temperature
(40 °C). However, none of the parameters analyzed in this study was
affected by the different temperature (24).
Immunoprecipitation and Immunoblotting--
Serum-deprived
cultures of WT and /
cells (~80% confluent) were stimulated with
insulin at various concentrations for 5 min. Cells were lysed in
detergent buffer containing 50 mM HEPES, pH 7.6, 150 mM NaCl, 1% Triton, phosphatase and protease inhibitors. The lysates were immunoprecipitated with the appropriate antibodies as
described. In vitro phosphorylation of the glycoprotein
fractions isolated from both cell types were carried out as described
elsewhere (13). Anti-phosphotyrosine antibodies were purchased from
Transduction Laboratories (Lexington, KY), insulin receptor and IGF-1
receptor antibodies from Calbiochem, and anti-IRS-1, anti-IRS-2,
anti-Gab-1, and anti-p85 antibodies were purchased from Upstate
Biotechnology (Lake Placid, NY).
Glucokinase Activity--
Cells were cultured in serum-free
medium overnight and incubated in the absence or presence of 100 nM insulin for 3 h. Thereafter, cells were homogenized
in buffer containing 50 mM Tris-HCl, pH 7.4, 0.3 M sucrose, 0.1 M KCl, 1 mM EDTA,
and 2.5 mM -mercaptoethanol. Following centrifugation at
15,000 rpm for 15 min, the post-mitochondrial supernatant was
centrifuged at 180,000 × g for 1 h to obtain the cytosolic fraction. Glucokinase activity was measured by a
spectrophotometric assay, in the presence of 45 mM
Tris-HCl, pH 7.4, 110 mM KCl, 8 mM
MgCl2, 0.5 mM NADP, 0.9 unit/ml
glucose-6-phosphate dehydrogenase, 100 mM glucose, and ATP
(0 or 5 mM). Glucokinase activity was calculated as the
ATP-dependent rate of NADPH formation, based on a
stoichiometry of 2 mol of NADPH formed per mol of glucose phosphorylated (25).
Hepatic Glucose Production--
Cells were cultured overnight in
-minimal essential medium supplemented with 0.25% bovine serum
albumin, and incubated in glucose- and serum-free
-minimal essential
medium supplemented with 16 mM lactate and 4 mM
pyruvate in the presence or absence of 100 nM insulin.
Aliquots of medium were removed at the indicated time points, and the
glucose concentration was measured with a glucose analyzer (Beckman)
(26).
Glycogen Content-- SV40-transformed cells were cultured in serum-free medium as indicated above in the presence or absence of 100 nM insulin. Thereafter, cells were homogenized in 0.6 N HClO4 and centrifuged, and aliquots of the supernatant were incubated with amyloglucosidase in acetate buffer as described previously (27). Released glucose was measured with a glucose analyzer.
Glycogen Synthase Activity-- Cells were incubated with glucose-free medium supplemented with dialyzed fetal calf serum for 3 h. Insulin was added for 30 min at 37 °C. The reaction was stopped by freezing cells in liquid nitrogen. The reaction was performed as described in buffer containing 40 mM Tris-HCl, 25 mM NaF, 20 mM EDTA, 10 mg/ml glycogen, 7.2 mM UDP-glucose disodium salt, with and without 6.7 mM glucose 6-phosphate, and 0.05 µCi/60 µl [14C]UDPG for 25 min at 37 °C (28). The incorporation of [14C]UDPG was determined in a liquid scintillation counter. The ratio of glucose 6-phosphate-independent (I) glycogen synthase activity was calculated as shown: % I = I-form/(I- + D-form) × 100.
DNA Synthesis--
Hepatocytes were grown to near confluence in
regular medium, incubated overnight in insulin-free medium, and placed
in medium containing increasing concentrations of insulin
(1010 to 10
7 M) or buffer alone
for 16 h. Thereafter, the medium was replaced by the same medium
supplemented with HEPES (25 mM, pH 7.4) and [3H]thymidine (500 Ci/ml, NEN Life Science Products) for
1 h. Cells were washed three times, solubilized, and precipitated
with 20% trichloroacetic acid. Radioactivity was measured in a liquid
scintillation counter (28).
Growth Curves-- Hepatocytes were plated at concentrations of 5 to 50 × 104 cells/ml and allowed to attach to the plates in complete medium. Thereafter, they were cultured for 96 h in regular medium or 1% bovine serum albumin with or without varying concentrations of insulin, IGF-1, or IGF-2. At the end of the incubation, medium containing 2.5% neutral red (Sigma) was added for 2 h, and the absorbance was measured at 570 nm. Cell growth was expressed as percentage of the absorbance values. Basal values are represented by the growth of cells incubated in 1% bovine serum albumin and maximal values by the growth observed in cells incubated with complete medium.
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RESULTS |
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Protein Phosphorylation in Insulin Receptor-deficient
Hepatocytes--
Initially, we analyzed 125I-insulin
binding to cells derived from insulin receptor-deficient mice
(hereafter referred to as /
cells). Binding competition curves
(Fig. 1a) and Scatchard
analysis (Fig. 1b) are consistent with the absence of high
affinity insulin binding sites in
/
cells. The residual
125I-insulin binding (ID50 = 100 nM) detected in
/
cells can be attributed to the
presence of IGF-1 receptors, as we have previously shown (17). Indeed,
the linear Scatchard plot of
/
cells is typical of binding to IGF-1
receptors (Fig. 1b). From the intercept of the slope on the
horizontal axis of the Scatchard plot, we estimate that WT cells
express ~105 insulin receptors/cell. Previously, we have
shown that the levels of expression of IGF-1 receptors are similar in
WT and
/
cells (~105 IGF-1 receptors/cell).
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IRS-1 and -2 Phosphorylation and PI 3-Kinase
Co-precipitation--
Next, we investigated the ability of insulin to
activate intracellular signaling pathways in both cell types. Insulin
stimulated tyrosine phosphorylation of IRS-1 in WT and /
cells to a
similar extent. In contrast, tyrosine phosphorylation of IRS-2 was
severely blunted in
/
cells compared with WT cells (Fig.
3). Upon tyrosine phosphorylation, IRS
molecules bind the regulatory (p85) subunit of PI 3-kinase, leading to
increased PI 3-kinase activity. Insulin treatment led to a 3.4-fold
increase in the amount of p85 co-precipitated with IRS-1 in WT cells,
and to a 5.3-fold increase in
/
cells (Fig.
4b). In contrast, the amount
of p85 detected in anti-IRS-2 immunoprecipitates was increased 3.3-fold
in WT cells, and 1.3-fold in
/
cells (Fig. 4c). Thus,
insulin can fully activate IRS-1 through IGF-1 receptors, whereas
activation of IRS-2 is substantially reduced. The decreased
co-precipitation of p85 with IRS-2 suggests that IRS-2-associated PI
3-kinase activity is reduced in
/
cells. In fact, it has been shown
that activation of PI 3-kinase by insulin requires binding of tyrosine
phosphorylated IRSs to the SH2 domains of p85 (29-31). Thus, this
experiment provides insight into activation of PI 3-kinase in response
to insulin.
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Investigation of Additional Insulin Receptor Substrates That
Associate with p85--
We then addressed whether activation of other
molecules important for insulin action is also blunted in /
cells.
Gab-1 is a member of the IRS family, phosphorylation of which is
stimulated by both insulin and IGF-1 (6). Tyrosine phosphorylation of gab-1 in insulin-treated WT and
/
cells was similar (data not shown). Two additional members of the IRS family have been described: IRS-3 and IRS-4 (4, 5). Of these, IRS-3 is known to be expressed in
liver (32), while no information is available on liver expression of
IRS-4 (5). Since we did not have specific antibodies against these two
IRSs, we asked whether we could detect them in co-immunoprecipitation assays with anti-p85 antibodies followed by anti-phosphotyrosine immunoblots. In both cell types, the main p85-bound phosphoproteins following insulin treatment are represented by two broad bands at
160-180 kDa and 125 kDa (Fig. 4a). Detailed analysis of
these patterns leads us to conclude that, among the major
p85-associated proteins, only IRS-2 co-immunoprecipitation is
significantly impaired in
/
cells compared with WT cells: (i) the
p160-180 band can be almost entirely immunodepleted by sequential
immunoprecipitation with IRS-1 and IRS-2 antibodies (compare the
intensity of the p160 band in Fig. 4, a and d,
with the intensity of the non-insulin-stimulated band present at the
top of each gel). A residual p160 band can be detected in extracts
immunodepleted of IRS-1 and IRS-2 (Fig. 4d). This 160-kDa
protein migrates slightly more slowly than IRS-1 and IRS-2, and does
not cross-react with either antibody on Western blots. It may represent
IRS-4, or a novel member of the IRS family. Co-precipitation of this
band is similar in both cell types. A minor band of molecular mass of
150 kDa is also present in WT cells, and completely absent in
/
cells. Co-precipitation of this tyrosine-phosphorylated protein is
modestly affected by insulin in WT cells. (ii) Co-precipitation of the
p125 band is only slightly decreased in
/
cells (Fig.
4a). This band probably contains multiple molecular species,
including gab-1. As stated above, phosphorylation of gab-1 is similar
in both cell types. (iii) Following immunodepletion of IRS-1 and IRS-2,
we observed a 60-kDa band, co-precipitation of which is modestly
affected by insulin in WT cells (~2-fold), but not in
/
cells
(Fig. 4e). It is highly likely that p60 represents IRS-3 (4,
32, 33).
Effects of Insulin on Glucose Metabolism Are Blunted in /
Hepatocytes--
Hepatocytes are an ideal model to study insulin
action, because insulin exerts both metabolic and growth-promoting
effects in these cells. We asked whether the differences observed in
the signaling pathway would be mirrored by changes in the ability of
/
cells to respond to insulin in assays of glucose metabolism. Insulin stimulated glucokinase (Fig.
5a) and glycogen synthase activities (Fig. 5b), and suppressed glucose production in
WT cells (Fig. 5c, left panel). In
/
cells,
basal rates of glucokinase activity and glycogen synthesis, as well as
glycogen content (not shown) were decreased compared with WT cells.
Glucose production rates, on the other hand, were about 2-fold higher
(Fig. 5c, right panel) in
/
cells. Moreover,
insulin was totally ineffective in modulating these metabolic
activities in
/
cells, despite the activation of IGF-1 receptors.
Thus, IGF-1 receptor-mediated activation of IRS-1 is not sufficient to
mediate insulin's metabolic actions in liver.
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Growth-promoting Actions of Insulin--
Insulin has a potent
effect to stimulate DNA synthesis and cell growth in hepatocytes (34).
In /
cells, thymidine incorporation in response to insulin was
decreased by >80% compared with normal cells, consistent with
impaired DNA synthesis (Fig.
6a). We also studied the
ability of WT and
/
cells to replicate (Fig. 6b). Both
cell types grew at similar rates in the presence of serum. Insulin and
IGF-2 were equally effective in stimulating growth of serum-deprived WT
cells and ~2-fold more potent than IGF-1. In
/
cells, insulin-
and IGF-2-dependent growth was decreased by ~50%
(p < 0.01), while no differences were observed in
IGF-1-dependent growth.
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DISCUSSION |
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In these investigations, we have analyzed the correlation between IRSs phosphorylation and insulin action in hepatocytes derived from insulin receptor-deficient mice. We provide evidence that impaired activation of IRS-2 in these cells is associated with failure of both metabolic and growth-promoting actions of insulin. An important result of the present study is that ablation of insulin receptors results in a selective loss of IRS-2 phosphorylation, in the absence of detectable changes in IRS-1 phosphorylation. Indirect evidence suggests that additional IRSs are minor components of the IRS signaling system in this cell type. Thus, these data support the notion that signaling from insulin receptors to IRS-2 is required for the characteristic actions of insulin in liver. Since our data were obtained in the context of a physiologic target cell of insulin action, we believe that they add significant new information to our understanding of the insulin signaling system.
The link between impaired phosphorylation of IRS-2 and impaired insulin
action is further demonstrated by the decrease in the formation of
IRS-2/PI 3-kinase complexes in /
cells. There is substantial
evidence that PI 3-kinase is required for many, if not all, of insulin
actions (35-46). While in this study we did not measure PI 3-kinase
activity directly, it is well established that insulin activates PI
3-kinase by causing IRSs to bind to the SH2 domains of the p85 subunit
(29-31). Thus, there is an excellent correlation between
co-precipitation of IRSs with p85 and PI 3-kinase activity. Our
analysis of the patterns of tyrosine-phosphorylated proteins detected
in p85 immunoprecipitates indicates that the main proteins bound to p85
in hepatocytes are indeed IRS-1 and IRS-2, and therefore account for
most of PI 3-kinase activity elicited by insulin in these cells.
Furthermore, we have recently been able to show that insulin fails to
stimulate Akt activity in
/
cells, which is consistent with an
impairment of PI 3-kinase activity2 (47-50). These
data provide support to the notion that impairment of IRS-2
phosphorylation in
/
cells is one of the mechanisms of the failure
of insulin action, and not an epiphenomenon.
The conclusion that IRS-2 plays an important role in mediating the metabolic actions of insulin is supported by several lines of independent evidence. IRS-2-deficient mice are diabetic as a result of combined insulin resistance and impaired insulin production (21), while IRS-1-deficient mice are growth retarded, but mildly insulin-resistant (18, 19). Furthermore, normal insulin action in liver of IRS-1-deficient mice is associated with increased phosphorylation of IRS-2 (51). Based on the phenotypes of insulin receptor-, IGF-1 receptor-, IRS-1-, and IRS-2-deficient mice (13-15, 18, 19, 21), one has to conclude that IRS-1 is predominantly an IGF-1 receptor substrate, and IRS-2 an insulin receptor substrate. This conclusion is strengthened by the present findings, indicating that insulin receptors are required for optimal IRS-2 phosphorylation. By suggesting that IRS-2 functions primarily as an insulin receptor substrate, our data provide a compelling explanation for the phenotype of mice lacking IRS-2 (21), and corroborate the findings of Bruning et al. that IRS-1 and IRS-2 are functionally distinct molecules (22). Substrate selection, however, is likely to be a more complex event in vivo, as indicated by studies of mice with combined null mutations of the insulin receptor and IRS-1 genes, which develop insulin resistant diabetes with significantly higher frequency than mice heterozygous for each individual mutation (20).
An important question raised from our studies is why IGF-1 receptors fail to phosphorylate IRS-2 as insulin receptors do. There is ample evidence that IRS-1 and IRS-2 utilize different mechanisms to interact with their receptor partners (52-54). This evidence, however, does not support a different mode of interaction of insulin and IGF-1 receptors with IRS-1 and IRS-2. An alternative possibility is that the subcellular localization of IRS-2 in hepatocytes prevents its efficient phosphorylation by IGF-1 receptors. A similar mechanism has been postulated to explain the differences between epidermal growth factor and insulin signaling (55), but there is no direct evidence that a similar mechanism may be at play in this instance.
The failure of insulin at high doses to activate metabolic responses through IGF-1 receptors is an important finding and deserves further comment. There has been considerable controversy over the ability of IGF-1 receptors to mediate metabolic actions (56). We have previously shown that IGF-1 receptors are weak mediators of metabolic effects in mice lacking insulin receptors (17). We postulated that IGF-1 may enhance peripheral glucose uptake in muscle and decrease hepatic gluconeogenesis. Based on those data, we proposed that IGF-1 may act on hepatic glucose production either directly through IGF-1 receptors, or indirectly through inhibition of glucagon secretion. The failure of IGF-1 receptors to impinge on hepatic glucose metabolism favors an indirect mechanism as a more likely explanation of our previous findings, but further studies comparing gluconeogenetic rates are required.
The impairment of insulin-mediated growth in /
cells correlated
with the loss of insulin receptor-mediated IRS-2 phosphorylation, suggesting that growth-promoting signaling of insulin receptors occurs
prevalently through IRS-2. On the other hand,
IGF-1-dependent growth occurred normally in
/
cells,
indicating that IGF-1 receptors signal through IRS-1, phosphorylation
of which is not affected in
/
cells. It is possible that some of
the effects of insulin or IGF-1 on growth are mediated by shc through
the grb-2/mSOS pathway to mitogen-activated protein kinase (41, 57,
58), although this point remains controversial (59-61). This
possibility is currently under investigation. However, it is
interesting to note that Bruning and co-workers have shown that
IGF-1-dependent growth of IRS-1-deficient fibroblasts is
impaired despite normal activation of mitogen-activated protein kinase,
and cannot be rescued by IRS-2, consistent with our model in which
IGF-1 receptors utilize primarily IRS-1 to mediate their actions on
cell growth (22). The impairment of IGF-2-mediated growth in
/
cells is consistent with previous evidence from our laboratory and
others that insulin receptors mediate the growth-promoting actions of IGF-2 (62, 63).
In conclusion, our data support a model in which the specificity of insulin signaling in liver is bestowed by the formation of a signaling complex between insulin receptors and IRS-2. It remains to be seen whether a similar mechanism operates in other cell types, for example in insulin-dependent translocation of glucose transporters in skeletal muscle and adipose tissue.
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ACKNOWLEDGEMENTS |
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We thank Janice Y. Chou for help in establishing the cell lines, Drs. Don Bottaro, Derek LeRoith, and Yoshiaki Kido for helpful discussions. We are indebted to Prof. Jesse Roth for countless insightful comments in the preparation of the manuscript.
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FOOTNOTES |
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* This work was supported in part by a generous gift from Sigma Tau Pharmaceuticals and by a research grant of the American Diabetes Association (to D. A.), by Telethon Grant E554 and European Community Grant BMH4-CT-0751 (to F. B.), and by a grant from Associazione Italiana Ricerca sul Cancro (to P. F.).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: Bldg. 10, Rm. 10D
18, National Institutes of Health, Bethesda, MD 20892-1862. Tel.:
301-496-9595; Fax: 301-402-0574; E-mail:
accilid{at}cc1.nichd.nih.gov.
1 The abbreviations used are: IGF, insulin-like growth factor; IRS, insulin receptor substrate; WT, wild type; PI, phosphatidylinositol.
2 B.-C. Park and D. Accili, unpublished observation.
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REFERENCES |
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