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Endocrinology Vol. 142, No. 8 3354-3360
Copyright © 2001 by The Endocrine Society

ARTICLES

Mitogenic and Metabolic Effects of Type I IGF Receptor Overexpression in Insulin Receptor-Deficient Hepatocytes

Jane J. Kim, Byung-Chul Park, Yoshiaki Kido and Domenico Accili

Naomi Berrie Diabetes Center and Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032

Address all correspondence and requests for reprints to: Domenico Accili, M.D., Russ Berrie Science Pavilion, 1150 St. Nicholas Avenue, New York, New York 10032. E-mail: da230{at}columbia.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that hepatocytes lacking insulin receptors (Ir-/-) fail to mediate metabolic responses, such as stimulation of glycogen synthesis, while retaining the ability to proliferate in response to IGFs. In this study we have asked whether overexpression of type I IGF receptors would rescue the metabolic response of Ir-/- hepatocytes. After IGF-I stimulation, insulin receptor substrate-1 and -2 phosphorylation and PI3K activity were restored to levels similar to or greater than those seen in wild-type cells. Rates of cell proliferation in response to IGF-I increased approximately 2-fold, whereas glycogen synthesis was restored to wild-type levels, but was comparatively smaller than that elicited by overexpression of insulin receptors. In summary, overexpression of IGF-I receptors in Ir-/- hepatocytes normalized insulin receptor substrate-2 phosphorylation and glycogen synthesis to wild-type levels, whereas it increased cell proliferation above wild-type levels. Moreover, stimulation of glycogen synthesis was submaximal compared with the effect of insulin receptor overexpression. We conclude that IGF-I receptors are more efficiently coupled to cell proliferation than insulin receptors, but are less potent than insulin receptors in stimulating glycogen synthesis. The data are consistent with the possibility that there exist intrinsic signaling differences between insulin and IGF-I receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INSULIN RECEPTOR and the IGF-I receptor promote growth and metabolism (1). However, despite a high degree of homology and the activation of similar signaling pathways, they exert distinct biological effects on target tissues. This is underscored by the divergent phenotypes of mice with targeted null mutations of the two genes. Mice lacking insulin receptors are born with near-normal body size, but die within a few days of diabetic ketoacidosis (2, 3). In contrast, mice lacking IGF-I receptors are small at birth and die of respiratory failure due to hypoplasia of intercostal muscles (4). These data support the classical paradigm, according to which the insulin receptor regulates metabolism, whereas the IGF-I receptor promotes growth (5). However, mice lacking both insulin and IGF-I receptors are smaller than mice lacking IGF-I receptors alone, indicating that insulin receptors can also promote fetal growth (6). Genetic (6) and biochemical evidence (7, 8) indicates that the fetal growth-promoting actions of insulin receptors are triggered by IGF-II.

It is not clear what determines the specificity of insulin vs. IGF-I signaling. In vivo, insulin and IGF-I concentrations differ by at least 2 orders of magnitude (9). However, IGF-I bioavailability is strictly determined by circulating IGF-binding proteins (10), whereas insulin secretion is under tight metabolic control, providing an initial mechanism to control activation of the respective receptors. Moreover, differences in tissue distribution and relative abundance of the two receptors are likely to determine some differences in their biological effects (11). In vitro studies, on the other hand, have provided inconclusive evidence as to whether the two receptors possess specific or promiscuous signaling capabilities. There is limited evidence for intrinsic differences in the kinase activity of the purified catalytic domains of the insulin or IGF-I receptor expressed as glutathione-S-transferase fusion proteins in Escherichia coli (12). Both receptors are able to activate similar signaling pathways, the insulin receptor substrate (IRS)-PI3K-Akt and the growth factor receptor binding protein 2 and Son of Sevenless-MAPK pathways (13, 14, 15). However, there is evidence for insulin receptor- or IGF-I receptor-specific substrates (16, 17, 18, 19, 20, 21, 22), leading to, for example, cell-specific protection from apoptosis (23, 24, 25).

Transfection experiments have indicated that both insulin and IGF-I receptors are capable of stimulating cell proliferation, although the relative potencies of the two receptors appear to differ (13, 15, 26). The ability of IGF-I receptors to mediate metabolic signaling remains unclear. Transfections of chimeric receptors into 3T3-L1 adipocytes have suggested that IGF-I receptors are less potent than insulin receptors in stimulating glycogen synthesis (27), although another study failed to replicate these findings (28). In vivo, we have previously reported that IGF-I has a hypoglycemic effect, but is unable to halt the progression of the diabetic ketoacidosis and rescue mice lacking insulin receptors from death (29). Based on these data, we have suggested that the IGF-I receptor is able to mediate some, but not all, of the metabolic actions of the insulin receptor in vivo. Further evidence to this effect comes from studies of hepatocytes derived from insulin receptor-deficient mice. These cells lack insulin receptors, but express IGF-I receptors (~105/cell), as expected of neonatal hepatocytes (30). As hepatocytes possess an intrinsic ability to respond to the metabolic actions of insulin, they enable us to circumvent limitations of prior studies, in which the effect of IGF-I receptor overexpression was confounded by the presence of endogenous insulin receptors (28). In previous work we have shown that these cells lack the ability to promote glucose phosphorylation and glycogen synthesis and to inhibit glucose production (31, 32). In the present study we show that overexpression of IGF-I receptors in Ir-/- cells restores IRS-2 phosphorylation and glycogen synthesis to wild-type levels, while increasing cell proliferation above wild-type levels. Moreover, we show that the effect of IGF-I receptors on glycogen synthesis is comparatively smaller than that of insulin receptors, suggesting that the IGF-I receptor is intrinsically less suited to metabolic signaling than the insulin receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and Western blotting
The following antibodies were used: antiphosphotyrosine antibodies from Transduction Laboratories, Inc. (Lexington, KY); antiinsulin receptor (C-19) and anti-IGF-I receptor (C-20) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-IRS-1, anti-IRS-2, and anti-p85{alpha} (PI3K) antibodies from Upstate Biotechnology, Inc. (Lake Placid, NY); and antiphospho-glycogen synthase kinase (GSK)-3{alpha}/ß (Ser21/9) from New England Biolabs, Inc. (Beverly, MA). Western blotting was performed according to standard techniques, followed by detection with enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK). The intensity of the bands corresponding to the various proteins was quantitated by autoradiography, followed by analysis with NIH Image 1.60 software.

Cell culture and transfections
The generation of permanent hepatocyte cultures from insulin receptor-deficient mice (Ir-/-) and normal controls [wild type (WT)] using viral transformation with a temperature-sensitive strain of simian virus 40 (SV40; tsA) has been described in previous publications (31, 32, 33, 34, 35). Cells were maintained in {alpha}MEM supplemented with 1 mM L-glutamine, 4 nM dexamethasone, and 4% FCS at 33 C. Cells were incubated at 37 C before the experiments.

Transfection of hepatocytes with pcDNA3-hIR, an expression plasmid encoding the WT human insulin receptor cDNA, was described previously (32). Transient overexpression of IGF-IR was similarly accomplished using a pcDNA3-hIGF-IR expression vector, derived by cloning the hIGF-IR cDNA into the EcoRI and XhoI sites of plasmid pcDNA3 (Invitrogen, Carlsbad, CA). Plasmid pBPV-hIGF-IR was used to achieve overexpression of IGF-I receptors after stable transfection of Ir-/- hepatocytes (36). Fifteen micrograms of purified plasmid DNA were mixed before transfection with 1 µg pTK-Hyg vector (CLONTECH Laboratories, Inc., Palo Alto, CA), which confers hygromycin resistance. Semiconfluent Ir-/- hepatocytes in 10-cm petri dishes were transfected using Lipofectamine Plus transfection reagent (GIBCO Life Technologies, Rockville, MD). Three days after transfection, selection medium containing 400 mg/liter hygromycin (Calbiochem, La Jolla, CA) was added. After culturing cells for 2 wk in selection medium, individual clones were isolated and expanded. Expression of IGF-I receptors in each clone was assessed by Western blotting with anti-IGF-I receptor antibody C-20.

Insulin- and IGF-I-dependent protein phosphorylation
Serum-deprived cultures of WT and Ir-/- cells (~80% confluent) were stimulated with insulin or IGF-I at different concentrations for various lengths of time. Thereafter, the reaction was stopped by freezing cells on a dry ice/ethanol bath, and cells were directly solubilized in detergent buffer containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 1% Triton X-100, phosphatase, and protease inhibitors. The lysates were immunoprecipitated with the appropriate antibodies and analyzed by SDS-PAGE followed by Western blot as previously described (31).

Measurements of PI3K activity
After immunoprecipitation with anti-phosphotyrosine antibodies, immune complexes were washed and resuspended in 20 µl phosphatidylinositol (0.5 mg/ml) sonicated with 25 mM HEPES (pH 7.1), 0.5 mM EGTA, and 0.5 mM sodium phosphate. The phosphorylation reaction was started by the addition of 10 µl 250 µM ATP containing 5µCi [{gamma}-32P]ATP and incubated for 6 min at room temperature. The reaction was stopped by adding 15 µl 4 N HCl. Phospholipids were extracted with 130 µl CHCl3/methanol (1:1), and 30 µl of the CHCl3 layer were resolved on TLC plates (37). Radiolabeled spots corresponding to PI3-monophosphate were quantitated with NIH Image 1.60 software.

Proliferation of transfected cells
Cells were plated in quadruplicate in 96-well plates at concentrations of 2–4 x 105/ml and allowed to attach to the plates in complete medium overnight. Thereafter, they were cultured for 72 h in serum-containing medium or in 0.1% BSA with or without 100 nM insulin or IGF-I. At the end of the incubation period, medium containing 2.5% neutral red (Sigma) was added for 2 h, and absorbance was measured at 595 nm. Absorbance at 655 nM was subtracted as background. Cell proliferation was determined from the absorbance values and plotted on a scale between 0–100%, in which proliferation in the presence of medium supplemented with 1% BSA represented the basal growth rate (0%), and proliferation in the presence of standard medium (containing 4% FBS) represented the maximal growth rate (100%) (31).

Measurements of glycogen synthesis
After transient transfection, confluent monolayers of hepatocytes in six-well plates were incubated overnight in medium containing 1% dialyzed FCS, then for 3 h in serum-free {alpha}MEM containing 5.5 mM glucose and 0.33 Ci/ml D-[U-14C]glucose in the presence or absence of 100 mM insulin or IGF-I. Glycogen synthesis was determined by measuring the incorporation of D-[U-14C]glucose into glycogen as described previously (32).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF-I receptor overexpression in SV40-transformed hepatocytes
To address whether overexpression of IGF-I receptors would restore metabolic responses in Ir-/- hepatocytes, cells were transfected with an expression vector encoding human IGF-I receptor cDNA. Western blot analysis was used to assess expression levels achieved after stable transfection in Ir-/- hepatocytes. Untransfected hepatocytes (either WT or Ir-/-) express approximately 105 IGF-I receptors/cell (31) (Fig. 1Go, lanes 1 and 2). After stable transfection with pBPV-hIGF-IR, the levels of IGF-I receptor ß-subunit increased between 2-fold (lane 7) and 5-fold (lanes 3–5 and 8). Subsequent experiments were performed using the stably transfected clone shown in lane 8 unless otherwise indicated. After transient transfection, IGF-IR expression was similarly increased (5-fold higher than in untransfected cells; data not shown).



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  		Figure 1. Expression of IGF-I receptors after stable transfection in Ir-/- hepatocytes. Detergent extracts were prepared from WT (lane 1) and Ir-/- cells (lanes 2–9) and immunoprecipitated with anti-IGF-I receptor antibody C-20. Immunoblotting was performed with the same antibody. Lane 1, Untransfected WT cells; lane 2, untransfected Ir-/- cells; lanes 3–9, seven different clones obtained after stable selection with hygromycin.

 
We next examined IGF-I receptor phosphorylation in transfected cells. After the addition of IGF-I to the culture medium, phosphate incorporation into the IGF-I receptor tyrosine residues increased in a dose-dependent manner and reached a maximum at 100 nM IGF-I. IGF-I caused greater receptor phosphorylation than insulin at equimolar ligand concentrations (Fig. 2Go, compare lanes 7 and 8).



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  		Figure 2. IGF-I- and insulin-induced phosphorylation of IGF-I receptors in hepatocytes. Confluent monolayers of Ir-/- hepatocytes were stimulated with various doses of IGF-I or insulin for 5 min. Thereafter, receptor phosphorylation was assessed by immunoprecipitation with antiphosphotyrosine antibody, followed by immunoblotting with anti-IGF-I receptor antibody C-20. The data were quantitated using scanning densitometry of the autoradiograms. A representative experiment is shown. Lanes 1–3, Untransfected cells; lanes 4–8, cells transfected with IGF-I receptor expression vector.

 
IGF-I receptor expression restores IRS-2 phosphorylation
We next studied the activation of intracellular signaling pathways. IGF-I increased IRS-1 phosphorylation about 2-fold in both WT cells and Ir-/- cells (Fig. 3Go, lanes 6–9). Likewise, IRS-1 tyrosine phosphorylation was stimulated in a dose-dependent manner up to approximately 2-fold in IGF-I-treated Ir-/- cells overexpressing IGF-I receptors (Fig. 3Go, lanes 1–4). Thus, there were no significant differences between untransfected and IGF-I receptor-transfected Ir-/- cells with respect to IRS-1 tyrosine phosphorylation. In contrast, IGF-I-stimulated phosphorylation of IRS-2 was decreased by about 70% in Ir-/- cells compared with WT cells (Fig. 4Go, compare lanes 7 and 9). This is consistent with our previous observations that IRS-1 appears to be phosphorylated more efficiently than IRS-2 in Ir-/- hepatocytes (31). Overexpression of IGF-I receptors in Ir-/- hepatocytes restored IRS-2 phosphorylation to levels slightly higher than those in WT cells. These data show that IGF-I receptors can efficiently phosphorylate IRS-2 when overexpressed and suggest that the decrease in IRS-2 phosphorylation in Ir-/- hepatocytes is not due to an intrinsic failure of IGF-I receptors to engage IRS-2.



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  		Figure 3. IGF-I- and insulin-induced IRS-1 phosphorylation in hepatocytes. WT and Ir-/- hepatocytes were incubated with various doses of IGF-I or insulin for 5 min. IRS-1 was immunoprecipitated with an antiserum raised against the PH domain of the human IRS-1 molecule, and tyrosine phosphorylation was detected with antiphosphotyrosine antibody (upper panel). The total amount of IRS-1 present on the filter was determined by reblotting the filter with anti-IRS-1 antibody (middle panel). A representative experiment is shown. In the lower panel, the mean ± SEM stimulation of IRS-1 phosphorylation was calculated from several independent experiments using scanning densitometry of the autoradiograms to quantitate the intensity of the bands.

 


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  		Figure 4. IGF-I- and insulin-induced IRS-2 phosphorylation in hepatocytes. Detection of IRS-2 tyrosine phosphorylation was carried out as described in Fig. 3Go, except that an antibody against human IRS-2 was employed for immunoprecipitation. Upper panel, Immunoblot with antiphosphotyrosine antibody; middle panel, immunoblot with anti-IRS-2 antibody to normalize protein levels. A representative experiment is shown. In the lower panel, the mean ± SEM IRS-2 phosphorylation was calculated from several independent experiments, using scanning densitometry of the autoradiograms to quantitate the intensity of the bands.

 
IGF-I receptor overexpression enhances PI3K activity in Ir-/- hepatocytes
We next measured stimulation of the lipid kinase activity of the enzyme PI3K, a key mediator of insulin signaling (38). After IGF-I stimulation, levels of PI3K activity increased approximately 10-fold and did not differ significantly between WT and Ir -/- cells. Overexpression of IGF-I receptors increased PI3K activity about 15-fold, consistent with the observed increase in IRS-2 phosphorylation (Fig. 5Go; P < 0.02).



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  		Figure 5. IGF-I-stimulated PI3K activity. Subconfluent monolayers of WT and Ir-/- cells were treated with 100 nM IGF-I for 5 min and quickly frozen. Thereafter, cells were solubilized and immunoprecipitated with antiphosphotyrosine antibody. PI3K activity was measured in the immunoprecipitates using phosphatidylinositol as a substrate (37 ). The reaction products were resolved on TLC plates. Radiolabeled spots corresponding to PI3-monophosphate were quantitated with NIH Image 1.60 software. *, P < 0.02, by one-factor ANOVA.

 
IGF-I receptor overexpression increases GSK-3{alpha} phosphorylation in Ir-/- hepatocytes
It has been proposed that phosphorylation of GSK-3 is one of the mechanisms by which insulin stimulates glycogen synthesis. Therefore, we examined GSK-3 phosphorylation after ligand stimulation by Western blot using an anti-pS21/9 antibody. We identified both the 51-kDa GSK-3{alpha} and 46-kDa GSK-3ß isoforms in SV40-transformed hepatocytes. A fainter band migrating at approximately 55 kDa, which probably corresponds to a recently described GSK-3{alpha} isotype (39), was also seen (Fig. 6Go). In Ir -/- cells, none of the three GSK-3 isoforms underwent phosphorylation after insulin or IGF-I treatment, consistent with our previous observations of GSK-3 activity (32). In contrast, in WT cells we observed a 5-fold rise in GSK-3{alpha} phosphorylation after insulin stimulation and a 1.8-fold increase after IGF-I stimulation (Fig. 6Go, inset). In Ir-/- cells overexpressing IGF-I receptors, phosphorylation of the 51-kDa GSK-3{alpha} increased 3.4-fold after insulin stimulation and 5-fold after IGF-I stimulation, similar to the increase in insulin-treated WT cells. The higher Mr isoform and the 47-kDa GSK-3ß isoform were not phosphorylated under our experimental conditions, consistent with other reports (Fig. 6Go) (39).



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  		Figure 6. GSK-3 phosphorylation. WT and Ir-/- cells were treated with insulin or IGF-I for 5 min and solubilized in buffer containing Triton X-100 as described in Materials and Methods. Total cellular lysates were resolved by SDS-PAGE and transferred to nylon membranes. The membranes were probed with anti-phospho-GSK-3 antibody. A representative autoradiogram obtained from WT cell is shown in the inset. Lane 1, Untreated cells; lane 2, insulin-treated cells; lane 3, IGF-I-treated cells. Bands corresponding to the GSK-3{alpha} 51-kDa subunit were quantitated by scanning densitometry. The results of these experiments are shown in the graph.

 
IGF-I receptor overexpression in Ir-/- hepatocytes confers IGF-dependent glycogen synthesis
Insulin increases hepatic glycogen synthesis by a variety of mechanisms (40). Although the magnitude of insulin’s effect is small, this increase in the hepatic glycogen pool has important physiological consequences, as hepatic glycogen represents a larger percentage of total body glycogen in rodents than in humans. To evaluate the role of IGF-I receptor in glucose metabolism, we studied glycogen synthesis in Ir-/- cells overexpressing IGF-I receptors. In this experiment we compared the effect of IGF-I receptor overexpression with that of insulin receptor overexpression (Fig. 7Go). In untransfected WT cells, insulin stimulation resulted in a near doubling of glycogen levels, whereas IGF-I stimulation of Ir-/- cells failed to elicit measurable increases in glycogen synthesis (32). After transfection of insulin receptors into Ir-/- cells, basal glycogen synthesis increased 60%, and insulin stimulation resulted in a 4-fold increase over the levels observed in WT cells. In contrast, overexpression of IGF-I receptor increased basal values by 30% and restored insulin- or IGF-I-dependent glycogen synthesis to the levels observed in untransfected WT cells (Fig. 7Go). The levels of insulin and IGF-I receptors in transfected cells were similar (data not shown).



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  		Figure 7. Glycogen synthesis. After transient transfection with expression plasmids encoding either insulin or IGF-I receptors, hepatocytes were labeled with 0.33 Ci/ml D-[U-14C]glucose in the presence or absence of 100 mM insulin or 100 mM IGF-I. Glycogen synthesis was determined by measuring the incorporation of D-[U-14C]glucose into glycogen as described previously (32 ).

 
IGF-I receptor overexpression promotes proliferation of Ir-/-hepatocytes
We next evaluated the mitogenic effects of IGF-I receptor overexpression by comparing the ability of transfected cells to grow in medium supplemented with IGF-I or insulin and comparing it to growth in the presence of FCS. In all cell types, IGF-I stimulated cell growth more potently than insulin (Fig. 8Go). In untransfected Ir-/- cells, incubation with insulin or IGF-I resulted in 2% or 42% of the growth effect of FCS, respectively. In contrast, WT cells grew at rates 18% and 51% of those for cells in serum-supplemented medium after incubation with insulin and IGF-I, respectively. Overexpression of IGF-IR increased the growth rates in response to insulin and IGF-I to 45% and 102% of the effect of FCS, respectively (Fig. 8Go).



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  		Figure 8. Cell growth. Cells were plated at different concentrations and allowed to attach to the plates in complete medium before being cultured in serum-containing medium or in 1% BSA with or without varying concentrations of insulin or IGF-I. At the end of the incubation period, medium containing 2.5% neutral red (Sigma) was added for 2 h, and absorbance was measured at 595 nm. Absorbance at 655 nm was subtracted as background. Cell growth is expressed as percentage of the absorbance values (31 ).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Insulin and IGF-I receptors are structurally very similar (41, 42). In addition to binding each other’s ligand with low affinity, they both bind IGF-II with high affinity (7, 8). Moreover, they form heterodimeric insulin/IGF-I receptors in cells where they are coexpressed (43, 44, 45). Finally, their signaling pathways are tantalizingly similar (46). For these reasons, the identification of the molecular basis of their distinct biological properties has proved to be an unrewarding task. The emerging paradigm from studies of genetically modified mice is that insulin receptors can mimic the growth-promoting actions of IGF-I receptors, whereas IGF-I receptors cannot fully mimic the metabolic actions of insulin receptors (6, 29, 47). This is based on three lines of evidence: 1) combined lack of Ir and Igf1r results in greater growth retardation than lack of either receptor alone (6); 2) Ir can promote the growth of mice with combined lack of Igf1r and Igf2r (47); and 3) IGF-I administration cannot fully reverse the metabolic phenotype due to lack of Ir (29). In this study we addressed whether this is the result of intrinsic differences in the signaling properties of the two receptors or is due to extrinsic differences, such as receptor number on the surface of target cells.

In prior studies we observed that the metabolic impairment observed in Ir-/- hepatocytes correlated with decreased IRS-2 phosphorylation (31). We proposed that IRS-2 is central to insulin signaling in liver, a conclusion borne out by independent studies of mice with targeted ablation of the Irs-2 gene (37, 48, 49) and from other models of insulin resistance (50). Moreover, we reported that the pathways leading to stimulation of glycogen synthesis differ in Ir-/- hepatocytes compared with WT hepatocytes (32), as do phosphorylation patterns of the FKHR transcription factor (35), a potential mediator of insulin effects on the expression of genes affecting glucose production and lipid synthesis (51).

In these investigations we asked whether IGF-I receptor overexpression would suffice to restore the metabolic response of Ir-/- hepatocytes. We show that increasing IGF-I receptor levels restores IRS-2 phosphorylation, suggesting that IGF-I receptors possess the ability to engage this crucial protein in insulin signaling. Moreover, increased IRS-2 phosphorylation is associated with levels of GSK-3 phosphorylation and glycogen synthesis similar to those of wild-type controls. These data are consistent with the idea that IRS-2 plays a key role in metabolic signaling in hepatocytes. The failure of IGF-I receptor overexpression to alter IRS-1 phosphorylation is not unexpected, as we previously showed that IRS-1 phosphorylation is unaffected in Ir-/- cells (31).

It is intriguing to note that the response of glycogen synthesis to IGF-I receptor overexpression appears to be less robust than the response to cell proliferation. In our experiments insulin receptors were consistently more potent than IGF-I receptors in stimulating glucose incorporation into glycogen. Experiments with chimeric receptors composed of the extracellular domain of the TrkC receptor fused to the kinase domain of the insulin or IGF-I receptor transfected into 3T3-L1 adipocytes have yielded inconclusive data on this point (27, 28). In view of the fact that GSK-3 phosphorylation in response to both receptors appears to be equivalent, the present data implicate additional steps, such as activation of glycogen synthase phosphatases, as key in insulin control of glycogen synthesis.

In conclusion, we show that IGF-I receptor overexpression restores glycogen synthesis to control levels while overstimulating cell growth in Ir-/- cells. The findings are consistent with a model in which insulin receptors mediate metabolic signaling more efficiently than IGF-I receptors, whereas the latter are more potent stimulators of cell proliferation. These observations, obtained in a physiological target cell for both receptors, suggest that intrinsic signaling properties account for the different biological roles of these two receptors.


   Acknowledgments
 
We thank Dr. Jun Nakae for helpful comments about the manuscript. We thank A. F. Parlow (NIDDK’s National Hormone & Pituitary Program) for the gift of rhIGF-1.


   Footnotes
 
This work was supported by NIH Grants DK-58282 and DK-57539. J.J.K. is supported by a mentor-based post-doctoral fellowship Award of the American Diabetes Association (to D.A.)

Abbreviations: GSK, Glycogen synthase kinase; IRS, insulin receptor substrate; SV40, simian virus 40; WT, wild-type.

Received January 8, 2001.

Accepted for publication April 17, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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