EPILOGUE
Alterations in the early steps of the insulin-signaling system in skeletal muscle of GH-transgenic mice

Fernando P. Dominici1, Debora Cifone1, Andrzej Bartke2, and Daniel Turyn1

1 Instituto de Química y Fisicoquímica Biológicas, University of Buenos Aires-Consejo Nacional de Investigaciones Científicas of Argentina, Facultad de Farmacia y Bioquímica, 1113 Buenos Aires, Argentina; and 2 Department of Physiology, School of Medicine, Southern Illinois University, Carbondale, Illinois 62901-6512


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Growth hormone (GH) excess is associated with insulin resistance, but the molecular mechanisms of this association are poorly understood. In the current work, we have examined the consequences of exposure to high GH levels on the early steps of the insulin-signaling system in the muscle of bovine (b) GH-transgenic mice. The protein content and the tyrosine phosphorylation state of the insulin receptor (IR), the IR substrate-1 (IRS-1), the association between IRS-1 and the p85 subunit of phosphatidylinositol (PI) 3-kinase, and the phosphotyrosine-derived PI 3-kinase activity in this tissue were studied. We found that in skeletal muscle of bGH-transgenic mice, exposure to high circulating GH levels results in 1) reduced IR abundance, 2) reduced IR tyrosine phosphorylation, 3) reduced efficiency of IRS-1 tyrosine phosphorylation, and 4) defective activation of PI 3-kinase by insulin. These alterations may be related to the insulin resistance exhibited by these animals.

insulin receptor; insulin receptor substrate-1; phosphatidylinositol 3-kinase; growth hormone; insulin resistance


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ELEVATION of circulating growth hormone (GH) levels causes hyperinsulinemia and insulin resistance (8). Acromegalic patients exhibit hyperinsulinemia and impaired glucose tolerance, which may lead to development of diabetes mellitus (12, 30). A significant impairment of glucose metabolism has also been observed after exogenous administration of GH in physiological or pharmacological doses in humans (6, 16, 21) and animals (1, 19). However, only a few reports have addressed the sequence of the molecular events that produce these changes.

Insulin binding to the extracellular alpha -subunit of the insulin receptor (IR) leads to autophosphorylation of tyrosine residues in the receptor beta -subunit and activation of the tyrosine kinase intrinsic to the beta -subunit (14). One of the major proteins that becomes phosphorylated by the IR upon insulin stimulation is the IR substrate-1 (IRS-1), a 185-kDa cytosolic protein with many tyrosine phosphorylation sites (28, 32). Tyrosine phosphorylation of IRS-1 generates, in the molecule, several binding sites for specific target proteins, such as the p85 subunit of phosphatidylinositol (PI) 3-kinase, growth factor receptor-bound protein 2 (GRB-2), an oncoprotein, nck, and SH2-containing phosphotyrosine phosphatase (SH-PTP2) (18, 32). When it is tyrosine phosphorylated, IRS-1 binds the p85 subunit of PI 3-kinase, thereby activating this enzyme (2). Activation of PI 3-kinase is necessary for insulin-stimulated glucose uptake and seems to be essential for regulation of metabolism by insulin (25).

Growth hormone signaling also involves engagement of IRS-1 and activation of PI 3-kinase activity (20, 33). Thus the insulin- and GH-signaling pathways may interact at these levels, and exposure to elevated circulating GH levels may affect these early events of insulin action. In the current work, we examined the effects of chronic elevation of circulating levels of GH on the insulin-signaling system with transgenic mice overexpressing bovine (b) GH. Earlier studies demonstrated that these animals are an adequate model to study the GH-induced insulin resistance (31). We have previously analyzed the concentration and binding properties of the IR, its tyrosine kinase and autophosphorylation activities, and its interaction with downstream components of the insulin-signaling system in the liver of these animals (4, 9; 9a). Data from those investigations led us to conclude that the insulin resistance was not located in the liver of GH-transgenic mice and that it could be confined to the muscle compartment. Therefore, in the current work we have evaluated the in vivo tyrosine phosphorylation of the IR and the IRS-1, the association between PI 3-kinase and IRS-1, and the phosphotyrosine-associated PI 3-kinase activity, after insulin administration in muscle of normal and bGH-transgenic mice. In addition, the abundance of these three proteins (IR, IRS-1, and PI 3-kinase) in this tissue was determined.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Materials. HEPES, Tris, phenylmethylsulfonyl fluoride (PMSF), aprotinin, ATP, phosphatidylinositol, phosphatidylinositol 4-monophosphate, Triton X-100, Tween 20, porcine insulin, and BSA (fraction V) were from Sigma (St. Louis, MO). Silica gel TLC plates were from Merck (Darmstadt, Germany). Protein A-Sepharose 6MB was from Pharmacia (Upsala, Sweden). 125I-protein A was purchased from ICN Biomedicals (Costa Mesa, CA). [gamma -32P]ATP was from Du Pont-NEN (Boston, MA). Immobilon P membranes were from Millipore (Bedford, MA). The reagents and apparatus for SDS-PAGE and immunoblotting were from Bio-Rad Laboratories (Richmond, CA). The monoclonal anti-phosphotyrosine antibody PY20 (alpha -PY) and the polyclonal anti-IR beta -subunit antibody (alpha -IR) were from Transduction Laboratories (Lexington, KY). The anti-rat carboxy-terminal IRS-1 antibody (alpha -IRS-1) and the antibody to the p85 subunit of PI 3-kinase (alpha -p85) were from Upstate Biotechnology (Lake Placid, NY).

Animals. Transgenic mice were originally produced by microinjection of the bGH gene fused to the control sequences of the rat phosphoenolpyruvate carboxykinase gene (PEPCK-bGH) into the pronuclei of fertilized eggs. The hemizygous transgenic mice used in the present study were derived from a founder male with ~25 copies of the hybrid gene and were produced by mating transgenic males with normal C57BL/6 × C3H F1 hybrid females. These matings produced approximately an equal number of transgenic mice and normal animals that were used as controls. Transgenic animals had markedly accelerated postweaning growth, leading to a significant increase in body weight at the age of 3-5 mo when these studies were conducted. Animals were housed 3-5 per cage in a room with controlled light (12:12-h light-dark cycle) and temperature (22 ± 2°C) with free access to food (Lab Diet, Formulab 5008, containing a minimum of 23% protein and 6.5% fat and a maximum of 4% fiber; PMI Feeds, St. Louis, MO) and tap water.

Measurement of plasma insulin and GH concentrations. Fasting plasma insulin concentration was determined with a solid-phase RIA kit from Diagnostic Products (Los Angeles, CA). Mouse and bGH levels were determined as previously described (4).

Experimental design and preparation of muscle extracts. The mice were starved overnight, and 15 min before the experiment they were anesthetized by the intraperitoneal administration of 100 mg of pentobarbital sodium/kg body wt. After anesthesia was induced, the portal vein was exposed and 10 IU of insulin/kg body wt in normal saline (0.9% NaCl) in a final volume of 0.1 ml were injected via this vein. Control and transgenic mice that had been injected only with diluent were used to evaluate changes under basal conditions. Approximately 2 min after injection, the hindlimb muscles were removed, minced coarsely, and homogenized in 10 vol of solubilization buffer A [1% Triton, 100 mM Tris (pH 7.4), 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM PMSF, and 0.1 mg/ml aprotinin] at 4°C. Muscle extracts were centrifuged at 40,000 rpm at 4°C in a Beckman 90 Ti rotor (Palo Alto, CA) for 40 min to eliminate insoluble material. Protein concentration was determined by the method described by Bradford (5), and the supernatants were subjected to immunoprecipitation with either alpha -IR or alpha -IRS-1.

Immunoprecipitation. Muscle homogenates containing 5 mg of total protein prepared as described in Experimental design and preparation of muscle extracts were incubated at 4°C overnight with alpha -IR or alpha -IRS-1. After incubation, 50 µl of protein A-Sepharose (50%, vol/vol) were added to the mixture. The preparation was further incubated with constant rocking for 2 h and centrifuged at 3000 g for 1 min at 4°C. The supernatant was discarded, and the precipitate was washed three times with a homogenizing buffer A. The final pellet was resuspended in 30 µl of reducing sample buffer [final concentrations: 62.5 mM Tris, 10% glycerol, 5% 2-mercaptoethanol, 2% SDS, 0.02% bromophenol blue], boiled for 5 min, and stored at -70°C until electrophoresis.

Immunoblotting. Samples were subjected to SDS-PAGE on a 6% polyacrylamide gel with a Bio-Rad Mini Protean apparatus (Bio-Rad Laboratories, Richmond, CA). Electrotransfer of proteins from the gel to Immobilon P membranes was performed for 2 h at 100 V (constant) with the Bio-Rad miniature transfer apparatus in 25 mM Tris, 192 mM glycine, 20% (vol/vol) methanol, and 0.02% SDS. To reduce nonspecific antibody binding, the membranes were incubated at 4°C overnight in a blocking buffer composed of Tris-buffered saline-Tween 20 (TBS-T) buffer [10 mM Tris · HCl (pH 7.6), 150 mM NaCl, and 0.02% Tween 20] containing either 3% BSA (for phosphotyrosine detection) or 5% nonfat dry milk (for protein detection). The membranes were then incubated for 4 h at room temperature with alpha -PY (1 µg/ml), alpha -IR (1 µg/ml), or alpha -IRS-1 (1 µg/ml) diluted in the corresponding blocking buffer. The membranes were subjected to four 5-min washes in TBS-T buffer and were then incubated with 3 µCi of 125I-protein A (30 µCi/µg) in 15 ml of blocking buffer for 1 h at room temperature and then washed again for 60 min as described previously. 125I-protein A bound to antibodies was detected by autoradiography with preflashed Kodak XAR film (Eastman Kodak, Rochester, NY) at -70°C for 6-72 h. Band intensities were quantitated by optical densitometry (model CS-930, Shimadzu, Japan) of the developed autoradiographs.

The amount of the p85 subunit of the PI 3-kinase in alpha -IRS-1 immunoprecipitates was evaluated by stripping the membranes and reblotting as follows: the blots that had been blotted with alpha -PY were rinsed, incubated in 2% SDS, 60 mM Tris (pH 6.7), and 100 mM 2-mercaptoethanol at 50°C for 30 min, and reprobed again, without the addition of extra primary antibody, to check that all antibody had been removed. The membranes were then blocked for 2 h at room temperature with 5% nonfat dry milk in TBS-T buffer and incubated with an antibody to the p85 subunit of PI-3-kinase (alpha -p85; 1:2,000 final concentration) in TBS-T plus 1% nonfat dry milk. Bound antibodies were detected by incubation with 125I-protein A as described previously.

To determine the abundance of p85 in muscle, equal amounts of solubilized proteins (200 µg) were denatured by being boiled in reducing sample buffer, resolved by SDS-PAGE, and subjected to immunoblotting with alpha -p85. Bound antibodies were detected by incubation with 125I-protein A. Quantitation of specific protein bands was performed by densitometry.

Measurement of PI 3-kinase activity in anti-phosphotyrosine immunoprecipitates. This determination was performed essentially as described by Carvalho et al. (7). Muscles of control and transgenic mice that had been injected with or without insulin as described in Experimental design and preparation of muscle extracts were extracted and homogenized in buffer B [50 mM HEPES (pH 7.4), 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 2 mM Na3VO4, 10 mM Na4P2O7, 100 mM NaF, 2 mM EDTA, 2 mM PMSF, 2 mM aprotinin, 2 mM leupeptin, 10% (vol/vol) glycerol, and 1% Triton X-100] at 4°C. The homogenate was centrifuged at 100,000 g for 1 h at 4°C. Muscle homogenates containing 5 mg of total protein were incubated overnight at 4°C with alpha -PY (5 µg). Immunocomplexes were collected by addition of 100 µl of a 50% (vol/vol) slurry of protein A-Sepharose in solubilization buffer and further incubation for 2 h at 4°C. After incubation, the immunoprecipitates were washed three times with ice-cold PBS (pH 7.4) containing 1% Triton X-100 and 100 µM Na3VO4, three times in 100 mM of ice-cold Tris (pH 7.5) containing 100 mM LiCl2 and 100 µM Na3VO4, and twice with 10 mM of ice-cold Tris (pH 7.5) containing 100 mM NaCl, 1 mM EDTA, and 100 µM Na3VO4. The precipitates were then resuspended in 50 µl of 10 mM ice-cold Tris (pH 7.5) containing 100 mM NaCl, 1 mM EDTA, 10 µl of 100 mM MgCl2,and 20 µg of phosphatidylinositol sonicated in 10 mM Tris (pH 7.5) and 1 mM EDTA. The reaction was started by the addition of 10 µl of 440 µM ATP containing 30 µCi of [gamma -32P]ATP (6,000 Ci/mmol). After 10 min at room temperature with constant shaking, the reaction was terminated by addition of 20 µl of 8 N HCl and 160 µl methanol-chloroform (1:1). Mixtures were centrifuged, and the lower organic phase was applied to silica TLC plates (Merck) coated with 1% (wt/vol) potassium oxalate. TLC plates were developed in CHCl3-CH3OH-H2O-NH4OH (60:47:11.3:2) and visualized by autoradiography. Phosphatidylinositol 4-phosphate, which comigrates with 3-phosphorylated phosphatidylinositol, was used as standard. 32P incorporated into phosphatidylinositol was quantitated by optical densitometry as described previously.

Statistical analysis. Results are expressed as means ± SE and were obtained by simultaneously processing samples from animals from all groups. For comparison, an unpaired Student's t-test was used. Data were considered significantly different at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Data on body weight and plasma glucose, insulin, and GH concentrations of the studied animals are summarized in Table 1. Overexpression of GH induced an insulin-resistant state in transgenic animals as evidenced by the concomitant presence of normal fasting glucose levels and substantially elevated plasma insulin concentration (Table 1).

                              
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Table 1.   Characteristics of experimental animals

IR and IRS-1 tyrosine phosphorylation and abundance in muscle. To determine the state of tyrosine phosphorylation of the IR in vivo in control and GH-transgenic mice, muscle homogenates were subjected to immunoprecipitation with alpha -IR and were immunoblotted with alpha -PY. The amount of tyrosine phosphorylation of the IR beta -subunit was quantitated by densitometry. For comparisons, the value of insulin-stimulated controls was considered as 100%.

In control animals, basal IR tyrosine phosphorylation in muscle was very low and was increased 12-fold (P < 0.001; n = 6) after 10 IU/kg insulin injection (Fig. 1, A and B). In muscle of GH-transgenic mice, basal IR tyrosine phosphorylation was increased by 170% (22 ± 12 vs. 8 ± 2%; n = 6/group), although this apparent difference did not reach statistical significance. In contrast to the results obtained in normal mice, only a slight increase in the amount of IR tyrosine phosphorylation was detected in transgenic mice after insulin stimulation (Fig. 1, A and B). The level of IR tyrosine phosphorylation reached after stimulation with insulin in GH-transgenic mice corresponded to 31 ± 7% of values measured in normal animals (P < 0.01; n = 6/group). To evaluate IR abundance in muscle, extracts from this tissue were immunoprecipitated with alpha -IR and immunoblotted with the same antibody. As shown in Fig. 1, C and D, IR abundance in skeletal muscle of transgenic mice was reduced to 39 ± 6% of normal values (P < 0.01; n = 12/group). When the amount of IR tyrosine phosphorylation was corrected by the amount of IR protein in skeletal muscle (phosphate-to-protein ratio), IR tyrosine phosphorylation after insulin stimulation in GH-transgenic mice was found to be 79% of normal values. This indicates that the decrease in IR protein concentration may be partially accountable for the decreased tyrosine phosphorylation of the IR in these animals.


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Fig. 1.   Insulin receptor (IR) tyrosine phosphorylation and abundance in muscle from control and GH-transgenic mice. Animals were anesthetized, and normal saline (-) or insulin (10 IU/kg) (+) was injected into portal vein. After 2 min, muscle was excised and extracts were prepared as described in MATERIALS AND METHODS. Equal amounts of solubilized muscle protein (5 mg) were immunoprecipitated (IP) with alpha -IR, separated by SDS-PAGE, and subjected to immunoblot analysis with anti-phosphotyrosine antibody PY20 (alpha -PY). Two animals/group were studied in each experiment (A). Quantitation of IR tyrosine phosphorylation was determined by scanning densitometry and is shown as means ± SE of 3 different experiments (n = 6/group). Data are relative to control by assigning a value of 100% to insulin-stimulated control mean (B). * P < 0.01 vs. insulin-treated control mice. Same samples were immunoprecipitated with alpha -IR and immunoblotted with same antibody as described in MATERIALS AND METHODS (C). Quantitation of IR abundance was performed by scanning densitometry. Data are means ± SE of 3 separate experiments (n = 12/group) (D). ** P < 0.01 vs. control mice.

To evaluate the extent of IRS-1 tyrosine phosphorylation under basal conditions and after insulin injection, muscle homogenates were immunoprecipitated with alpha -IRS-1 and subjected to immunoblotting with alpha -PY. Scanning densitometry revealed that in control animals basal IRS-1 phosphorylation was barely detectable (Fig. 2, A and B). In skeletal muscle from GH-transgenic mice, basal IRS-1 tyrosine phosphorylation was increased by ~300% (35 ± 4 vs. 9 ± 3 %; P < 0.01; n = 6/group) compared with that of controls (Fig. 2, A and B). After insulin administration, the tyrosine phosphorylation of IRS-1 increased 11-fold in control mice (P < 0.01) and only 2.8-fold in GH-transgenic mice. Despite this diminished response to insulin in transgenic mice, a similar level of IRS-1 tyrosine phosphorylation was reached in both groups of animals (Fig. 2, A and B). IRS-1 protein content was evaluated in alpha -IRS-1 immunoprecipitates from muscle by immunoblotting with the same antibody. As shown in Fig. 2, C and D, there was a significant increase in IRS-1 protein levels in skeletal muscle from transgenic mice (209 ± 21% of controls; P < 0.05; n = 12/group). When the data of IRS-1 phosphorylation were corrected for the abundance of IRS-1 protein in muscle, the phosphate-to-protein ratio of IRS-1 after insulin stimulation in skeletal muscle from GH-transgenic mice was reduced to 41% of normal values.


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Fig. 2.   IR substrate (IRS)-1 tyrosine phosphorylation and abundance in muscle from control and growth hormone (GH)-transgenic mice. Animals were anesthetized, normal saline (-) or 10 IU/kg of insulin (+) were injected into portal vein, and 2 min later muscle was excised and extracts were prepared as described in MATERIALS AND METHODS. Equal amounts of solubilized muscle protein (5 mg) were immunoprecipitated (IP) with alpha -IRS-1, separated by SDS-PAGE, and subjected to immunoblot analysis with alpha -PY. Two animals/group were studied in each experiment (A). Quantitation of IRS-1 tyrosine phosphorylation was determined by scanning densitometry and is shown as means ± SE of 3 different experiments (n = 6/group). Data are expressed as relative to control by assigning a value of 100% to insulin-stimulated control mean (B). * P > 0.01 vs. saline-treated control mice. Same samples were immunoprecipitated with alpha -IRS-1 and immunoblotted with same antibody as described in MATERIALS AND METHODS (C). Quantitation of IRS-1 levels was performed by scanning densitometry. Data are means ± SE of 3 separate experiments (n = 12) (D). ** P < 0.05 vs. control mice.

p85 association with IRS-1 and phosphotyrosine immunoprecipitable PI 3-kinase activity in muscle. To evaluate the association of the p85 subunit of PI 3-kinase with IRS-1 in muscle, samples from this tissue that had been previously immunoprecipitated with alpha -IRS-1 and immunoblotted with alpha -PY were reblotted with alpha -p85 (Fig. 3, A and B). Comparison of the immunoreactivity detected in basal conditions suggested that in transgenic animals there was a substantial increase in the association of p85 with IRS-1 compared with the control group (105 ± 36 vs. 28 ± 13%; n = 6/group). However, this apparent change was not significant because of the large variability of p85-IRS-1 association detected in these mice in the basal state (Fig. 3, A and B). After insulin administration in vivo, the intensity of the 85-kDa band corresponding to the regulatory subunit of PI 3-kinase increased by three- to fourfold in control animals (P < 0.001). In contrast, no changes were detected in GH-transgenic mice (Fig. 3, A and B).


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Fig. 3.   Association of p85 subunit of phosphatidylinositol (PI) 3-kinase with IRS-1 in muscle from control and GH-transgenic mice. Proteins from muscle were isolated as described in MATERIALS AND METHODS. A: after centrifugation, aliquots from supernatants containing equal amounts of protein were immunoprecipitated with alpha -IRS-1 and immunoblotted with alpha -p85. Two animals/group were studied in each experiment. B: scanning densitometry was performed on autoradiograms from 3 separate experiments. Data are means ± SE (n = 6/group).

Growth hormone overexpression led to changes on the PI 3-kinase activity in muscle. As shown in Fig. 4, A and B, basal activity was increased by 140% in transgenic mice (52 ± 5 vs. 22 ± 7%; n = 6; P < 0.05) compared with controls. In muscle of control mice, insulin injection led to an important increase in the phosphotyrosine-associated PI 3-kinase activity (4.5-fold increase), whereas a similar treatment led to only a slight increase in the PI 3-kinase activity in GH-transgenic mice (Fig. 4, A and B). Comparison of the intensity of spots corresponding to phosphatidylinositol 3-phosphate detected after insulin injection revealed that the level of PI 3-kinase activity after insulin stimulation was reduced to 67 ± 13% of normal values in muscle of GH-transgenic mice (P < 0.05; n = 6; Fig. 4, A and B).


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Fig. 4.   PI 3-kinase activity (phosphotyrosine immunoprecipitable) and p85 abundance in muscle from control and GH-transgenic mice. Proteins from muscle were isolated as described in MATERIALS AND METHODS. After centrifugation, aliquots from supernatants containing equal amounts of protein were incubated with alpha -PY and protein A-Sepharose. PI 3-kinase was assayed in immunoprecipitates in presence of phosphatidylinositol and [gamma -32P]ATP as described in MATERIALS AND METHODS. Lipid products were separated by TLC (A). Spots that comigrated with a PI 4-phosphate standard (PIP) were quantitated by densitometry (B). Two animals/group were studied in each experiment. Values are expressed as relative to control, setting mean densitometric units of insulin-stimulated control mice at 100%. Data are means ± SE of 3 separate experiments (n = 6/group). *P < 0.05 vs. saline-treated control mice; ** P < 0.05 vs. insulin-treated control mice. Aliquots from total muscle extracts containing equal amounts of protein (200 µg) were separated by SDS-PAGE and immunoblotted with alpha -p85 as described in MATERIALS AND METHODS (C). Quantitation of p85 abundance was performed by scanning densitometry; data are means ± SE of 3 separate experiments (n = 6/group) (D). # P < 0.01 vs. control mice.

To determine whether GH overexpression was associated with changes in the abundance of the p85 subunit of PI 3-kinase, total skeletal muscle homogenates were subjected to immunoblotting with alpha -p85. The representative autoradiogram in Fig. 4C shows the p85 protein from two control and two transgenic mice. The p85 content was significantly increased in transgenic mice (138 ± 8% of the levels in control mice; P < 0.01; n = 6/group) as determined by densitometric analysis (Fig. 4D).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have demonstrated that both acute elevation and chronic elevation of circulating GH levels lead to insulin resistance and hyperinsulinemia in humans and animals (1, 6, 8, 12, 19). In an attempt to identify the mechanisms involved, we have investigated the effects of long-term exposure to elevated GH levels on the early steps of the insulin-signaling system with a transgenic model of mice expressing high levels of bGH. A state of insulin resistance was demonstrated in these animals by a modest glucose intolerance, an exaggerated increase in insulin levels after administration of a high carbohydrate diet, a decrease in the expression of several proteins of importance in carbohydrate metabolism, as well as decreases both in the insulin-mediated activation of glycogen synthase in liver and muscle and in the activation of glycogen phosphorylase in both tissues (31).

We have previously reported that overexpression of bGH is associated with a significant decrease in the concentration of the IR in the liver of these mice and that this is concomitant with increased tyrosine kinase activity, as well as increased receptor autophosphorylation (4, 9). These results together with data from studies of components of the insulin signaling downstream from the IR in liver of these animals (9; 9a) fail to account completely for the observed insulin resistance. This prompted us to investigate if in these animals there were alterations in the insulin- signaling system in the muscle, another important insulin target tissue. Therefore, in the present study we have analyzed the IR and IRS-1 tyrosine phosphorylation, the association of IRS-1 and the p85 subunit of PI 3-kinase, and the PI 3-kinase activity in basal conditions and after acute insulin stimulation in vivo in the muscle of GH-transgenic mice.

Consistent with results of our previous studies in liver (4, 9), we detected a significant reduction in the IR abundance in skeletal muscle of GH-transgenic mice. Previous studies evaluating the effect of elevated GH levels on insulin binding in different tissues produced contradictory results (6, 12, 15, 17, 21). Growth hormone itself has been shown not to directly affect insulin binding in studies performed in vitro (15). In vivo, GH elevation has been associated with decreased (6, 17) or unaltered (12, 21) insulin binding. Hyperinsulinemia characterizes states of GH excess (8), and we have previously demonstrated that the diminished IR number in the liver of GH-transgenic mice reflects downregulation of the receptor due to hyperinsulinemia rather than an effect of the elevation in GH levels (9). Therefore, we suspect that a similar situation may exist in skeletal muscle from these animals. Because the reduction in the abundance of skeletal muscle IR was more pronounced than that previously detected in the liver of these mice, the skeletal muscle may be more sensitive than the liver to chronic exposure to high insulin plasma levels.

Two recent studies reported dissimilar effects of GH treatment on the amount of IR in the muscle of rats; although Thirone et al. (29) reported no significant alterations in IR levels, Smith et al. (26) found an increase in the IR abundance in muscle from similarly treated rats. The divergence between the current data and those findings may arise from the different dynamics of serum GH levels in GH-treated rats and GH-transgenic mice. Transgenic animals display continuously high levels of circulating GH with minor fluctuations. Conversely, the exogenous administration of GH by injection would induce major fluctuations in the serum concentration of this hormone. The duration of exposure to high GH levels may be an additional difference between these two models.

Basal IR tyrosine phosphorylation was increased in the muscle of GH-transgenic compared with normal mice, although an important individual variability was detected (Fig. 1, A and B). However, tyrosine phosphorylation of the muscle IR after insulin stimulation was lower in transgenic than in normal mice, suggesting that overexpression of GH leads to an impairment of the insulin-signaling system in this tissue. This alteration may be a contributing factor to the insulin-resistant state of this mouse model. These results are in agreement with previous studies in rats in which GH administration produced insulin resistance (26, 29).

A diminished response to insulin injection in terms of IRS-1 tyrosine phosphorylation was observed in vivo in muscle of transgenic mice. An increase in the basal tyrosine phosphorylation of IRS-1 was responsible for this alteration. Recent studies (20, 33) have demonstrated that GH is able to stimulate tyrosine phosphorylation of IRS-1 both in cells in culture and in the liver of the intact rat. Furthermore, it was reported that GH induced an increase in the basal tyrosine phosphorylation of IRS-1 in skeletal muscle, when it was administered to rats in an attempt to induce an insulin-resistant state (26). These observations support the notion that the increased tyrosine phosphorylation of this substrate detected in GH-transgenic mice under basal conditions may be related to elevated plasma GH levels in these animals. Signaling via the insulin-like growth factor I (IGF-I) receptor may also be relevant to the current observations. Like the IR receptor, the IGF-I receptor is able to engage IRS proteins upon IGF-I stimulation (18, 32). We have previously demonstrated that circulating IGF-I levels are elevated in GH-transgenic mice (27), and thus in these mice elevated IGF-I concentrations may also have a role in the elevation of the tyrosine phosphorylation of IRS-I under basal conditions. Moreover, hyperinsulinemia may result in the activation of IGF-I receptors and/or in the activation of IR-IGF-I receptor hybrids, the presence of which in skeletal muscle has been recently described (3). This may be an additional factor contributing to the increase detected in the basal phosphorylation of IRS-1 in skeletal muscle of GH-transgenic mice.

The large increase in the protein content of IRS-1 in muscle of GH-transgenic mice (Fig. 2, C and D) could represent a compensatory change in response to the diminished amount and tyrosine phosphorylation of the IR in this tissue or a direct effect of GH on IRS-1 expression in muscle. The mean stoichiometry of phosphorylation of the IRS-1 in muscle (calculated by dividing tyrosine phosphorylation after insulin stimulation by protein content) was significantly reduced (41% of normal values) in GH-transgenic mice. If the additional IRS-1 in GH-transgenic mice was fully accessible for tyrosine phosphorylation, this is consistent with less phosphate incorporation per IRS-1 molecule. However, it is also possible that this excess of IRS-1 was not fully accessible to the IR tyrosine kinase, and thus in skeletal muscle of GH-transgenic mice tyrosine phosphorylated IRS-1 molecules could coexist with nontyrosine phosphorylated IRS-1 molecules. In either of these cases, it is worth noticing that this observation correlated well with the reduced tyrosine phosphorylation of the IR observed in this tissue and supports results from previous studies that have shown that IRS-1 phosphorylation depends more on IR kinase activity than on IRS-1 protein content (23).

One of the earliest events that can be detected after the binding of insulin to its receptor and the receptor autophosphorylation is the activation of PI 3-kinase (22). Activation of PI 3-kinase activity by insulin is mediated by the interaction between the p85 subunit of this enzyme and IRS-1 (2). The available information suggests that insulin stimulation of PI 3-kinase is essential for the activation of glucose transport and glycogen synthase in muscle (25). Therefore, to extend the characterization of the effect of GH excess on components of the insulin-signaling cascade, we were interested in measuring the association of the p85 subunit of PI 3-kinase with IRS-1 and the corresponding activity of this enzyme in muscle of GH-transgenic mice. The association of IRS-1 with the p85 subunit was greatly increased in muscle of transgenic mice under basal conditions (375% of normal values). As was discussed previously, this alteration could be ascribed to the elevated insulin levels as well as to elevated GH levels. Insulin administration in vivo produced no further changes, suggesting that the IRS-1-p85 association in muscle of GH-transgenic mice was maximal in the basal state. In support of this possibility, we found an increase in the basal activity of PI 3-kinase in phosphotyrosine immunoprecipitates from transgenic mice (230% of normal values). However, the increase in the activity of PI 3-kinase after insulin administration was very modest and thus the final value was reduced by 33% compared with the control group. These findings together with the observation that p85 protein concentration was increased in this tissue suggest that there is a defect in the activation of PI 3-kinase by insulin in skeletal muscle from GH-transgenic mice and that this defect is not due to a decrease in the cellular content of p85. Moreover, the impairment detected in the activation of this enzyme by insulin is not the result of defective interaction of the p85 subunit of PI 3-kinase with IRS-1 as shown by the results concerning IRS-1-p85 association.

Insulin activation of PI 3-kinase can also be mediated by the interaction of tyrosine-phosphorylated IRS-2 with the p85 subunit of this enzyme (25). Thus the PI 3-kinase activity associated with anti-phosphotyrosine immunoprecipitates reflects the interaction of p85 with not only the tyrosine-phosphorylated IRS-1 but also with other proteins such as IRS-2 (25). Therefore, although IRS-1-p85 association was increased in skeletal muscle of GH-transgenic mice, the decrease in the phosphotyrosine-derived PI 3-kinase activity detected in this tissue could be due to a defective interaction of the regulatory subunit of PI 3-kinase with other substrates apart from IRS-1 such as IRS-2.

It was reported previously that insulin stimulation of PI 3-kinase is defective in liver and muscle in several models of insulin resistance, including ob/ob mice (10), dexamethasone-treated rats (24), and the gold thioglucose-induced obese mice (13). This appears also to be the case in skeletal muscle from obese subjects (11). From these observations and the present data, we can conclude that alterations at the level of PI 3-kinase may be a common feature of insulin-resistant states.

From the current results, it can be postulated that in skeletal muscle of GH-transgenic animals the resistance to insulin appears to be a consequence of two principal events: the first a decrease in IR abundance, and the second an increase in the basal phosphorylation of the IRS-1, in the basal association of p85 with IRS-1 and also in the basal activity of the PI 3-kinase in this tissue. The latter alterations would result in a reduced sensitivity to insulin. The chronic basal hyperinsulinemia exhibited by these animals could be a contributing factor in the development of the insulin-resistant state. Chronic exposure to high GH and/or IGF-I levels could also be involved because recent evidence has demonstrated that IRS-1 can be phosphorylated in response to both of these proteins (18, 20, 32, 33).

In summary, in the current study we have shown that in muscle from GH-transgenic mice, GH overexpression was associated with decreased IR abundance, decreased insulin-induced IR phosphorylation, decreased efficiency of IRS-1 phosphorylation, and decreased activation of PI 3-kinase by insulin. These alterations may contribute to the insulin resistance detected in GH-transgenic mice.


    ACKNOWLEDGEMENTS

Transgenic and normal mice used in this work were derived from animals kindly provided by Dr. Thomas E. Wagner and Jeung S. Yun (Ohio University, Athens, OH). We thank Dr. A. F. Parlow, Pituitary Hormones and Antisera Center, and National Institute of Diabetes and Digestive and Kidney Diseases for reagents for mouse GH and bGH RIAs. We are grateful to J. Caramelo and S. Catz for advice about the PI 3-kinase assays.


    FOOTNOTES

Daniel Turyn is a Career Investigator from Consejo Nacional de Investigaciones Científicas of Argentina (CONICET) and received grant support from the University of Buenos Aires, CONICET, and Agencia Nacional de Promoción Científica y Tecnológica. Support for these studies was also provided by National Institute of Child Health and Human Development Grants HD-20001 and HD-20033.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. Bartke, Dept. of Physiology, Southern Illinois Univ., Carbondale, IL, 62901-6512 (E-mail: abartke{at}som.siu.edu).

Received 13 November 1998; accepted in final form 22 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 277(3):E447-E454
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