©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Impaired Insulin Signaling in Skeletal Muscles from Transgenic Mice Expressing Kinase-deficient Insulin Receptors (*)

Pi-Yun Chang (1), Laurie J. Goodyear (2), Heike Benecke (1)(§), Jeffrey S. Markuns (2), David E. Moller (1)(¶)

From the (1) Charles A. Dana Research Institute and Harvard-Thorndike Laboratory of Beth Israel Hospital, Department of Medicine, Beth Israel Hospital and the (2) Research Division, Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts 02215

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transgenic mice which overexpress kinase-deficient human insulin receptors in muscle were used to study the relationship between insulin receptor tyrosine kinase and the in vivo activation of several downstream signaling pathways. Intravenous insulin stimulated insulin receptor tyrosine kinase activity by 7-fold in control muscle versus 1.5-fold in muscle from transgenic mice. Similarly, insulin failed to stimulate tyrosyl phosphorylation of receptor -subunits or insulin receptor substrate 1 (IRS-1) in transgenic muscle. Insulin substantially stimulated IRS-1-associated phosphatidylinositol (PI) 3-kinase in control versus absent stimulation in transgenic muscles. In contrast, insulin-like growth factor 1 modestly stimulated PI 3-kinase in both control and transgenic muscle. The effects of insulin to stimulate p42 mitogen-activated protein kinase and c-fos mRNA expression were also markedly impaired in transgenic muscle. Specific immunoprecipitation of human receptors followed by measurement of residual insulin receptors suggested the presence of hybrid mouse-human heterodimers. In contrast, negligible hybrid formation involving insulin-like growth factor 1 receptors was evident. We conclude that (i) transgenic expression of kinase-defective insulin receptors exerts dominant-negative effects at the level of receptor autophosphorylation and kinase activation; (ii) insulin receptor tyrosine kinase activity is required for in vivo insulin-stimulated IRS-1 phosphorylation, IRS-1-associated PI 3-kinase activation, phosphorylation of mitogen-activated protein kinase, and c-fos gene induction in skeletal muscle; (iii) hybrid receptor formation is likely to contribute to the in vivo dominant-negative effects of kinase-defective receptor expression.


INTRODUCTION

Insulin's diverse actions are initiated by binding to its specific transmembrane receptor. The intracellular -subunits of the insulin receptor possess intrinsic protein tyrosine kinase activity which is stimulated by insulin binding to the extracellular -subunits and is augmented by -subunit tyrosyl autophosphorylation. The preponderance of current data suggests that most, if not all, of insulin's biological effects are mediated by the insulin receptor tyrosine kinase (1) . However, several mutated versions of the insulin receptor with impaired tyrosine kinase activity (toward exogenous substrates) reportedly retain the capacity to mediate signaling toward one or more biological actions of insulin (2, 3, 4, 5, 6) . In addition, certain monoclonal anti-insulin receptor antibodies have been reported to mimic the actions of insulin without activation of the receptor tyrosine kinase (7, 8) , although these findings have subsequently been questioned by others (9) .

Activation of the insulin receptor and other growth factor-activated receptor tyrosine kinases including the insulin-like growth factor 1 (IGF-1)() receptor is associated with stimulation of several common intracellular signaling pathways. Specific intermediates that participate in these pathways include phosphatidylinositol (PI) 3-kinase, p70 S6 kinase, p21 ras, and mitogen-activated protein (MAP) kinases (1, 10, 11, 12) . Receptor-mediated tyrosyl phosphorylation of a major 185-kDa insulin receptor substrate, IRS-1, plays an important role in mediating these insulin signaling pathways since phosphorylated IRS-1 binds to a number of Src homology 2 (SH2) domain-containing proteins including the p85 subunit of PI 3-kinase, GRB-2, nck, and SH-PTP2 (syp) (1, 13, 14) . In addition, recent studies have shown that insulin can activate both PI 3-kinase and the ras-MAP kinase cascade in mice lacking IRS-1 (15, 16) . This IRS-1-independent signaling may involve receptor-mediated phosphorylation of Shc (17) or a newly identified substrate now referred to as IRS-2 (16) .

Previous studies have demonstrated that kinase-deficient insulin receptors when overexpressed in cultured cells have an impaired ability to mediate IRS-1 phosphorylation, PI 3-kinase activation, and activation of the ras-MAP kinase cascade (1, 2, 4, 11, 18, 19, 20, 21) . However, the relationship between the receptor tyrosine kinase and activation of these (or other) cellular signaling intermediates in any in vivo target tissues for insulin has not been directly addressed. We have recently generated lines of transgenic mice which express a dominant-negative mutant human insulin receptor (Ala Thr) in skeletal and cardiac muscle (22) . These mice are mildly insulin resistant as determined by hyperinsulinemia and reduced glycemic responsiveness to exogenous insulin (22) . Furthermore, skeletal muscle from these transgenic mice displayed reduced insulin-stimulated insulin receptor tyrosine kinase activity (22) . Therefore, this animal model provides a unique opportunity to study the effects of altered insulin receptor tyrosine kinase in an important insulin target tissue.

In the present study, we investigated the consequences of impaired insulin receptor kinase activity for the ability of insulin administered in vivo to regulate molecules that participate in several important signaling pathways (IRS-1, PI 3-kinase, MAP kinase, and c-fos mRNA) in muscle. We also provide evidence which supports the hypothesis that the mechanism(s) responsible for trans-dominant effects of kinase-deficient insulin receptor expression involves the formation of hybrid heterodimer complexes between endogenous murine receptors and overexpressed mutant human receptors.


EXPERIMENTAL PROCEDURES

Transgenic Mice

The construction of transgenic mice with muscle-specific overexpression of a mutant human insulin receptor (Ala Thr) was described previously (22) . Mice (inbred FVB-NJ strain) used in the present study were 8-12-week-old hemizygous transgenics derived from two independent lines of transgenic mice. Similar numbers of mice from both lines were used for experiments described below. Age- and sex-matched littermate control mice were used for each experiment.

Assessment of Muscle Insulin Receptor Expression

The level of muscle insulin receptor expression in control versus transgenic mice was determined by immunoblotting with a nonspecies-specific anti-insulin receptor antibody (IR-C) directed against the C terminus of the receptor -subunit (kindly provided by B. Cheatham and C. R. Kahn, Joslin Diabetes Center, Boston). Muscle protein lysates were prepared from samples of powdered frozen muscle as described below. Protein concentration was determined using the Bradford dye binding assay kit (Bio-Rad) or BCA protein assay reagent (Pierce). Aliquots of muscle lysate containing solubilized protein (300 µg) were separated by SDS-polyacrylamide gel electrophoresis, followed by transfer to nitrocellulose membranes. Membranes were blocked with 5% nonfat dried milk in TNA (20 mM Tris-HCl, pH 7.8, 150 mM NaCl, 0.01% sodium azide) with the addition of 0.05% Tween 20 for 2 h at room temperature followed by the addition of the IR-C antibody (1:500) for an additional 2 h. After removal of unbound antibody, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG for 1 h followed by washing for 2 10 min in TNA plus 0.05% Nonidet P-40 and 2 10 min in TNA with 0.1% Tween 20. Detection was accomplished using enhanced chemiluminescence (Amersham).

Measurement of Skeletal Muscle Insulin Receptor Tyrosine Kinase Activity

The procedure for measurement of muscle insulin receptor tyrosine kinase activity was similar to previously described methods (22, 23). In brief, fasted mice (overnight) were anesthetized with Avertin (Tribromoethanol, tertamyl alcohol, Aldrich) and kept on a warming pad. Samples of gluteal or gastrocnemius muscle (50-200 mg) were removed 2 min following intravenous injection (tail vein) of the indicated doses of insulin or recombinant IGF-1 (Genentech, South San Francisco, CA) or saline. Muscle samples were snap-frozen in liquid nitrogen and Polytron homogenized in ice-cold Buffer A containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM sodium pyrophosphate, 2 mM NaVO, 1 mM MgCl, 1 mM CaCl, 10 mM NaF, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1% Nonidet P-40, and 10% glycerol. After incubation for 30 min and brief centrifugation, supernatant containing 100 µg of soluble protein was incubated overnight at 4 °C in microtiter wells coated with a nonspecies-specific insulin receptor antibody (AB-3, Oncogene Science). Tyrosine kinase activity of the bound insulin receptors was measured by adding 20 µl of poly-Glu-tyr (4:1) (Sigma) (4 mg/ml in water) and 20 µl of reaction mixture containing 10 mM MgCl, 5 mM MnCl, 0.5 µM ATP, and 7 µCi of [-P]ATP. After 20 min of incubation at 20 °C, the reaction was stopped by spotting onto filter paper (Whatman 3MM). Filter papers were washed with 10% trichloroacetic acid and 10 mM sodium pyrophosphate. Radioactivity was determined by Cerenkov counting.

Tyrosyl Phosphorylation of Insulin Receptor and IRS-1

Muscle lysates were prepared as described above. Solubilized proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with TNA containing 5% albumin for 2 h at 37 °C. The membranes were then incubated with affinity-purified anti-phosphotyrosine antibody (PY provided by R. J. Smith, Joslin Diabetes Center, 24) in TNA plus 5% bovine serum albumin for 12-16 h at 4 °C. Membranes were washed 2 5 min in TNA plus 0.05% Nonidet P-40 and 1 5 min in TNA plus 0.1% Tween 20. Bound antibodies were detected by incubation with I-Protein A (1 µCi/ml) (ICN Biomedical, Costa Mesa, CA) for 1 h at room temperature. Bands corresponding to phosphorylated IRS-1 or insulin receptor -subunit were quantitated using a Molecular Dynamics PhosphorImager. The level of IRS-1 protein expression was assessed by immunoblotting of mouse muscle lysate with anti-IRS-1 antibody (gift of Morris White, Joslin Diabetes Center) using methods described above.

Measurement of Muscle PI 3-kinase Activity

Gluteal and gastrocnemius muscle lysates were prepared as described above after 2 min intravenous stimulation with insulin, IGF-1, or saline. Cleared muscle lysates were assayed for PI 3-kinase activity that was measured in immunoprecipitates obtained with antibodies to IRS-1 (IRS-1, affinity purified polyclonal antibody prepared by injecting rabbits with a synthetic peptide corresponding to the last 14 amino acids in the C-terminal region of rat IRS-1, provided by R. J. Smith, Joslin Diabetes Center). Enzyme activity in reconstituted immunoprecipitates was assayed as described previously (25) , with some modification.

Muscle lysate containing 3 mg of solubilized protein was incubated with IRS-1 and protein A-Sepharose (Pierce). Immune complexes were washed three times with phosphate-buffered saline containing 1% Nonidet P-40 and 100 µM NaVO, three times with 100 mM Tris-HCl, pH 7.5, containing 500 mM LiCl, and twice with 10 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl, 1 mM EDTA, and 100 µM NaVO. The pellets were resuspended in 50 µl of the Tris-NaCl buffer containing 12 mM MgCl and 10 µg of phosphatidylinositol (Avanti Polar Lipids Inc., Alabaster, AL). The PI 3-kinase reaction (room temperature) was initiated by adding 10 µl of 440 µM ATP containing 30 µCi of [P]ATP. After 10 min of vigorous vortexing, the reaction was stopped by the addition of 20 µl of 8 N HCl and 160 µl of chloroform/methanol (1:1, v/v). The phases were separated by centrifugation, and the lower organic phase was spotted onto thin layer chromatography plates (25) . Lipids were resolved by chromatography in CHOH/CHCl/HO/NHOH (60:47:11.3:2) and visualized by autoradiography. Radioactivity in the spots which co-migrated with a PI-3-P standard was quantitated using a PhosphorImager.

Assessment of p42/p44 Map Kinase Phosphorylation

Mice were fasted for 48 h, followed by anesthesia with Avertin and placement on a warming pad. Following intravenous administration of the indicated insulin doses or saline, mice were sacrificed at the indicated time points and muscle was removed and snap-frozen in liquid nitrogen. Muscle was homogenized and solubilized in a buffer containing 20 mM HEPES, pH 7.0, 5 mM EDTA, 10 mM EGTA, 10 mM MgCl, 50 mM -glycerophosphate, 1 mM NaVO, 2 mM dithiothreitol, 2 µg/ml leupeptin, 5 µg/ml aprotinin, 40 µg/ml phenylmethylsulfonyl fluoride, and 1% Nonidet P-40 followed by incubation for 30 min at 4 °C, and brief centrifugation. The phosphorylation state of p42 and p44 MAP kinases was assessed by electrophoretic mobility as follows. Aliquots of muscle lysate containing 70 µg of solubilized protein were separated by 10% SDS-polyacrylamide gel electrophoresis followed by electrophoretic transfer to nitrocellulose membranes. Membranes were probed with an anti-ERK1/ERK2 antibody (-C2, provided by John Blenis, Harvard Medical School). The conditions for immunoblotting, washing, and detection by enhanced chemiluminescence are described above.

Measurement of c-fos mRNA Expression

For c-fos mRNA expression, fasted mice (overnight) were anesthetized with Avertin, followed by intraperitoneal injection of 10 milliunits/g insulin as a combination of regular (Humulin R, Lilly) and long-acting (Humulin N) insulin (26) . A dose of intraperitoneal glucose (1.0 mg/g body weight) that was empirically determined to prevent the development of hypoglycemia was also administered. After 1 h, mice were sacrificed, and a sample of muscle was removed and snap-frozen in liquid nitrogen. Twenty µg of total RNA (27) was separated by electrophoresis on 1.2% formaldehyde-agarose gels followed by transfer to nylon membranes. Membranes were hybridized (22) with a random-primed [P]dCTP-labeled fragment of rat c-fos cDNA (provided by M. Jakubowski, Beth Israel Hospital, Boston). After washing with high stringency (28) , membranes were subjected to autoradiography as described previously (28) . A random-primed labeled [P]dCTP -actin cDNA probe was also prepared and used as described previously (28) . The bands corresponding to c-fos and -actin transcripts were quantitated by PhosphorImager.

Insulin Receptor Immunoprecipitation: Assessment of Potential Hybrid Receptor Formation

Muscle lysates from control and transgenic mice were prepared using Buffer A as described above. Aliquots of lysate (500 µl) containing 200 µg of solubilized protein were immunoprecipitated with a monoclonal human-specific anti-insulin receptor antibody (83-14, kindly provided by K. Siddle, Cambridge University, U.K.) and Protein A-Trisacryl beads (Pierce) for 2 h at 4 °C. After immunoprecipitating three times, aliquots of the supernatant were used for an insulin receptor binding assay and immunoblotting with nonspecies-specific anti-insulin receptor antibody (IR-C). Immunoprecipitated proteins (present in the pellet) were also analyzed by immunoblotting with IR-C. For the insulin binding assay, procedures were as described previously (22) . For Western blotting of insulin receptors present in the pellet or supernatant, proteins were separated, blotted, and probed with IR-C exactly as described above (``Quantitation of Muscle Insulin Receptor Expression''). IGF-1 receptors present in solubilized muscle lysates (and in 83-14 immunoprecipitates from muscle lysates) were detected by immunoblotting with a polyclonal anti-mouse IGF-I receptor antibody (-IGFR, provided by L. H. Wang, Mount Sinai, New York). Protein lysates prepared from CHO cells which overexpress human IGF-I receptors (CHO-IGFR, provided by Richard Roth, Stanford University) and CHO cells which overexpress human insulin receptors (CHO-IR, 4) were used as controls in the above experiments.


RESULTS

Overexpression of Mutant Human Insulin Receptors in Transgenic Mice

Two independent lines of transgenic mice which overexpress mutant Thr human insulin receptors in skeletal and cardiac muscle were generated and characterized as described previously (22) . In order to verify that mutant human receptors were overexpressed in muscles that were subsequently studied, solubilized gluteal and gastrocnemius muscle proteins were separated by SDS-PAGE followed by immunoblotting with antibody IR-C which recognizes both murine and human insulin receptors. As shown in Fig. 1, analysis of these muscles confirmed that the level of mutant receptor expression in both lines of transgenic mice was severalfold higher than the expression of endogenous insulin receptors in control mice.


Figure 1: Overexpression of mutant human insulin receptors in skeletal muscle of transgenic mice. Solubilized muscle proteins (300 µg) prepared from control and transgenic mice were fractionated by 7% SDS-PAGE followed by immunoblotting with a non-species-specific anti-insulin receptor antibody. This example shows results obtained with gluteal muscle derived from three control and three transgenic mice. The arrow indicates a 95 kDa protein band which corresponds to the insulin receptor -subunit.



Insulin Receptor Tyrosine Kinase Activity Following in Vivo Insulin Stimulation

We previously showed that gluteal muscle insulin receptor tyrosine kinase activity was markedly impaired in transgenic mice after administration of insulin by intraperitoneal injection (22) . In the present study, insulin was administered intravenously followed by removal of gluteal or gastrocnemius muscle samples. Equal aliquots of solubilized muscle proteins were applied to microwells coated with the nonspecies-specific insulin receptor antibody (AB-3), and in vitro insulin receptor kinase activity toward poly-Glu-Tyr was measured. Experiments conducted in control mice established that maximal insulin receptor kinase activity was achieved with insulin doses greater than 5 milliunits/g (Fig. 2A). Thus, subsequent experiments were performed using this maximally effective insulin dose. Fig. 2B shows that insulin stimulated receptor phosphotransferase activity by 5-6-fold over basal in control gluteal and gastrocnemius muscles. In contrast, insulin-stimulated receptor tyrosine kinase activity in muscles from transgenic mice was markedly impaired (less than 1-fold over basal).


Figure 2: Insulin receptor tyrosine kinase activity after in vivo insulin stimulation. Tyrosine kinase activity toward poly-Glu-Tyr was determined using gluteal or gastrocnemius muscles obtained 2 min after intravenous insulin (or saline) injection. A, dose-response curve generated using intravenous injection of increasing insulin doses into control mice. Each point represents mean ± S.E. of results obtained with eight mice. Data are expressed as nanomoles of ATP transferred to the substrate per milligram of gluteal muscle protein. B, insulin receptor tyrosine kinase activity obtained using muscle from control (C) and transgenic (TG) mice with (+) or without (-) in vivo insulin stimulation (5 milliunits/g body weight). The results are expressed as percentage of basal control and represent mean ± S.E. of data from two experiments (n = 6 controls, n = 6 transgenic mice in each experiment). * = transgenic versus insulin-stimulated control gluteal muscle, p < 0.0001. ** = transgenic versus insulin-stimulated control gastrocnemius muscle, p < 0.01).



Effect of the Transgene on Insulin-stimulated Receptor and IRS-1 Tyrosyl Phosphorylation

Tyrosyl phosphorylation of insulin receptor -subunits and IRS-1 was assessed by direct immunoblotting of solubilized muscle proteins with antiphosphotyrosine antibody. First, dose-response curves were generated using gluteal muscle derived from control mice (Fig. 3). For both insulin receptor and IRS-1 phosphorylation, the results paralleled the effect of insulin to stimulate receptor tyrosine kinase activity toward the exogenous substrate since partial stimulation was evident with 1.0 milliunits/g and maximal stimulation was achieved with 5.0-50 milliunits/g. For subsequent experiments 5 milliunits/g insulin was used.


Figure 3: Tyrosyl phosphorylation of insulin receptor and IRS-1: insulin dose-response. Gluteal muscles were obtained from control mice after injection of saline or increasing insulin doses. Muscle proteins were resolved by SDS-PAGE followed by immunoblotting with anti-phosphotyrosine antibody. Tyrosyl phosphorylated proteins detected using I-Protein A were quantitated by PhosphorImager analysis. A, dose-response curve for insulin-stimulated receptor -subunit phosphorylation in control mice. B, dose-response curve for insulin-stimulated IRS-1 tyrosyl phosphorylation in control mice. Each point represents mean ± S.E. of data obtained from four mice.



Insulin increased receptor autophosphorylation by 200% in gluteal and 182% of basal in gastrocnemius muscles from control mice (Fig. 4, A and B). Similarly, insulin stimulated IRS-1 phosphorylation by 297% in control gluteal and 208% of basal in control gastrocnemius muscles (Fig. 4, A and C). In contrast, in both sets of muscle derived from transgenic mice there was no detectable insulin stimulation of either receptor or IRS-1 tyrosyl phosphorylation (Fig. 4). In separate experiments we determined that the level of IRS-1 protein expression in transgenic gluteal muscle was comparable to the level of expression in control mice (not shown).


Figure 4: Effect of mutant insulin receptor transgene on receptor and IRS-1 tyrosyl phosphorylation. A, this example autoradiogram shows basal and maximal insulin-stimulated phosphorylation of IRS-1 (185 kDa band) and insulin receptor -subunit (95 kDa band) in control (C) and transgenic (TG) gluteal muscles. B, quantitation of insulin receptor autophosphorylation in gluteal and gastrocnemius muscles following saline (-) or 5 milliunits/g insulin (+) injection. The results represent the mean ± S.E. of data obtained in three separate experiments (n = 6 controls, n = 6 transgenic mice in each experiment) and are expressed as percentage of basal control. * = transgenic versus insulin-stimulated control gluteal muscle, p < 0.005. ** = transgenic versus insulin-stimulated control gastrocnemius muscle, p < 0.05. C, quantitation of IRS-1 phosphorylation. Results represent mean ± S.E. of data obtained in three separate experiments (n = 6 control, n = 6 transgenic mice in each experiment). * = transgenic versus insulin-stimulated control gluteal muscle, p < 0.0001. ** = transgenic versus insulin-stimulated control gastrocnemius muscle, p < 0.001.



Effect of the Transgene on Insulin and IGF-1-stimulated PI 3-Kinase Activity

To investigate whether the defect involving insulin-stimulated IRS-1 phosphorylation in transgenic muscle was associated with impairment of PI 3-kinase activation, PI 3-kinase activity in anti-IRS-1 immunoprecipitates was assayed. Initially, we found that the in vivo insulin dose-response curve for PI 3-kinase in control muscle paralleled insulin's effects on receptor kinase activity or IRS-1 phosphorylation (Fig. 5A). Subsequently, we compared control muscles to transgenic muscles after treatment with 5 milliunits/g intravenous insulin. PI 3-kinase activity in control gluteal and gastrocnemius muscles was increased by 37- and 16-fold of basal, respectively (Fig. 5, B and C). In contrast, there was no detectable insulin stimulation of PI 3-kinase activity in transgenic gluteal and gastrocnemius muscles (Fig. 5, B and C).


Figure 5: Insulin-stimulated PI-3-kinase activity. Muscle proteins obtained from insulin, or saline-treated mice were immunoprecipitated with IRS-1 antibody. PI 3-kinase activity in immunoprecipitates was assayed by measurement of P incorporation into PI-3-P after thin layer chromatography. A, dose-response curve generated by injection of increasing insulin doses into control mice. Each point represents mean ± S.E. of results obtained from four mice. B, this example autoradiogram shows basal and insulin-stimulated PI-3-P phosphorylation in anti-IRS-1 immunoprecipitates from control (C) and transgenic (TG) gluteal muscle. C, quantitation of PI-3-kinase activity in gluteal and gastrocnemius muscles from mice treated with saline (-) or 5 milliunits/g insulin (+). The results represent mean ± S.E. of data obtained in three separate experiments (n = 6 control mice, n = 6 transgenic mice in each experiment). * = transgenic versus insulin-stimulated control gluteal muscle, p < 0.02. ** = transgenic versus insulin-stimulated control gastrocnemius muscle, p < 0.005.



In order to determine whether overexpression of mutant insulin receptors would exert transdominant effects on IGF-1 receptor-mediated signaling, we measured muscle PI 3-kinase activation after in vivo IGF-1 administration. We first established that intravenous injection of IGF-1 was capable of exerting a biologic response. Thus, doses of 0.5 µg/g body weight or more provoked hypoglycemia in control mice. Using a high dose of IGF-1 (2.0 µg/g), PI 3-kinase activity in control mouse muscle was substantially augmented (974 ± 193% of basal, n = 7 mice). In contrast, stimulated transgenic muscle PI 3-kinase activity was only 231 ± 33% of basal (n = 8 mice). However, we found that this dose of IGF-1 was associated with persistent activation of muscle insulin receptor tyrosine kinase in control mice (not shown). Therefore, in subsequent experiments we used 0.5 µg/g of IGF-1, a dose that was not associated with activation of muscle insulin receptors. This lower IGF-1 dose resulted in a similar degree of modest PI 3-kinase stimulation in both control and transgenic muscle (274.5 ± 46% of basal versus 244 ± 33%, respectively, not significant). Thus, the ability of IGF-1 to stimulate PI 3-kinase exclusively through its own receptors was apparently preserved in transgenic muscle.

Insulin Stimulation of Muscle MAP Kinase Phosphorylation

In order to explore the effect of impaired muscle insulin receptor tyrosine kinase activity on the ability of insulin to activate MAP kinases, we used an electrophoretic mobility shift assay to assess the phosphorylation state of p42 (ERK2) and p44 (ERK1) MAP kinases. Preliminary experiments showed that maximal MAP kinase phosphorylation occurred with insulin doses 10 milliunits/g (not shown). As depicted in Fig. 6A, insulin administration to control mice resulted in time-dependent phosphorylation of muscle p42 MAP kinase with maximal effects occurring by 5 min. This time- and dose-dependent phosphorylation was also seen with p44 MAP kinase, although this isoform is apparently not as abundantly expressed in skeletal muscle. In comparing the molecular weight shift of MAP kinase in gluteal muscles derived from transgenic versus control mice, we noted that insulin stimulated substantial phosphorylation of p42 MAP kinase in control muscle with absent or minimal stimulation of a p42 MAP kinase-phosphorylated band shift in muscle from transgenic mice (Fig. 6B).


Figure 6: Insulin-stimulated phosphorylation of MAP kinase. After intravenous injection of saline or 10 milliunits/g insulin, muscle proteins were resolved by 10% SDS-PAGE followed by immunoblotting with anti-MAP kinase antibody. Bands corresponding to dephosphorylated (lower) and phosphorylated (upper) p42 and p44 MAP kinase were detected using enhanced chemiluminescence. A, time course generated by obtaining gluteal muscle samples from five different mice at the indicated time points after insulin administration. B, comparison of saline (-) and insulin-stimulated (+) MAP kinase phosphorylation in control (C) versus transgenic (TG) gluteal muscle obtained 5 min after injection. This example shows results obtained with two control and two transgenic mice. Similar results were obtained with seven additional mice from each group.



Insulin Stimulation of c-fos mRNA Expression

To study whether the marked impairment of muscle insulin receptor kinase in transgenic mice would affect insulin's ability to stimulate the expression of immediate early genes, we assessed the level of c-fos mRNA by Northern blot analysis after in vivo insulin stimulation. Preliminary experiments demonstrated that c-fos mRNA levels in muscle from control mice were maximally elevated 1 h after intraperitoneal insulin administration and returned to basal levels by 6 h, while -actin mRNA levels remained constant (not shown). In addition, we determined the dose-response characteristics of insulin-stimulated c-fos mRNA expression. As shown in Fig. 7A, maximal stimulation of c-fos expression was achieved with insulin doses of 10 milliunits/g or greater. When transgenic and control mice were treated with 10 milliunits/g of insulin, the level of c-fos mRNA expression in gluteal muscle was 664 ± 118% of basal in control mice versus 276 ± 56% in transgenics (Fig. 7B). Similar impairment of insulin-stimulated c-fos expression in transgenic mice was observed using gastrocnemius muscles, although the degree of stimulation in control mice was less in this muscle (Fig. 7B).


Figure 7: Insulin-stimulated induction of c-fos mRNA expression. Sixty min after intraperitoneal injections of saline or insulin (and glucose), muscle was excised and total RNA was prepared. A, dose-response curve generated using increasing insulin doses administered to control mice. Each point represents mean ± S.E. of 3 mice; the relative level of c-fos mRNA was determined by Northern blotting followed by PhosphorImager quantitation and is expressed as a percentage of the basal c-fos mRNA level. B, quantitation of insulin-stimulated c-fos mRNA expression in muscle from control (C) and transgenic (TG) mice with (+) or without (-) in vivo insulin stimulation (10 milliunits/g). The results are expressed as percentage of basal control and represent mean ± S.E. of data from three separate experiments (n = 8 controls, n = 8 transgenic mice in each experiment). * = transgenic versus insulin-stimulated control gluteal muscle, p < 0.0005. ** = transgenic versus insulin-stimulated control gastrocnemius muscle, p < 0.01.



Assessment of Potential Hybrid Receptor Formation

To explore potential mechanisms for the dominant-negative effects of mutant receptor expression, we investigated whether hybrid receptors composed of mouse-human insulin receptor halves or mouse IGF-1 receptor-human insulin receptor halves were present in muscle from transgenic mice. Since no mouse-specific anti-insulin receptor antibody exists, we could only indirectly address the possible formation of hybrid mouse-human insulin receptors. Muscle protein lysates from control and transgenic mice were immunoprecipitated with a human-specific monoclonal anti-insulin receptor antibody) followed by measurement of total insulin receptors remaining in the supernatant and receptors present in the pellet. Preliminary experiments established that three rounds of immunoprecipitation were sufficient to deplete more than 95% of human receptors present in lysates from transgenic muscle (not shown). As shown in Fig. 8A, 83-14 immunoprecipitated human receptors from transgenic muscle but failed to immunoprecipitate mouse insulin receptors from control muscle lysate as expected. After immunoprecipitation with 83-14, less insulin receptors (mouse) remained in the supernatants from transgenic muscle compared with supernatants from control muscle lysates exposed to the same conditions. The reduced abundance of mouse insulin receptors in transgenic muscle after depletion of human receptor protein was determined by immunoblotting (Fig. 8B) and by insulin binding assays performed after immobilizing insulin receptors in post-immunoprecipitation lysates on microtiter wells coated with the nonspecies-specific insulin receptor antibody (Fig. 8C). Thus, specific insulin binding in transgenic lysates was reduced to 0.35 ± 0.1 pmol of I-insulin bound/mg protein compared to 2.36 ± 0.45 in protein lysates derived from control muscle (Fig. 8C). Assuming that the underlying level of endogenous mouse receptor protein expression is not reduced in transgenics, the above results suggest that the majority of mouse insulin receptors present in transgenic muscle had formed hybrid complexes with mutant human receptors.


Figure 8: Assessment of potential hybrid mouse-human insulin receptors. Solubilized muscle (gluteal, 200 µg) proteins derived from control or transgenic mice were subjected to quantitative immunoprecipitation with a human-specific anti-insulin receptor antibody (83-14). Immunoprecipitated proteins present in the pellet (A) and remaining proteins present in the supernatant (B) were resolved by SDS-PAGE followed by transfer to nitrocellulose membranes and immunoblotting with a nonspecies-specific anti-insulin receptor antibody. C, quantitation of mouse insulin receptors present in solubilized gluteal muscle proteins (supernatant) after depletion of human insulin receptors by quantitative immunoprecipitation. Insulin receptors were immobilized on microtiter wells coated with a nonspecies-specific anti-insulin receptor antibody (AB-3) followed by incubation with I-insulin, washing, and measurement of bound insulin. The results represent mean ± S.E. of data from 4 control and 8 transgenic mice. Insulin binding was expressed as pmole of -I-insulin bound/mg protein after subtracting values corresponding to nonspecific binding (<5%).



A similar approach was taken to assess whether mouse IGF-1 receptor-human insulin receptor hybrids were present in transgenic muscle. Following quantitative immunoprecipitation of transgenic muscle lysates with 83-14, we used an anti-IGF-1 receptor antibody (IGFR) to determine by immunoblotting that a small amount of IGF-1 receptor -subunit (a 100 kDa band) could be immunoprecipitated with human insulin receptor protein (not shown). In contrast, no IGF-1 receptor protein was detectable in 83-14 immunoprecipitates from control mouse muscle. However, in comparison with the total amount of IGF-1 receptor present in control or transgenic muscle, less than 10% of IGF-1 receptor protein present in transgenic mouse muscle could be immunoprecipitated with 83-14. These results suggest that there was only a small degree of hybrid formation between murine IGF-1 receptors and mutant human insulin receptors in transgenic mouse muscle.


DISCUSSION

Skeletal muscle is a key site for insulin-mediated in vivo glucose disposal (29) . Furthermore, insulin- or IGF-1 receptor-mediated cellular signaling plays a major role in normal muscle differentiation and development (30) , promotes amino acid uptake (31), and inhibits muscle proteolysis (32) . In addition, impaired insulin stimulation of muscle insulin receptor tyrosine kinase activity has been demonstrated in humans with insulin-resistant non-insulin-dependent diabetes mellitus and in similarly affected rodent model systems (33, 34) . The present studies used an in vivo model to directly investigate the consequences of impaired skeletal muscle insulin receptor tyrosine kinase activity on downstream insulin signaling events in this important target tissue.

We previously demonstrated that transfected mutant Thr insulin receptors expressed in CHO cells had severely impaired insulin-stimulated phosphotransferase activity and failed to mediate several biological responses to insulin (4) . Subsequently, we reported that overexpression of these mutant insulin receptors in the muscle of transgenic mice was associated with a state of mild in vivo insulin resistance and impaired muscle insulin receptor tyrosine kinase activity (22) . In the current study, we used gluteal and gastrocnemius muscles from these mice where high levels of the kinase-deficient dominant-negative receptors were expressed. Importantly, total insulin-stimulated insulin receptor autophosphorylation and phosphotransferase activity toward poly-Glu-Tyr was severely reduced in both transgenic gluteal and gastrocnemius muscles. This occurred with the use of a maximally stimulating insulin dose as assessed by dose-response curves generated using control mouse muscle. Thus, overexpression of mutant receptors exerted a transdominant effect to abrogate insulin stimulation of endogenous insulin receptors via impaired autophosphorylation and activation of the tyrosine kinase.

In contrast to the impairment of receptor autophosphorylation which we observed in transgenic muscle, overexpression of human insulin receptors with mutated ATP-binding sites (A/K 1018) in rodent fibroblasts (35) or cultured adipocytes (36) reportedly did not reduce the net amount of insulin-stimulated receptor autophosphorylation. The reason for this discrepancy is not clear although it might relate to differences in the ability of mutant receptor -subunits to undergo trans-phosphorylation mediated by residual wild type -subunits (36-38). Reduced muscle insulin receptor autophosphorylation in our mouse model is consistent with the finding that insulin receptors derived from a heterozygous patient with the Thr receptor allele (where the ratio of mutant/normal receptor protein is presumed to be 1:1) exhibited a 50% decrease in maximal insulin stimulation of both receptor autophosphorylation and tyrosine kinase activity (39, 40) . Although total insulin-stimulated phosphorylation of an exogenous substrate (histone) was not impaired in fibroblasts overexpressing A/K 1018 receptors (35) , expression of these same mutant receptors in 3T3-L1 cells (36) did inhibit net insulin-stimulated poly-Glu-Tyr phosphorylation in agreement with our results using muscle from transgenic mice and the same insulin receptor kinase substrate.

Studies performed in cultured cell systems have suggested that normal insulin-mediated receptor autophosphorylation and tyrosine kinase activity is required for insulin-stimulated phosphorylation of IRS-1 and activation of PI 3-kinase (1, 18, 19, 20) . Furthermore, in obese insulin-resistant mice, reduced insulin receptor autophosphorylation was associated with similarly reduced IRS-1 phosphorylation (41) . However, glucocorticoid treatment of rats was reported to impair insulin receptor tyrosine kinase activity without an apparent reduction in IRS-1 phosphorylation (42) . Importantly, our results clearly show that the severe reduction in maximal insulin-stimulated receptor tyrosine kinase activity (toward poly-Glu-Tyr) in transgenic muscles resulted in proportional defects involving in vivo insulin-stimulated IRS-1 tyrosyl phosphorylation and insulin-stimulated PI 3-kinase activity in IRS-1 immunoprecipitates. This is consistent with results recently reported by Wilden and Kahn (20) who studied these effects of insulin using different insulin receptor mutants with progressive tyrosine kinase defects which were expressed in CHO cells. Experiments that we performed with control mice also showed that the insulin dose-response for receptor kinase, IRS-1 phosphorylation, and PI 3-kinase were nearly identical. These data confirm findings recently observed in the rat (43) and with isolated mouse soleus muscles (44) . The ability of kinase-deficient human receptors to impair muscle IRS-1 or PI 3-kinase activation mediated by endogenous mouse receptors is consistent with the observation that kinase-deficient insulin receptors transfected into cultured cells inhibit the ability of endogenous (35, 36) or co-expressed (38) normal insulin receptors to mediate trans-phosphorylation of IRS-1.

Like other growth factors, insulin stimulates the activation of numerous cellular Ser/Thr kinases including members of the MAP kinase (or ERK) family. Current evidence suggests that an important role of MAP kinases in growth factor or insulin signaling involves regulation of both cell proliferation (G/G transition) and differentiation (45) . One aspect of these effects involves stimulated expression of the immediate early gene, c-fos, which is also regulated by insulin (46, 47) . MAP kinase may participate in c-fos gene induction via phosphorylation of p62- or via p90-mediated phosphorylation of the serum response factor since these transacting factors form a complex that activates the c-fos promoter (45) . Recent reports showed that in vivo insulin administration to rats stimulates p42 and p44 MAP kinase activation (12, 48) in skeletal muscle. Thus, insulin or IGF-1-mediated muscle differentiation and development may involve the activation of MAP kinases.

We and others previously suggested that kinase-deficient insulin receptors expressed in transfected cells are unable to promote MAP kinase activation (11, 20) . However, these results were based on phosphorylation of myelin basic protein or MAP-2 by total cell lysates. Furthermore, no information is available concerning the relationship between the receptor kinase and c-fos gene induction. Interestingly, kinase-negative EGF receptor mutants have been reported to be capable of normally activating MAP kinase (49) and c-fos gene expression (50) . By using an electrophoretic mobility assay, we determined that insulin stimulated the phosphorylation of p42 (and p44) MAP kinase in a time- and dose-dependent manner in normal muscle. The ability of insulin to induce c-fos gene expression in control mouse muscle was similar to results recently reported by Olson and Pessin (26) using rat adipose and cardiac tissue. The fact that insulin-stimulated p42 MAP kinase phosphorylation and c-fos mRNA accumulation in transgenic muscle were markedly impaired demonstrates that these effects are dependent upon the receptor tyrosine kinase in this important target tissue.

Since a number of patients with the phenotype of severe insulin are simple heterozygotes for insulin receptor alleles which encode kinase-deficient receptor mutants (51) , it is important to characterize the mechanisms responsible for the dominant-negative properties of such receptor variants. Mutant forms of several other receptor tyrosine kinases including EGF (52) , platelet-derived growth factor (53) , fibroblast growth factor (54) , and keratinocyte growth factor (55) receptors also possess dominant-negative properties. The formation of nonfunctional heterodimers with wild type receptors has been demonstrated in several of these cases (52, 53, 54) . In the case of the insulin receptor, hybrid heterodimers composed of one mutant half-receptor and one wild type half-receptor are functionally inactive after assembly in vitro(37) . Thus, hybrid formation has been invoked as a potential mechanism for the transdominant effects of kinase-deficient insulin receptors. Previous reports have also suggested that transfected mutant human insulin receptors do form hybrids with endogenous insulin receptors in cultured rodent cells (36, 56) , although other investigators have failed to detect hybrid formation when mutant and (truncated) wild type receptors were co-transfected (38) .

In the present study, we demonstrated that the number of residual mouse insulin receptors present in solubilized muscle proteins from transgenic mice after immunodepletion of human insulin receptors was substantially lower than the level of endogenous receptors in control muscle analyzed under the same conditions. These results provide indirect evidence that mouse insulin receptors formed hybrid receptor complexes with mutant human receptors in vivo. Alternatively, it is also possible that the level of endogenous muscle insulin receptor expression in transgenic mice was lower as a consequence of forced overexpression of human receptors.

Using a similar immunoprecipitation approach, we were able to determine that only a small proportion of endogenous IGF-1 receptors could be isolated from transgenic muscle by the human-specific anti-insulin receptor antibody. This may serve to explain the fact that low dose IGF-1 was able to provoke IRS-1-associated PI 3-kinase activation in transgenic muscle to a degree that was similar to that observed in control muscle. Thus, the lack of substantial mutant insulin-IGF-1 receptor hybrids was associated with the absence of transdominant inhibition of an effect mediated by endogenous IGF-1 receptors. In contrast, overexpression of A/K 1018 insulin receptors in rat 1 fibroblasts was reportedly associated with impairment of IGF-1-mediated IRS-1 phosphorylation and mitogenesis (57) . Since we have observed that overexpression of human insulin receptors in CHO cells is associated with substantial formation of hybrids with endogenous IGF-1 receptors (58), it is logical to conclude that the stoichiometry of transfected insulin receptors (300,000/cell) versus endogenous IGF-1 receptors (10,000/cell) favors hybrid formation and may thus contribute to dominant-negative impairment of IGF-1 action in cultured cells.

In summary, we have used a transgenic mouse model characterized by overexpression of tyrosine kinase-deficient human insulin receptors in muscle to determine that activation of the insulin receptor tyrosine kinase is required for stimulation of several known insulin signaling pathways in this tissue. The presence of mutant human insulin receptors exerts dominant-negative effects at the level insulin-stimulated receptor autophosphorylation and tyrosine kinase activation. Furthermore, our findings suggest that the formation of hybrid mutant-normal receptor heterodimers contributes to mechanisms which underlie the dominant-inhibitory effects of mutant receptor expression for in vivo insulin signaling.


FOOTNOTES

*
This work was supported by grants from the American Diabetes Association and NIH-NIDDK RO1 45874-01 (both to D. E. M.) and by NIAMS Grant R29-AR42238 and a grant from the Juvenile Diabetes Foundation (both to L. J. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Medicine, University of Hamburg, Hamburg 20246, Germany.

To whom correspondence should be addressed: Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-2151; Fax: 617-667-2927; E-mail: dmoller@bih.harvard.edu.

The abbreviations used are: IGF-1, insulin-like growth factor 1; MAP, mitogen-activated protein; IRS-1, insulin receptor substrate 1; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We are grateful for the technical assistance of Luigi Gnudi (Beth Israel Hospital, Boston), for advice provided by Harald H. Klein (University of Lubeck, Lubeck, Germany), and for valuable reagents provided by Robert J. Smith, Bentley Cheatham, C. Ronald Kahn, and Morris White (Joslin Diabetes Center, Boston); Ken Siddle (Cambridge University, UK), John Blenis (Harvard Medical School), Richard Roth (Stanford University, Palo Alto, CA), and Lu-Hai Wang (Mount Sinai, New York, NY).


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