Increased IGFR activity and glucose transport in cultured skeletal muscle from insulin receptor null mice

Liat Shefi-Friedman1, Efrat Wertheimer2, Shlomzion Shen1, Asia Bak1, Domenico Accili3, and Sanford R. Sampson1

1 Gonda-Goldschmeid Diagnostic Center, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900; 2 Department of Pathology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel; and 3 Columbia University/ Berrie Research Pavilion, New York, New York 10032


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have studied the role of the insulin receptor (IR) in metabolic and growth-promoting effects of insulin on primary cultures of skeletal muscle derived from the limb muscle of IR null mice. Cultures of IR null skeletal muscle displayed normal morphology and spontaneous contractile activity. Expression of muscle-differentiating proteins was slightly reduced in myoblasts and myotubes of the IR null skeletal muscle cells, whereas that of the Na+/K+ pump appeared to be unchanged. Insulin-like growth factor receptor (IGFR) expression was higher in myoblasts from IR knockout (IRKO) than from IR wild-type (IRWT) mice but was essentially unchanged in myotubes. Expression of the GLUT-1 and GLUT-4 transporters appeared to be higher in IRKO than in IRWT myoblasts and was significantly greater in myotubes from IRKO than from IRWT cultures. Consistent with GLUT expression, both basal and insulin or insulin-like growth factor I (IGF-I)-stimulated glucose uptakes were higher in IR null skeletal myotubes than in wild-type skeletal myotubes. Interestingly, autophosphorylation of IGFR induced by insulin and IGF-I was markedly increased in IR null skeletal myotubes. These results indicate that, in the absence of IR, there is a compensatory increase in basal as well as in insulin- and IGF-I-induced glucose transport, the former being mediated via increased activation of the IGF-I receptor.

myoblasts; myotubes; insulin receptor; insulin-like growth factor I receptor; GLUT; insulin receptor null mouse


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE INSULIN RECEPTOR (IR) and insulin-like growth factor I (IGF-I) receptor (IGFR) are tyrosine kinases that mediate insulin and IGF-I signaling (33). Both receptors have similar heterotetrameric structures consisting of two largely extracellular alpha -subunits and two transmembranal beta -subunit tyrosine kinases (27, 32). In addition, each receptor can bind both ligands, although with appropriately lower affinity for the heterologous ligand (13). It is also believed that the two receptors activate common intracellular signal transduction pathways (11, 24, 25, 37, 38). One of the major metabolic effects of insulin after stimulation of its receptor and activation of downstream signaling elements is stimulation of glucose uptake (38). This is accomplished by the translocation of specific glucose transporters from intracellular pools to the plasma membrane (38, 40). IGF-I, on the other hand, is considered to act mainly as a growth factor (5).

Several studies have been undertaken in an attempt to elucidate the role of various elements in the IR- and IGFR-mediated signaling pathways. One approach taken has been the disruption of specific genes for proteins in the signaling pathway, including insulin (14), IGF-I (21, 28), IGF-II (12), IR substrate (IRS)-1 (1, 3), IRS-2 (1, 39), IRS-3 (22), IRS-4 (15), and specific glucose transporters (16, 18). In addition, studies have been reported on transgenic mice in which the IGFR gene (21) or IR gene (2, 17) has been specifically disrupted. At birth, IR knockout (IRKO) mice are virtually indistinguishable from their littermates. After a few hours the mice develop detectable hyperglycemia, and within 1-2 days there is noticeable growth retardation. Subsequently, the mice develop diabetic ketoacidosis and die within a few days (2, 17). This is a major limitation for biochemical and physiological studies on the consequences of a lack of IR.

Skeletal muscle is one of the major insulin target organs involved in the regulation of plasma glucose, and better understanding of insulin signaling in this tissue is of major importance. Furthermore, study of IRKO tissues could shed light on cellular function in states of extreme insulin resistance. The problems of short life span of IRKO animals appear to have been overcome by the development of a nonlethal, transgenic muscle-specific IRKO (MIRKO) mouse (10). These animals develop certain metabolic features of diabetes and have severely impaired insulin-stimulated glucose transport by skeletal muscle, and yet they display normal glucose tolerance. Inasmuch as insulin can bind to and phosphorylate IGFR, it is surprising that this effect did not occur in MIRKO skeletal muscle (10). It is thus not clear whether the lack of effect of insulin is because of the lack of IR or due to other factors, perhaps secondary to the metabolic changes in these animals.

The purpose of this study was to investigate the importance of IR in mediation of the glucose uptake induced by insulin in skeletal muscle. To accomplish this, we studied the effect of IR lack on glucose transport mechanisms in a model system of IRKO skeletal muscle in primary culture. Primary cultured skeletal muscle cells have been found to be an excellent model for the study of effects of hormones, drugs, and other factors on muscle membrane transport systems (8, 9, 30, 34, 35). These cells, plated initially as individual myoblasts, align and fuse by days 3-4 in vitro into multinucleated myotubes that display spontaneously occurring action potentials and contractile activity and are readily maintained for up to 8-10 days. Moreover, the pattern of development of several membrane proteins has been shown to closely resemble that observed in vivo. This is in contrast to the various immortalized skeletal muscle cell lines that are deficient in many of these membrane properties. The results will show that lack of IR expression in skeletal myotubes is associated with compensatory increases in both IGFR autophosphorylation and basal and insulin-stimulated glucose transport.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Tissue culture media and serum were purchased from Biological Industries (Beit HaEmek, Israel). The following antibodies to various proteins were used: anti-GLUT-1 and anti-GLUT-4 [a gift from Dr. S. Cushman (National Institutes of Health) or a purchase from Santa Cruz Biotechnology, Santa Cruz, CA]; anti-MyoD and anti-myogenin (Santa Cruz Biotechnology); anti-IRbeta and anti-IGFR (Transduction Laboratories, Lexington, KY; preliminary studies established that there is no detectable cross-reaction between the anti-IR and anti-IGFR antibodies and the IR and IGFR receptors); anti-Na+/K+ beta -subunit (Upstate Biotechnology, Lake Placid, NY); and anti-phosphotyrosine (Upstate Biotechnology). Leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), orthovanadate, and pepstatin were purchased from Sigma Chemical (St. Louis, MO). Insulin (Humulin; recombinant human insulin) was purchased from Eli Lilly (Fergersheim, France), and IGF-I was obtained from Cytolab (Rehovot, Israel). Protein A/G PLUS agarose was purchased from Santa Cruz Biotechnology. Enhanced chemical luminescence (ECL) was performed with antibodies (horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG) purchased from Bio-Rad (Hercules, CA) and with reagents from Sigma Chemical.

Genotyping of animals. The animals used for establishing the colony of mice heterozygous for the IR null gene were obtained from the original colony, as previously described (2). Determination of the genotype of newborn mice was done by PCR according to the method already described (2, 26).

Preparation of muscle cell cultures. IR null animals were identified by analysis of urinary glucose levels utilizing Clinistix test strips (Bayer, Tarrytown, NY). Urinalysis was performed 24-36 h after birth. Skeletal muscle cultures were prepared from thigh muscles obtained from 1- to 2-day neonatal IR wild-type (IRWT) or IRKO animals as previously described (8, 9, 30, 34, 35). Briefly, muscles were removed from the limbs of 10-15 newborn pups of each genotype separately, combined, and washed in PBS (pH 7.35) to remove excess blood cells and then transferred to a Ca2+-free, 0.25% trypsin solution containing EDTA (1 mM) for incubation with continuous stirring at 37°C. Cells were collected after serial trypsinization (successive 10-min periods until all tissue was dispersed) and then centrifuged for 5 min at 500 g. Pellets were resuspended in growth medium and preplated for 20-30 min to reduce the number of fibroblasts. The myoblasts were diluted with growth medium [83% DMEM-high glucose (25 mM), 15% horse serum, 2% chick embryo extract] to a concentration of 0.8 × 106 cells/ml for plating in collagen-coated 10-cm plastic tissue culture (10 ml/dish) or 24-well plates (400 µl/well). Cultures were prepared weekly and grown in a water-saturated atmosphere of 95% air-5% CO2 at 37°C. On day 4 in culture, myotubes were transferred to low-glucose (4.5 mM), serum-free DMEM containing 1% bovine serum albumin for 24 h before study. On the day of study, cells were transferred to PBS containing 2 mM glucose for 10 min before addition of insulin (70-100 nM) or IGF-I (10 nM).

Treatment of cells. Cultured cells, 4-5 days of age, were transferred to serum-free DMEM for 18-20 h before addition of insulin or IGF-I. On the day of study, the cells were again transferred to serum-free PBS for 15 min at 37°C, after which insulin or IGF-I was added for various periods of time as designated in the appropriate description in RESULTS. Preliminary dose-effect studies established that maximum effects of insulin and IGF-I on glucose transport were obtained at concentrations of 10-8 and 10-9 M, respectively. The concentration of insulin is of the same order of magnitude as that used in studies on isolated muscle from MIRKO mice (10). These concentrations were used in all experiments.

Cell fractionation. Crude membrane preparations were prepared from cell cultures as described previously (6, 7). For preparation of internal and plasma membrane fractions, culture dishes containing the myotubes were washed with Ca2+/Mg2+-free PBS and then mechanically detached with a rubber policeman in Ca2+/Mg2+-free PBS containing 2 mM EDTA. The cells were pelleted by centrifugation at 500 g for 10 min at 4°C. The pelleted cells were resuspended in sonication buffer (in mM: 50 Tris · HCl, pH 7.4; 150 NaCl; 2 EDTA; 1 EGTA) containing 20 µg/ml leupeptin, 10 µg/ml aprotinin, 0.1 mM PMSF, 1 mM DTT, 200 µM orthovanadate, and 2 µg/ml pepstatin. The suspension was then homogenized in a Dounce glass homogenizer (30 strokes) and centrifuged at 1,100 g for 5 min. The supernatant was then centrifuged at 100,000 g for 1 h; the supernatant from this centrifugation step was retained as the internal membrane fraction. The pellet was resuspended with the sonication buffer containing 1% Triton, vortexed, and sonicated (intermediate setting) four times for 5 s. The preparation was again vortexed until the suspension was uniform and was centrifuged at 23,000 g for 15 min. The supernatant from this step was designated the plasma membrane fraction. The internal and plasma membrane fractions were frozen at -70°C until used. The purity of the membrane preparations was confirmed by identification of specific membrane markers (Na+/K+-pump subunits).

Immunoprecipitation. To 0.3 ml of cell lysate, 25 µl of protein A/G Sepharose were added, and the suspension was rotated continuously for 30 min at 4°C. The preparation was then centrifuged at 20,000 g at 4°C for 10 min, and 30 µl of A/G Sepharose were added to the supernatant along with specific monoclonal antibodies to IR, IGFR, or to anti-phosphotyrosine (dilution 1:100). This was rotated overnight at 4°C. The suspension was then centrifuged at 20,000 g for 10 min at 4°C, and the pellet was washed twice as above with RIPA buffer [(in mM: 50 Tris · HCl, pH 7.4, 150 NaCl, 1 EDTA, 10 NaF, 1% Triton X-100, 0.1% SDS, 1% Na deoxycholate) containing a cocktail of antiproteases (20 µg/ml leupeptin, 10 µg/ml aprotinin, 0.1 mM PMSF, 1 mM DTT) and antiphosphates (200 M orthovanadate, 2 µg/ml pepstatin)]. The beads were eluted with 25 µl of sample buffer (0.5 M Tris · HCl, pH 6.8; 10% SDS; 10% glycerol; 4% 2-beta -mercaptoethanol; and 0.05% bromophenol blue). The suspension was again centrifuged at 15,000 g (4°C for 10 min) and washed two times in HEPES buffer containing 150 mM NaCl, followed by one wash in HEPES containing 80 mM NaCl and a final wash in NaCl-free HEPES. Sample buffer was added, and the samples were boiled for 10 min and then subjected to Western blot analysis.

Western blot analysis. Aliquots of cell lysates containing 20-25 µg protein were electrophoresed through SDS-polyacrylamide gels (7.5 or 10%) and electrophoretically transferred onto Immobilon-P (Millipore) membranes. After transfer, the membranes were subjected to standard blocking and incubation procedures and were incubated with appropriate antibodies to specific proteins. The membranes were washed four times for 15 min in Tris-buffered saline with Triton X-100 (TBST) and then further incubated for 20 min at room temperature with horseradish peroxidase-labeled secondary antibody diluted 1:10,000 in blocking buffer. After three washes (1 × 15 min and 2 × 5 min) in TBST, the membranes were treated with an enhanced chemiluminescence reagent for 1 min and then exposed on Kodak X-ray film for the required times (5-30 s) and developed.

Glucose uptake. Glucose transport was evaluated by measuring 2-deoxy-D-glucose (2-DG) uptake as described (6-8). After appropriate treatment, cells were washed three times with 0.5 ml PBS, the final wash being replaced immediately with 0.5 ml PBS containing glucose at a concentration of 2 mM and tracer amounts (1 µCi/ml) of 2-[3H]DG. Cells were then incubated for 15 min at 37°C, washed four times with 0.5 ml of cold PBS, and then detached from the wells by addition of 300 µl Triton X-100 (1%) and incubation for 30 min. The contents of each well were transferred to counting vials, and 3.5 ml of scintillation fluid were added to each vial. Samples were counted in the 3H window of a Tricarb scintillation counter. Nonspecific uptake was determined in the presence of excess (100 mM) D-glucose. Experiments were carried out in triplicate. Net specific uptake was then calculated as the difference between the total and nonspecific values. Baseline glucose uptake values under control conditions ranged from 14 to 20 nmol · min-1 · mg protein-1.

Statistical analysis. Quantitative data on glucose uptake and densitometry measurements of Western blots were analyzed by the Student's t-test (for data comparing 2 groups) or by ANOVA (for data comparing 3 or more groups), utilizing the InStat program (GraphPad, San Diego, CA).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Knockout of IR gene in skeletal myotubes. To confirm the lack of IR gene in skeletal muscle, we performed PCR in an attempt to identify the IR gene. As shown in Fig. 1A, whole limb muscle from candidate IR-/- newborn mice did not carry the intact wild-type IR gene. This was further confirmed by Western blot analysis of protein lysates of IRKO primary cell cultures (Fig. 1B).


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Fig. 1.   Identification of the insulin receptor (IR) gene. A: PCR analysis of IR gene in a whole limb muscle derived from wild-type (IR+/+) and IR knockout (IR-/-) newborn mice. PCR was performed using a set of primers specific for the mouse IR sequence (bottom) or the neomycin resistance gene sequence (Neo; top). B: Western blot analysis of skeletal myotube lysates from IR wild type (IRWT) and IR knockout (IRKO) mice. Cell lysates were immunoprecipitated with anti-IR antibody and probed with anti-IR.

Skeletal muscle from IRKO mice differentiates in culture. Insulin is an essential growth factor for proliferation and differentiation of many cells. We therefore examined whether myoblasts lacking IR expression retain their ability to differentiate into myotubes in vitro. First, we followed the morphology of the cells. As can be seen (Fig. 2A), myoblasts from IRWT and IRKO mice morphologically appear very similar to one another. Furthermore, myoblasts of IRWT and IRKO genotypes fused into large, multinucleated myotubes (Fig. 2B), and both IRWT and IRKO myotubes displayed spontaneous contractile activity. Next, we followed induction of expression of various proteins that normally are identified in primary muscle cultures in a differentiation-dependent manner. These include glucose transporters GLUT-1 and GLUT-4, IGFR, and the beta -subunit of the Na+/K+ pump, as well as myogenin and MyoD, which are proteins involved in muscle cell differentiation. Western blot analyses of total cell lysates from IRWT and IRKO skeletal myotubes showed that all proteins examined were expressed, as expected, in higher amounts in myotubes than in myoblasts (Fig. 3A). Of the proteins examined, the most striking differences between IRWT and IRKO skeletal muscle were observed in expression of the GLUT-1 and GLUT-4 glucose transporters. In total cell lysates, both these transporters were detected in greater amounts in preparations from IRKO than from IRWT cultures. This increase was also seen in both internal and plasma membrane fractions prepared from the lysates of IRWT and IRKO myotubes (Fig. 3B; see also Fig. 5). Expression of IGFR in the IRKO myoblasts was strikingly higher than in IRWT myoblasts. On the other hand, expression of this protein in IRKO myotubes appeared to be essentially the same as that in IRWT myotubes. There appeared to be no difference between IRWT and IRKO cultures with regard to expression levels of the beta -subunit of the Na+/K+ pump either in myoblasts or in myotubes. Myogenin was not detected in either IRWT or IRKO myoblasts, but high levels of this protein were seen in myotubes, expression being significantly higher in IRWT than in IRKO cultures (Fig. 3C; P < 0.05). Both myoblasts and myotubes from IRWT cultures expressed significantly higher amounts of MyoD than did those from IRKO cultures (myoblasts, P < 0.01; myotubes, P < 0.05).


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Fig. 2.   Characterization of cultured skeletal muscle myoblasts and myotubes prepared from IRWT and IRKO mice. Cells were isolated and maintained as described in METHODS. A: phase contrast photomicrographs of myoblasts (2-3 days in culture) from IRWT and IRKO mice. B: phase contrast photomicrographs of myotubes (5-6 days in culture) from IRWT and IRKO mice.



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Fig. 3.   Comparison of the expression of various proteins in cultured skeletal myotubes from IRWT and IRKO mice. A: Western blot analysis of total cell lysates, performed as described in METHODS, showing expression of the following proteins as evaluated using specific antibodies: GLUT-1 and GLUT-4, insulin-like growth factor I (IGF-I) receptor (IGFR), beta -subunit of the Na/K pump. IRWT and IRKO myoblasts (MB) and myotubes (MT) were isolated as described in METHODS. B: distribution of the GLUT-1 and GLUT-4 transporters in internal membrane (IM) and plasma membrane (PM) fractions of skeletal muscle myotubes from IRWT and IRKO mice. Myotubes were fractionated to PM and IM membranes as described in METHODS. Equal amounts (20 µg) of protein were subjected to Western blot analysis by use of anti-GLUT-1 or anti-GLUT-4 antibodies. Blots shown in both A and B are representative of 3 different experiments from 3 separate cultures. C: Western blots (top) and densitometry analyses (bottom) of myogenin and MyoD in whole cell lysates of MB and MT from IRWT and IRKO mice. Values are means ± SE of densitometry measurements done on bands from 3 separate experiments and analyzed statistically by ANOVA. Expression of myogenin was significantly higher in myotubes from IRWT than from IRKO cultures (*P < 0.05). Expression of MyoD was higher in both myoblasts (*P < 0.01) and myotubes (*P < 0.05) from IRWT than from IRKO cultures.

Basal and insulin- or IGF-I-stimulated glucose transport values are higher in IRKO than in IRWT skeletal myotubes. Next, it was important to determine whether the lack of IR imparts any physiological defects in the tissue. In view of the increased expression of the glucose transporter proteins in IRKO compared with IRWT skeletal myotubes, particularly in the plasma membrane fractions, it might be expected that basal and glucose transport would be different in IRKO compared with IRWT skeletal myotubes. Accordingly, we first compared basal glucose uptake in IRWT and IRKO skeletal myotubes. Basal glucose uptake in skeletal myotubes from IRKO mice was slightly but significantly higher than that in skeletal myotubes from IRWT mice (Fig. 4). Thus basal 2-DG uptake in IRWT cells was 18 ± 1.3 pmol · mg protein (P)-1 · min-1, whereas that from IRKO cells was 23 ± 2 pmol · mg P-1 · min-1, an increase of nearly 30% [mean values of 3 values in each of 5 experiments (n = 15) on 5 separate cultures; P < 0.05].


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Fig. 4.   Basal and insulin- and IGF-I-induced glucose transport in IRWT and IRKO skeletal myotubes. Myotubes from IRWT and IRKO were transferred to serum-free, low-glucose medium for 24 h. Cells were either untreated (basal uptake, BL) or treated with insulin (Ins; 10-8 M) or IGF-I (IGF; 10-9 M) for 30 min at 37°C. Glucose transport was evaluated by measuring 2-deoxy-[3H]glucose uptake (2-DG) for 15 min. Data are presented as % of basal WT control (18 ± 1.3 pmol · mg P-1 · min-1), which was set at 100%. The basal level of untreated KO myotubes was 23 ± 2 pmol · mg P-1 · min-1, 128% that of WT (P < 0.05). Each bar represents mean ± SE of triplicate values in each of 5 experiments performed on separate cultures (n = 15). Effects of insulin and IGF-I in IRKO skeletal myotubes were significantly higher than in IRWT skeletal myotubes (*P < 0.05; **P < 0.01; ***P < 0.001).

The main role of insulin in muscle tissue is regulation of glucose transport and metabolism by translocation and activation of glucose transporters, mainly GLUT-4. Muscle cells lacking the expression of IR could be utilized, therefore, to follow changes in these processes under conditions of insulin resistance. Thus, in another series of experiments, we examined the effects of lack of IR expression on insulin- and IGF-I-induced glucose transport in primary cultures of skeletal myotubes. In this series, the difference in basal glucose uptake between IRWT and IRKO skeletal muscles was essentially the same as in the previously described results. Somewhat surprisingly, both insulin and IGF-I caused a greater increase in glucose uptake in IRKO skeletal myotubes than in IRWT skeletal myotubes (Fig. 4). Thus, in both IRWT and IRKO myotubes, insulin over the range of 10-9 to 10-7 M caused an increase in glucose uptake, and at each concentration, glucose uptake was higher in IRKO than in IRWT myotubes. Data from experiments in which an insulin concentration of 10-8 M was used are summarized in Fig. 4. In myotubes from IRWT mice, the mean (±SE) glucose uptake after insulin stimulation was 28 ± 1.3 pmol · mg P-1 · min-1, an increase of 40% (P < 0.001). In contrast, insulin increased glucose uptake by >70% (to 40 ± 2.5 pmol · mg P-1 · min-1) in IRKO skeletal myotubes above IRKO basal levels (P < 0.001). Similarly, in IRWT myotubes, IGF-I caused glucose uptake to increase by 32% to 23.7 ± 1.7 pmol · mg P-1 · min-1 (P < 0.05). As in the case of insulin, IGF-I caused a greater increase in glucose uptake in IRKO skeletal myotubes (40% above IRKO basal levels) to 35 ± 2 pmol · mg P-1 · min-1 (P < 0.01). Compared with IRWT basal levels, glucose uptake in IRKO skeletal myotubes in response to insulin was nearly 250% higher, and glucose uptake in IRKO skeletal myotubes in response to IGF-I was nearly 200% higher; in both cases these effects were statistically significant (P < 0.05).

Insulin induction of glucose transport in muscle is accomplished, as mentioned, via translocation of glucose transporters GLUT-1 and GLUT-4 from intracellular pools to the plasma membrane. To examine the mechanisms underlying the increase in insulin and IGF-I induction of glucose uptake in the IRKO myotubes, we followed the effects of insulin- and IGF-I-induced translocation of GLUT-1 and GLUT-4 transporters. As can be seen in Fig. 5A, the amount of GLUT-4 in the plasma membrane under basal conditions was greater in IRKO than in IRWT myotubes. In addition, insulin and IGF-I each translocated GLUT-4 from the internal to plasma membrane fraction in both IRWT and IRKO skeletal myotubes. Densitometry analyses of the blots (Fig. 5B) show that the amount of GLUT-4 translocated to the plasma membrane in IRWT myotubes by insulin was statistically significant, whereas that by IGF-I was only marginally significant. In contrast, the absolute amount of GLUT-4 translocated to the plasma membrane in IRKO myotubes by both insulin and IGF-I was significant. As reported for primary cultures of rat skeletal myotubes (7), insulin did not induce translocation of GLUT-1 in skeletal myotubes from IRWT mice. This was also true for skeletal myotubes from IR null mice, as shown in Fig. 5, C and D. Although IGF-I appeared to translocate GLUT-1 in both IRWT and IRKO skeletal myotubes, this effect was not significant when analyzed by quantitative densitometry.


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Fig. 5.   Effects of insulin and IGF-I stimulation on translocation of glucose transporters (GLUT-4 and GLUT-1) in IRWT and IRKO skeletal muscle myotubes. Cells were grown and treated as detailed in Fig. 4. Thereafter, cells were either untreated (C) or treated for 30 min at 37°C with insulin (Ins; 10-8 M) or IGF-I (IGF; 10-9 M). PM and IM membrane fractions from cultures of IRWT and IRKO were analyzed by Western blotting with anti-GLUT-4 antibody (A) or anti-GLUT-1 antibody (C). Blots are representative of 3 different experiments on different cultures. Relative optical density (OD) measurements made from Western blots of glucose transporters in the 3 experiments are shown in graphs below each blot (B, GLUT-4; D, GLUT-1). In these graphs, the relative OD value of PM fraction of control IRWT was defined as 100%. In each case, OD measurements were made over the entire band. The amount of GLUT-4 in the PM under basal conditions was greater in IRKO than in IRWT myotubes (P < 0.05). The amount of GLUT-4 translocated to the PM in IRWT myotubes by insulin was statistically significant (P < 0.05), whereas that by IGF-I was only marginally significant (P = 0.06). Translocation of GLUT-4 to the PM in IRKO myotubes by both insulin and IGF-I was significant (P < 0.01). Insulin did not induce translocation of GLUT-1 in skeletal myotubes from either IRWT or IR null mice. Translocation of GLUT-1 in both IRWT and IRKO skeletal myotubes was not significant (WT, P > 0.9; KO, P > 0.15).

Autophosphorylation of IGFR induced by either insulin or IGF-I is increased in IRKO skeletal myotubes. The results so far demonstrate that, despite the lack of IR expression, not only was there no decrease in insulin- or IGF-I-induced glucose transport in skeletal myotubes from the IRKO mouse, but uptake in response to these hormones was even greater in IRKO than in IRWT skeletal myotubes. This indicates that insulin might be acting via a different receptor, the most likely candidate being IGFR, which is known to be cross-activated by insulin. To examine this possibility, we studied the autophosphorylation of IR and IGFR in control and insulin- or IGF-I-stimulated IRWT and IRKO myotube cultures. The results of these studies are exemplified in Fig. 6. In IRWT skeletal myotubes, insulin (10-9 to 10-7 M) strongly phosphorylated IR and had a weaker but detectable effect on IGFR. In contrast, in IRKO skeletal myotubes, insulin in the same concentration range induced a striking phosphorylation of IGFR, markedly greater than that observed in IRWT skeletal myotubes. Similarly, IGF-I caused a readily detectable tyrosine phosphorylation of both IGFR and IR in IRWT skeletal myotubes, the effect on the former being greater than on the latter, as expected. However, the induction of IGFR phosphorylation by IGF-I in IRKO skeletal myotubes was noticeably greater than in IRWT skeletal myotubes.


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Fig. 6.   Effects of insulin and IGF-I stimulation on tyrosine phosphorylation of the IR and IGFR. Cells were treated for 1 min with insulin (Ins; 10-9-10-7 M) or IGF-I (IGF; 10-9 M) or remained as untreated control. Cell lysates from cultures of IRWT (top) and IRKO (bottom) mice were immunoprecipitated (IP) with antibodies against the IR or IGFR and immunoblotted (IB) with anti-phosphotyrosine (alpha PY) antibody or anti-IR and anti-IGFR antibodies. Blots are representative of 4 different experiments from 4 separate cultures.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this report, we have shown that skeletal myotubes from IRKO mice can be grown for several days in culture. Skeletal muscle myoblasts from both IRWT and IRKO mice fused into myotubes at essentially the same time, and both sets of myotubes displayed spontaneous muscle contractions. This indicates that membrane properties related to both maintenance of membrane potential and generation of action potentials (8, 9, 30, 34, 35) were intact in the IRKO skeletal myotubes. Consistent with the fact that IRKO myotubes were morphologically indistinguishable from IRWT cells, evaluation of the expression of the muscle differentiation markers MyoD and myogenin indicated that the differentiation process of the muscle cells was unimpaired. Thus, even though expression of each of these proteins was lower in myotubes from IRKO mice than in IRWT mice, differentiation into myotubes was similar in both IR muscle cell types. Expression of these differentiation proteins was, therefore, sufficient for the differentiation process. Nonetheless, the slight decrease in their expression could support the notion that IR does play a role in differentiation. Indeed, a role for insulin and its receptor in differentiation has been suggested in various cells. We have recently shown that insulin enhances skin differentiation and that lack of IR expression is associated with reduced differentiation (36). We did observe an increase in expression of IGFR in cultured IRKO myoblasts compared with IRWT, consistent with earlier studies on IRKO mouse embryos (26). With maturation into myotubes, however, this increase disappeared.

Other findings of particular interest were those pertaining to the glucose uptake system in IRKO skeletal myotubes. First, skeletal myotubes from IRKO mice consistently displayed about 30% higher basal rates of glucose uptake than did the WT controls. This increase, though slight, was statistically significant. The elevated basal uptake was associated with an increase in the expression of GLUT-4 and GLUT-1, the major glucose transporters of muscle cells. This could explain the increase in basal glucose uptake seen in IRKO skeletal myotubes. Second, and somewhat surprisingly, the increase in rate of glucose uptake in response to both insulin and IGF-I was greater in IRKO than in IRWT skeletal myotubes. Here again, the increase in glucose transport was associated with a higher absolute amount of GLUT-4 translocating to the plasma membrane from the intracellular pool. One reason for these findings could be the increase in total GLUT-4 expression in IRKO skeletal myotubes. We have confirmed (6, 7) in mouse skeletal myotubes (both IRWT and IRKO) that insulin does not induce translocation of the GLUT-1 glucose transporter. Thus the elevated insulin-induced glucose transport in IRKO skeletal myotubes is best accounted for by the increased GLUT-4 translocation from intracellular to plasma membrane fractions. IGF-I also induced translocation of the GLUT-4 transporter but did not cause significant translocation of GLUT-1 in either IRWT or IRKO myotubes. The effect of IGF-I on GLUT-4 was greater in IRKO than in IRWT cells. We therefore conclude that the increased glucose transport in response to IGF-I in IRKO skeletal myotubes is accounted for by the effect on the GLUT-4 transporter. However, even though the amounts of translocated GLUT-4 appeared to be similar in response to both hormones, the increase in glucose uptake in response to insulin was consistently and significantly greater than to IGF-I. This might indicate that the hormones may differentially influence translocation and activation of the transporters. Consistent with our findings, it was reported that insulin and IGF-I caused strong increases in glucose uptake in primary cultures of fibroblasts from IRKO mice (19, 20). However, the responses in IRKO fibroblasts were not compared with those in WT mice, so it is uncertain whether the effects of insulin and IGF-I were different from control.

Another contributing factor to increased IGF-I and insulin-induced glucose transport is provided by the results of studies on autophosphorylation of the IGFR in response to each hormone. We found that IGFR autophosphorylation induced by IGF-I was higher in skeletal myotubes from IRKO than from IRWT mice. The increase in IGFR autophosphorylation in IRKO skeletal myotubes was also seen in response to insulin, although the amount of phosphorylation in each case was lower in response to insulin than to IGF-I. Clearly, however, insulin induced strong phosphorylation of IGFR at lower concentrations in IRKO than in IRWT skeletal muscle. It is possible that, in myotubes from IRWT mice, the autophosphorylation of both IR and IGFR induced by insulin and IGF-I may be explained by the presence of IR/IGFR hybrids, which are reported to represent the major form of IGFR in human and rabbit skeletal muscle (4). The proportion of IGFR present as IR/IGFR hybrids in mouse skeletal muscle is not known. Nonetheless, the finding that insulin induced IGFR autophosphorylation in IR null myotubes strongly indicates that insulin is able to signal metabolic responses via the IGFR, and that IGF-I stimulates glucose uptake in skeletal myotubes by activation of both IGFR and IR. In the absence of IR, IGF-I as well as insulin stimulates glucose uptake by higher activation of IGFR in compensation for the lack of IR activity.

According to our findings on tyrosine phosphorylation of IGFR in IRKO skeletal myotubes, this effect most likely occurs via insulin interaction with IGFR, but tyrosine phosphorylation of IGFR by insulin in IRKO fibroblasts was not reported (19, 20). Physiological support for the compensatory role of IGFR in lack of IR can be found in studies showing that IGF-I caused a rapid and sustained decrease of plasma glucose in IRKO mice (13a). It was suggested there that this effect was due to IGF-I stimulation of glucose uptake in skeletal myotubes. Although for technical reasons experiments could not be undertaken to test this proposal, our findings on cultured IRKO skeletal myotubes support this idea.

Our results contrast with those (10) on the MIRKO mouse. Skeletal muscle from these animals, whose blood glucose and plasma insulin levels were indistinguishable from control animals, displayed considerable insulin resistance. Moreover, there were no detectable increases in IGFR expression, and, in contrast to our findings in vitro, insulin did not cause any detectable tyrosine phosphorylation of IGFR in skeletal muscle of MIRKO animals. Indeed, insulin did not appear to activate any proteins (IRS-1, phosphatidylinositol 3-kinase) in the insulin-signaling pathway. MIRKO animals did, however, have elevated levels of free fatty acids and serum triglycerides, findings that are associated with the metabolic syndrome associated with insulin resistance (29). In this regard, it was recently reported in studies on C2C12 myotubes that free fatty acids could alter insulin-stimulated events downstream of IRS-1 (31). It is not known whether these factors might influence insulin signaling through the IGF receptor in IR null tissues. Interestingly, despite the various defects in the insulin-signaling pathway, MIRKO animals were able to clear a bolus injection of glucose with the same efficiency as WT mice. The possibility that this specific effect might be mediated by insulin or IGF-I interaction with IGFR was not examined. Our results would indicate that insulin released by glucose acts on IGFR in skeletal muscle to stimulate glucose uptake. In addition, it should be emphasized that the MIRKO study examined autophosphorylation of the IGFR after the hormones were injected into the animals, which were fasted overnight. These factors could indeed have altered the outcome of the experiments. It was also recently reported that the synergistic activation of glucose transport between insulin and exercise persists in MIRKO mice (40), but the possibility of IGFR participation in these animals did not appear to be considered.

The results presented in this study indicate that the phenomenon of insulin resistance may have important components downstream of IR at the level of other signaling elements. Thus our findings demonstrate that insulin can signal via IGFR in the case of nonexistent IR, and it is possible that this might also be the case in the presence of defective, but partially functional, IR. Although in general it seems that IGFR may compensate for a lack of IR, it may be assumed that insulin does not promote glucose uptake via IGFR stimulation as effectively in IR null animals as it does in isolated muscle cells and fibroblasts lacking IR. This can be concluded from the reported high plasma insulin and glucose levels in the IRKO mice (22, 23). Another possible explanation for the development of hyperglycemia in IRKO mice could be that the compensatory increase in IGFR accompanying the lack of IR found in muscle may not exist in other tissues involved in regulation of blood glucose levels, such as liver and fat.

In summary, skeletal myotubes from IRKO mice grown in culture possess an intact insulin- and IGF-I-responsive glucose uptake system. Nevertheless, clear differences in effects of these growth factors in IRKO compared with IRWT animals indicate that the absence of IR imposes changes in the signaling mechanism normally exerted via IGFR.


    ACKNOWLEDGEMENTS

This study was supported in part by the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities, a grant from the Sorrell Foundation, the Ben and Effie Raber Research Fund, and The Harvett-Aviv Neuroscience Research Fund. E. Wertheimer is a recipient of a Career Development Award from the Juvenile Diabetes Foundation International. S. R. Sampson is the incumbent of the Louis Fisher Chair in Cellular Pathology of Bar-Ilan University. This study represents an essential portion of the thesis submitted by L. Shefi-Friedman in partial fulfillment of the requirements for the PhD degree at Bar-Ilan University.


    FOOTNOTES

Address for reprint requests and other correspondence: S. R. Sampson, Faculty of Life Sciences, Bar-Ilan Univ., Ramat-Gan 52900, Israel (E-mail: sampsos{at}mail.biu.ac.il).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 August 2000; accepted in final form 21 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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