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Endocrinology Vol. 140, No. 9 4244-4250
Copyright © 1999 by The Endocrine Society

ARTICLES

Insulin-Like Growth Factor I Circumvents Defective Insulin Action in Human Myotonic Dystrophy Skeletal Muscle Cells1

Denis Furling2, André Marette and Jack Puymirat

Laboratory of Human Genetics, Department of Medicine (D.F., J.P.), and Lipid Research Unit, Department of Physiology (A.M.), Laval University Medical Research Center, University Hospital Center of Québec , Ste-Foy, Québec, Canada G1V 4G2

Address all correspondence and requests for reprints to: Dr. Jack Puymirat, Human Genetics Laboratory, Laval University Medical Research Center, University Hospital Center of Québec, 2705 boulevard Laurier, Ste-Foy, Québec, Canada G1V 4G2. E-mail: jack.puymirat{at}crchul.ulaval.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Primary human skeletal muscle cell cultures derived from muscles of a myotonic dystrophy (DM) fetus provided a model in which both resistance to insulin action described in DM patient muscles and the potential ability of insulin-like growth factor I (IGF-I) to circumvent this defect could be investigated. Basal glucose uptake was the same in cultured DM cells as in normal myotubes. In DM cells, a dose of 10 nM insulin produced no stimulatory effect on glucose uptake, and at higher concentrations, stimulation of glucose uptake remained significantly lower than that in normal myotubes. In addition, basal and insulin-mediated protein synthesis were both significantly reduced compared with those in normal cells. In DM myotubes, insulin receptor messenger RNA expression and insulin receptor binding were significantly diminished, whereas the expression of GLUT1 and GLUT4 glucose transporters was not affected. These results indicate that impaired insulin action is retained in DM cultured myotubes. The action of recombinant human IGF-I (rhIGF-I) was evaluated in this cellular model. We showed that rhIGF-I is able to stimulate glucose uptake to a similar extent as in control cells and restore normal protein synthesis level in DM myotubes. Thus, rhIGF-I is able to bypass impaired insulin action in DM myotubes. This provides a solid foundation for the eventual use of rhIGF-I as an effective treatment of muscle weakness and wasting in DM.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MYOTONIC DYSTROPHY (DM), the most common form of inherited neuromuscular disease in adults, affects 1 in 8000 individuals worldwide. DM is an autosomal dominant neuromuscular disease characterized by myotonia, progressive muscle wasting and weakness, cardiac conduction defects, cataracts, male baldness, and other manifestations. Variability of clinical severity and anticipation are characteristics of DM (1). Anticipation describes the tendency of an inherited disease to occur at an earlier age of onset, to present ever worsening symptoms, or both as it is passed on from generation to generation. The genetic defect has been identified as an unstable elongation of a trinucleotide sequence (CTG)n located in the 3'-untranslated region of a gene coding for a putative serine/threonine protein kinase, dystrophy myotonic protein kinase (DMPK) (2, 3, 4). Reduced DMPK levels and altered RNA metabolism, consequent to nuclear retention of mutant CTG-DMPK transcripts, are considered prime suspects in the etiology of DM disease (5, 6, 7).

Insulin resistance is the principal metabolic abnormality associated with the pathology of DM. Even though DM patients have normal basal insulin levels and normal glucose tolerance, they present marked hyperinsulinemia after oral glucose tolerance testing (8, 9, 10, 11). Impaired glucose utilization has been reported in tissues such as brain, cardiac muscle, and skeletal muscle in DM subjects (12, 13, 14). Intrabrachial arterial infusion of insulin in patients with DM demonstrated that despite administration of insulin from low to supraphysiological doses, glucose uptake in the forearm muscle remains 3 times lower in DM patients than in normal subjects (15). The mechanisms that underlie insulin resistance in skeletal muscles of DM subjects remain obscure, although both receptor and postreceptor defects are suggested (16, 17, 18, 19).

Abnormal regulation of protein synthesis is another important characteristic of this disorder. Excretion of 3-methylhistidine, an accurate indicator of muscle catabolism (20), is normal in patients with DM (after correction for muscle mass); however, studies of leucine incorporation in skeletal muscle have revealed a sharp decrease in protein synthesis (21). This led to the hypothesis that muscle wasting in DM may result from diminished muscle anabolic processes rather than from increased catabolism. The events that lead to decreased muscle protein synthesis in DM have yet to be elucidated, but a disruption in insulin-mediated protein synthesis could be involved.

Insulin-like growth factor I (IGF-I) is an anabolic hormone that exerts metabolic effects similar to those of insulin. Most of the biological effects of IGF-I are mediated through activation of the IGF receptor (22, 23). IGF-I stimulates intracellular amino acid transport, glucose transport, and protein synthesis in muscle rich in IGF-I receptor (24, 25). Interestingly, one clinical trial indicated that administration of recombinant human IGF-I (rhIGF-I) improved metabolism and function in the muscle of DM patients (26). IGF-I administration also improved metabolism, morphology, and function in the muscle of a murine model of muscular dystrophy (27).

In the present study, we investigate resistance to insulin action using primary human skeletal muscle cell cultures derived from DM muscles. Insulin-stimulated glucose transport and protein synthesis were examined in DM myotubes as well as insulin receptor and glucose transporter levels. Finally, the effects of rhIGF-I on glucose transport and protein synthesis were evaluated in DM myotubes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primary human muscle cell cultures
Normal 15-week-old human fetal muscle was obtained (Clonetics, Palo Alto, CA), and human skeletal muscles were biopsied from a 12-week-old aborted DM fetus. The protocol was approved by the Laval University Hospital Center ethical review board, and a signed consent was obtained from the mother for the use of fetal muscle tissues for research. Myoblasts were purified as previously described (28) and were further expanded at 37 C in the proliferative medium MCDB120 supplemented with 15% FBS. The medium was subsequently changed to a differentiating medium ({alpha}MEM supplemented with 5% horse serum and 0.5 mg/ml BSA). Cells were grown in 5% CO2 humidified. Normal and DM cells were used between the fourth and sixth passages. The number of passages refers to the total number of passages from the time of isolation of the initial myoblast population.

[2-3H]Deoxyglucose glucose transport
2-Deoxyglucose uptake was measured according to the procedure described by Sarabia et al. (29). Myotubes were preincubated for 24 h in serum-free medium containing 5.5 mM glucose. The cells were then incubated for 45 min at 37 C with or without adding either insulin or rhIGF-I. The cells were washed once with glucose-free HEPES-buffered saline solution and then incubated with 10 µM 2-deoxyglucose containing 0.3 µCi/ml 2-deoxy-[3H]glucose in the same buffer. Glucose uptake was measured at 8 min because the rate of 2-deoxyglucose uptake in fused myotubes from control subjects was linear for up to 20 min at room temperature (not shown). After washing, cells were lysed in 0.05 N NaOH, and radioactivity was measured with a scintillation counter. Noncarrier-mediated uptake was determined in parallel in the presence of 10 µM cytochalasin B and was subtracted from all experimental values. Protein content was determined by the Bradford method (30), using a Bio-Rad reagent (Bio-Rad Laboratories, Inc., Richmond, CA). Transport is expressed throughout as picomoles per min/mg protein.

Determination of protein synthesis
Myotubes were cultured in differentiating medium, which was changed for serum-free medium 48 h before beginning the experiments. Cells were incubated with or without insulin or rhIGF-I in the presence of 2 µCi/well [3H]phenylalanine (132 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL) as described by Frost et al. (31). The cells were then washed in 0.9% saline and lysed with 0.1 N NaOH. DNA content was read at 260 nm. Proteins were precipitated overnight at 4 C with 10% trichloroacetic acid and solubilized in 0.05 N NaOH. Radioactivity was measured in a liquid scintillation counter. Protein synthesis is expressed as nanomoles of [3H]phenylalanine incorporated per µg DNA.

Western blot analysis
Crude membranes were prepared from fused human cells according to the procedure described by Sarabia et al. (29). Cells were rinsed in 0.9% saline and harvested with a rubber scraper. Cells were centrifuged at 1,000 rpm, followed by homogenization of the cell pellet in homogenization buffer [20 mM HEPES-Na (pH 7.4), 250 mM sucrose, 2 mM EGTA, 5 mM NaN, 0.2 mM PMSF, 10 mM E64, 1 mM pepstatin A, and 1 mM leupeptin] using a Dounce homogenizer (Kontes Co., Vineland, NJ). The homogenates were centrifuged for 3 min at 6,000 rpm to remove nuclei and mitochondria. The supernatant was further centrifuged for 70 min at 200,000 x g to yield a total membrane pellet. The membranes were resuspended in Laemmli’s buffer (32), the protein content was determined, and samples were stored at -80 C until analysis. Proteins were separated by SDS-PAGE using a 7.5% polyacrylamide resolving gel slab and were then transferred to polyvinylidene difluoride membranes (Immobilon-P Millipore, Bedford, MA). After blocking nonspecific sites, the transferred proteins were probed with the polyclonal antibodies anti-GLUT1 (East-Acres Biologicals, Southbridge, MA) and anti-GLUT4 (Biogenesis, Bournemouth, UK). The membrane was washed, and incubated with a horseradish peroxidase-conjugated antirabbit IgG. Immune complexes were detected using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech).

Insulin receptor binding assay
Crude membranes from myotubes were prepared as described above. Sixty micrograms of crude membrane extracts were incubated for 30 min at 4 C in a buffer containing 25 mM Tris (pH 7.4), 10 mM MgCl2, 0.1% BSA, 1 mM phenylmethylsulfonylfluoride, and 1% Triton X-100 and then overnight after the addition of [125I]insulin (50,000 cpm). Four volumes of a buffer containing 25 mM Tris (pH 7.4), 10 mM MgCl2, 0.125% {gamma}-globulin, and 25% polyethylene glycol were added, and the samples were incubated for 10 min at 4 C and then centrifuged for 30 min at 1900 x g. The pellet’s radioactivity was measured by a Wallac, Inc. {gamma}-counter (1270 Rackgamma II, LKB, Rockville, MD). All results were corrected for nonspecific binding by subtracting the amount of [125I]insulin bound in presence of excess (3.6 x 10-8 M) unlabeled insulin.

RNA extraction and RT-PCR
Total RNA was isolated from fused human cells with Trizol reagent (Life Technologies, Inc., Grand Island, NY) and treated with deoxyribonuclease (Promega Corp., Madison, WI) following the manufacturer’s instructions. One microgram of RNA was reverse transcribed into complementary DNA using a reaction mixture of 200 U Moloney murine leukemia virus reverse transcriptase (Promega Corp.), 1 x RT-PCR reaction buffer, 10 mM of each deoxy-NTP, 20 U RNAguard (Pharmacia Biotech, Piscataway, NJ), and 0.5 µg random hexamers (Pharmacia Biotech) or 90 ng of each sequence-specific primer (using a mixture of antisense primers directed against the distinct genes described below). Complementary DNA was synthesized at 37 C for 1 h. Subsequently, 1/10th of the RT reaction was used as a template for PCR analyses. Oligonucleotide primer sequences were as follows: insulin receptor, F-5'-GAG-ATG-GAG-TTT-GAG-GAC-ATG and R-5'-GTG-TAA-GGG-ATG-TGT-TCC-TCG-TAG; dystrophin, F-5'-CCA-AAC-TAG-AAA-TGC-CAT-CTT-C and R-5'-TCT-GAA-TTC-TTT-CAA-TTC-GAT; glucose transporter (GLUT4), F-5'-CAT-AGG-AGC-TGG-TGT-GGT-CA and R-5'-CAA-ATA-GAA-GGA-AGA-CGT-AG; and glyceraldehyde-3-phosphate dehydrogenase (GADPH), F-5'-GAT-GAC-AAG-CTT-CCC-GTT-CTC-AGC-C and R-5'-TGA-AGG-TCG-GAG-TCA-ACG-GAT-TTG-GT. Standard PCR reaction mixture conditions containing 250 µM deoxy-NTPs, 2.5 U Taq DNA polymerase (QIAGEN, Chatsworth, CA), 1 x PCR reaction buffer, and 70 ng of each primer set were used. Cycle characteristics for these primers were 94 C for 1 min, 60 C for 1 min, and 72 C for 1 min. The number of cycles permitting amplification within a linear range was used for each set of primers. The PCR amplification products were resolved on a 3% agarose gel slabs stained with ethidium bromide. Peak areas associated with DNA bands were determined using the AlphaImager scan (Alpha Innovatech Corp., San Leandro, CA).

Southern blot analysis
Genomic DNA was extracted from skeletal muscle or tissue cultures using a genomic DNA isolation kit (Gentra Systems Inc., Minneapolis, MN) according to the manufacturer’s specifications. Ten milligrams of DNA were digested with EcoRI, separated by electrophoresis, and transferred onto a nylon membrane (Hybond, Amersham Pharmacia Biotech). Membranes were hybridized with 32P-labeled pGB2.2 probe and visualized by autoradiography (4).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Muscle cell cultures
Myoblasts were first labeled with neural cell adhesion molecule and then purified by cell sorting. More than 95% of cells in cultures were desmin positive, indicating their myogenic phenotype. The growth capacities of normal and DM cells were very similar. The doubling time of normal and DM myoblasts was about 36 h. When cultures were switched from permissive to nonpermissive conditions, DNA synthesis ceased in the majority of cells by 48 h. Myotube formation began within 3 days of switching to nonpermissive conditions, and myotubes continued to increase in size and number for several days. By day 5, the percentage of fusion was about 40–50%. Southern blot analyses were performed to determine whether myoblasts from DM patients retained the molecular defect (Fig. 1Go). An EcoRI digestion of genomic DNA isolated from normal blood produced the typical 9- and 10-kb bands. Analysis of DM myoblast DNA revealed a band of 12.2 kb whose higher mol wt corresponds to an expansion of about 750 CTG repeats. The number of repeats in DM myoblasts and that in DM skeletal muscle used for cultures were similar (not shown). Control myoblasts showed only a 10-kb band, indicating that these cells were homozygous for the probe.



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  		Figure 1. Determination of the number of (CTG)n repeats in myoblasts from DM (MB DM) by Southern blot analysis. Control myoblasts (MB) were also analyzed, and normal blood was used as a standard. Ten micrograms of genomic DNA were digested with EcoRI, electrophoresed, transferred onto nylon membrane, and hybridized with 32P-labeled pGB2.2 DNA probe. Membranes were then autoradiographed.

 
Metabolic studies
Resistance to insulin action in human myotubes was first investigated at the level of glucose uptake. Figure 2AGo shows the insulin dose-response curve of 2-deoxyglucose uptake in cells derived from normal muscle. A typical sigmoidal curve was obtained, with maximal stimulation at 10-6 M insulin. Half-maximal stimulation of glucose uptake occurred at 1.55 x 10-8 M insulin. The mean basal transport rate was 29.3 ± 1.4 pmol/min per mg protein. At 10-8 and 10-7 M, insulin induced significant 20% and 41% increases in glucose uptake (P < 0.005, by Student’s t test), whereas maximal stimulation (47%) was observed at 10-6 M insulin. Noncarrier-mediated uptake (i.e. the cytochalasin B-insensitive component) was not affected by insulin (data not shown). The stimulatory effect of half-maximal and maximal insulin concentrations on glucose uptake was then investigated in DM myotubes (Fig. 3AGo). Basal glucose uptake was unaltered in DM myotubes (28.5 ± 1.5 vs. 29.3 ± 1.4 pmol/min per mg protein, P > 0.05). Treatment of DM myotubes with 10-8 M insulin had no significant effect on glucose uptake (27.5 ± 1.7 vs. 28.5 ± 1.5 pmol/min per mg protein; P > 0.05), whereas at 10-7 and 10-6 M, insulin increased glucose uptake by 17% and 30%, respectively (P < 0.01). Compared with control muscle cells, glucose uptake in cells from DM subjects was unstimulated by a 10-nM dose of insulin and remained low even at higher concentrations (reduction of 24% at 100 nM and 17% at 1 µM compared with controls; P < 0.02).



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  		Figure 2. A, Dose-response curve of insulin action of 2-deoxyglucose uptake in human myotubes. Cells were depleted of serum in {alpha}MEM containing 5.5 mM glucose for 24 h, incubated in the absence or presence of increasing concentrations of insulin for 45 min at 37 C, and then 2-deoxyglucose uptake was measured. B, Dose-response curve of insulin action of protein synthesis in human myotubes. Cells were depleted of serum in {alpha}MEM without insulin and containing 5.5 mM glucose for 48 h and then incubated in the absence or presence of the indicated concentrations of insulin and rhIGF-I for 5 h at 37 C. Results are the mean ± SD of three different myoblast cultures. Each experiment was performed in triplicate.

 


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  		Figure 3. A, Comparison of glucose transport activity in human skeletal muscle cells from normal and DM subjects. Cells were incubated with {alpha}MEM depleted of insulin for 24 h and then incubated in the absence or presence of insulin and rhIGF-I. Cells were then washed, and 2-deoxyglucose uptake was measured. B, Comparison of protein synthesis activity in human skeletal muscle cells from normal and DM subjects. Cells were incubated with {alpha}MEM depleted of insulin for 48 h and then incubated in the absence or presence of insulin and rhIGF-I for 5 h at 37 C. Cells were then washed, and protein synthesis was measured. Results are the mean ± SD of three different myoblast cultures derived from the same muscle. Each experiment was performed in triplicate. *, P < 0.05; **, P < 0.005 (by t test).

 
The effect of insulin on protein synthesis was then investigated in human myotubes from control and DM subjects. Figure 2BGo represents the insulin dose-response curve of protein synthesis, as measured by [3H]phenylalanine incorporation. Treatment of normal cells with 10-8 M insulin induced a significant 15% increase in protein synthesis (87.7 ± 6.6 vs. 76.3 ± 7.3 nmol [3H]phenylalanine incorporated/µg DNA; P < 0.02), and this effect rose to 22% and 30% (P < 0.005) at 10-7 and 10-6 M insulin, respectively. Half-maximal stimulation occurred at 1.1 x 10-8 M insulin. In DM myotubes, the basal rates of protein synthesis (54.2 ± 6.2 [3H]phenylalanine incorporated/µg DNA) dropped by 29% (P < 0.005). No stimulatory effect was observed at 10-8 M insulin, whereas insulin stimulated protein synthesis by 56% at 10-6 M (84.7 ± 3.5 [3H]phenylalanine incorporated/µg DNA; P < 0.005; Fig. 3BGo). Compared with control myotube values, insulin-mediated protein synthesis was lower at both concentrations tested in cells from DM subjects (15% reduction; P < 0.01).

Because IGF-I is associated with metabolic effects similar to those of insulin, we examined whether rhIGF-I could exert its insulin-like properties in DM myotubes. The optimal concentration of rhIGF-I (13 nM) that can induce maximal stimulation of glucose uptake was determined in preliminary experiments. In normal muscle cells, rhIGF-I (13 nM) induced a significant 49% increase in glucose uptake (P < 0.005), which is nearly the same as the value obtained during maximum stimulation by insulin (47%). In DM myotubes, rhIGF-I stimulates glucose uptake to the same extent (51%; P < 0.005). Thus, glucose uptake was increased by 21% compared with the highest dose of insulin (30% at 1 µM). The stimulatory effect of rhIGF-I on protein synthesis was measured in DM and normal myotubes. rhIGF-I induced a significant 33% rise in protein synthesis in normal myotubes (101.78 ± 4.78 [3H]phenylalanine incorporated/µg DNA; P < 0.005). In DM myotubes, rhIGF-I stimulated protein synthesis by 61% (87.4 ± 9.32 [3H] phenylalanine incorporated/µg DNA; P < 0.005), but protein synthesis levels remained 14% lower (P < 0.05) than that in normal IGF-I-treated myotubes.

Levels of insulin receptors, and GLUT1 and GLUT4 transporters in DM myotubes
The first step in the insulin signaling cascade is binding of insulin to its cognate receptor. We therefore measured these two activities: insulin receptor messenger RNA (mRNA) expression and binding of insulin to insulin receptor, in DM and normal myotubes. Steady state levels of insulin receptor mRNA transcription were statistically lower in DM compared with those in normal myotubes (Fig. 4Go). A significant 31% reduction in the content of insulin receptor mRNA relative to that of dystrophin mRNA (P < 0.01) was detected in DM myotubes using RT-PCR amplification. Insulin receptor binding on total membranes further confirmed impaired expression of the receptor in DM myotubes. A 14% reduction in the incidence of specific insulin binding was observed in DM myotubes compared with that in normal cells (0.058 ± 0.02 vs. 0.068 ± 0.04 pmol/mg protein; P < 0.02).



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  		Figure 4. Multiplex RT-PCR analysis of insulin receptor (InR) mRNA compared with those of dystrophin (Dys) in normal and DM myotubes. The graph shows the ratio of InR mRNA vs. Dys mRNA. Results are the mean ± SD of three different cultures. **, P < 0.01 (by t test).

 
Glucose transport is facilitated by the glucose transporter proteins GLUT1 and GLUT4 in skeletal muscle, so the expression of these glucose transporters was analyzed in myotubes from DM and normal muscle (Fig. 5AGo). Western blot analysis with specific antibodies directed against GLUT1 and GLUT4 revealed the presence of bands migrating at 45,000 and 47,000 Mr, respectively. No difference was observed in the levels of either glucose transporter protein between normal and DM myotubes. RT-PCR analysis of GLUT4 (relative to GADPH mRNA) confirmed the normal expression of the mRNA encoding this transporter in DM myotubes (Fig. 5BGo).



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  		Figure 5. A, Expression of GLUT1 and GLUT4 proteins in myotubes of human skeletal muscle cells from normal and DM subjects. Thirty micrograms of crude membrane protein were loaded. B, Levels of GLUT4 and GADPH mRNA in normal and DM myotubes in three different cultures evaluated by RT-PCR as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
When characterized in human muscle cells, glucose uptake was found to be saturable, carrier mediated, and regulated by insulin (29, 33). In the present study, we report the successful establishment of human muscle cell cultures grown from satellite cells taken from normal and DM fetal muscles. Because the expression of GLUT4 protein is detectable only in differentiated myotubes (34), it is considered a good indicator of differentiated skeletal muscle in culture. The finding that levels of expression of GLUT4 are similar in normal and DM myotubes provides convincing evidence that the observations noted in DM myotubes reflect true insulin resistance and are not the consequence of retarded growth or reduced differentiation of DM myotubes grown in culture. Furthermore, these results confirm that primary skeletal muscle tissue cultures are appropriate for investigating the role of insulin in glucose transport and protein synthesis in DM.

The characteristics of the glucose transport system in human fetal muscle cells in culture were investigated at the stage of fused myotubes. We found that in normal myotubes insulin stimulated glucose uptake by 47% at a maximal insulin concentration, which concurs with the range of values reported by others (29, 33, 35). Skeletal muscle cell cultures from a DM subject had decreased glucose transport activity only in the presence of insulin. The ability of insulin to increase glucose uptake was significantly reduced by 24% in DM myotubes. It is important to mention that human myotubes contain specific high affinity receptors for both insulin and IGF-I and that the number of IGF-I receptors in myotubes exceeds that for insulin (36, 37). It is therefore possible that the higher concentration of insulin used in this study mediated its effect in part through the IGF-I receptor. Because it was shown that insulin does not displace IGF-I-binding sites at 10 nM (38), it is unlikely that IGF-I receptors were involved in the effect of insulin at this concentration. This suggests that the absence of insulin action at 10 nM is a dependable indicator of insulin resistance in DM myotubes.

Our study also revealed that skeletal muscle cell cultures from a DM subject present impaired protein synthesis. The basal rate of protein synthesis dropped by 29% in DM myotube cultures, suggesting a defect in anabolic processes in DM. At 10 nM, insulin significantly increased protein synthesis in normal, but not in DM, myotubes. At a higher concentration (1 µM), insulin stimulated protein synthesis in both normal and DM myotubes, but the levels of protein synthesis remained 25% lower in DM myotubes than in normal myotubes. As mentioned above, it is also possible that IGF-I receptors are involved in the effect of high insulin concentrations. These results indicate that the reduction in protein synthesis observed in DM muscle in vivo is also retained in cultured muscle cells. This is the first hint of the implication of insulin resistance in the impaired protein synthesis observed in skeletal muscle of DM subjects. The fact that a lower rate of protein synthesis was observed in the absence of insulin indicated that another insulin-independent alteration as well is involved in impaired protein synthesis of DM muscle cells.

The beneficial effect of rhIGF-I in DM was described in a previous clinical study (26). In rhIGF-I-treated DM patients, the researchers observed an increase in protein synthesis, glucose disposal rate, neuromuscular function, and muscle strength. Here, we demonstrate that rhIGF-I is able to circumvent insulin resistance in DM skeletal muscle cells. Glucose uptake rose to a level similar to that in normal rhIGF-I-treated myotubes, indicating that rhIGF-I can sustain normal glucose transport in DM muscle cells and that insulin-stimulated glucose transport deficiency in DM myotubes can be bypassed by rhIGF-I. Protein synthesis in DM muscle cells was stimulated by rhIGF-I to levels comparable to basal levels in untreated normal myotubes, but remained 14% lower than that observed in normal cells treated with rhIGF-I. Moreover, restoration of normal protein synthesis by rhIGF-I is consistent with the implication of insulin resistance in impaired protein synthesis in DM muscle cells. Thus, our results can explain in part the beneficial effect of rhIGF-I treatment in DM patients because the IGF-I receptor is present in high concentrations in muscle, and IGF-I acts through activation of its receptor to correct insulin resistance (22). Taken together, these results support the further development of IGF-I as a treatment of muscle wasting in DM disease.

Both receptor and postreceptor defects have been implicated in the development of insulin resistance in muscle of DM subjects (16, 17, 18, 19). The possibility that reduced insulin receptor binding may be involved in insulin resistance of DM muscles is still controversial, because both normal (39) and diminished (17) receptor levels have been reported. In the present study, we found a small (14%), but significant, reduction of total insulin receptor binding in DM myotubes compared with that in normal cells. This decrease is consistent with the 31% reduction in insulin receptor mRNA measured in the same cells. These results are in line with those obtained by Hoffman’s group (17), who reported a similar reduction in the expression of the insulin receptor in DM muscle in vivo, and they lend support to the dominant negative RNA hypothesis suggested by several groups (5, 40, 41, 42, 43). The presence of the CTG expansion could alter the RNA metabolism of a number of genes involved in the biochemical abnormalities observed in DM. This reduction in insulin receptor binding may in part explain the absence of an effect by insulin on both glucose uptake and protein synthesis at 10 nM. However, this small decline in total receptor binding is not likely to be responsible for the significant reduction in maximal insulin responsiveness in DM myotubes. This defect probably results from some postreceptor defect in the insulin signaling cascade. Moreover, if a postreceptor mechanism is involved in insulin resistance in DM cells, our results suggested that IGF-I and insulin should differ in some of their postreceptor mechanisms.

Insulin binds to its receptor and activates the intrinsic tyrosine kinase activity of the transmembrane ß-subunits. This leads to the phosphorylation of insulin receptor substrate (IRS)-proteins (IRS-1 and IRS-2) on tyrosine residues (44). Phosphorylated IRS proteins can then associate with the regulatory subunit of phosphatidylinositol 3-kinase (PI-3K), thereby activating the catalytic subunit of this lipid kinase. Activation of PI-3K represents a key step for insulin action on glucose uptake (45). Activation of PI-3K has been shown to induce redistribution of GLUT4 from an intracellular storage vesicular compartment to the cell surface, where glucose uptake takes place. We thus examined whether GLUT4 expression is altered in DM myotubes. GLUT4 was expressed at similar levels in normal and DM skeletal muscle cells, indicating that impaired glucose uptake attributed to insulin resistance cannot be explained by defective synthesis of the transporter. The levels of GLUT1, the other transporter isoform expressed in the myotubes, were not affected in DM muscle cell either. These results suggest that impaired action of insulin during glucose uptake may be explained by both a more upstream signaling defect in insulin signalization and reduced translocation of GLUT4 to the cell surface. More studies will be needed to identify the precise signaling step affected in DM muscle cells.

DMPK is a serine/threonine kinase that shares 72% sequence homology with Rho-associated kinase (46). Indeed, the small GTP-binding protein Rho has been recently suggested to play a role in the regulation of glucose uptake by insulin (47, 48). Although the cellular targets of DMPK are still unknown, it may be possible that altered expression of this kinase is involved in the defective action of insulin in DM myotubes. Whether DMPK, alteration of RNA metabolism, or both are linked to insulin action (thus explaining the occurrence of insulin resistance in DM muscle cells) is presently under investigation.

In summary, the present study demonstrated that the reported defects in insulin-stimulated glucose metabolism and protein synthesis in muscle of DM patients are conserved in cultured skeletal muscle cells. This in vitro model will be useful in further characterization of the molecular defects underlying insulin resistance in DM. Finally, we have shown that rhIGF-I is able to circumvent the defects that block insulin action in DM myotubes. These results provide firm support that rhIGF-I could be an effective treatment of muscle weakness and wasting in DM.


   Acknowledgments
 
We thank Dr. J. Mathieu for providing the DM muscle sample, and Dr. R. Faure for helpful assistance with the insulin receptor binding assay.


   Footnotes
 
1 This work was supported by a grant from the Association Française de Lutte contre les Myopathies. Back

2 Recipient of the Association Française de Lutte contre les Myopathies. Back

Received March 17, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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