Insulin binding and internalization in cultured fibroblasts from myotonic muscular dystrophy

Journal of the Neurological Sciences, 1987, 80:229-235

229

Elsevier JNS 02858

Insulin binding and internalization in cultured fibroblasts from myotonic muscular dystrophy Chizuko N a k a n o and K o u s a k u Ohno Division of Child Neurology, Institute of Neurological Sciences, Tottori University School of Medicine, Yonago (Japan)

(Received 24 October, 1986) (Revised, received30 March, 1987) (Accepted 30 March, 1987)

SUMMARY The binding and internalization of insulin receptors were investigated in 8 adult form and 6 early-onset myotonic muscular dystrophy (MMD) patients and in age- and sex-matched controls, using cultured skin fibroblasts to avoid the in vivo milieu of donors. The specific insulin binding in the presence of [ 125I]insulin alone at pH 7.4 and pH 8.0 in MMD patients was not significantly different from that of the controls. The competition curves of insulin binding and the Scatchard plots in MMD and control fibroblasts were similar. Insulin receptor affmity in MMD patients was not different from that in the controls. In the presence of chloroquine, a lysosomotropic agent, the rate of increase in cell-associated radioactivity was similar in both MMD groups and controls. Thus, the normal binding and internalization of the insulin receptors in cultured skin fibroblasts from MMD patients suggest that the insulin receptors are not determined by the pathological genetic factors in MMD. Furthermore, the abnormal insulin binding to freshly isolated cells, reported previously, may be a reflection of environmental factors rather than a genetically determined cellular abnormality in MMD.

Key words: Myotonic dystrophy; Adult form; Early-onset form; Insulin binding; Internalization; Cultured fibroblasts

Correspondenceto: Dr. ChizukoNakano, Divisionof Child Neurology,Instituteof Neurological Sciences, Tottori UniversitySchoolof Medicine,86, Nishi-machi,Yonago683, Japan. 0022-510X/87/$03.50 © 1987Elsevier Science Publishers B.V.(BiomedicalDivision)

230 INTRODUCTION

Myotonic muscular dystrophy (MMD) is an autosomal dominant, multisystemic disease frequently associated with peripheral insulin resistance (Tevaarwerk and Hudson 1977). Several studies on insulin binding to freshly isolated monocytes from MMD patients have been reported, but the results somewhat differ from each other, that is, decreased affinity (Festoffand Moore 1979; Tevaarwerk et al. 1979; Stuart et al. 1983) or reduced concentration (Festoff and Moore 1979) of insulin receptors, abnormal regulation of insulin affinity following glucose ingestion (Moxley et al. 1981), or no significant abnormality of insulin binding (Kobayashi et al. 1977). It is unknown whether these various results of insulin binding in MMD reflect a primary or secondary defect. Therefore, in order to study the insulin binding under an isolated condition that is not influenced by the patient' physiological status and to determine whether the insulin binding in MMD patients is affected by genetic or environmental factors, we studied the binding of insulin receptors and furthermore the internalization, using cultured skin fibroblasts that express genetically determined donor characteristics without any influence of the in vivo milieu. MATERIALS AND METHODS

Cells and cell cultures

Skin biopsies were performed on 3 male and 5 female patients with the adult form of MMD, ranging in age from 32 to 46 years, 2 male and 4 female children with early-onset MMD, ranging from 0 to 14 years, and 14 adults and 15 children as sexand age-matched controls. Fibroblast strains were established and cultured as described previously (Ohno et ai. 1984). Unless otherwise specified, cells were grown in Dulbecco's modification of Eagle's medium (DEM) at a glucose concentration of 1 g/1 (GIBCO, Berkeley, CA), containing 500 IU/ml penicillin G, 100 #g/ml streptomycin sulfate and 10~o fetal bovine serum (FBS) (M.A. Bioproducts, Walkersville, MD). Cultures were fed twice a week. Insulin binding assay

The binding assay was done in triplicate for each measurement according to the methods described previously (Schilling et al. 1979; Hidaka et al. 1981; Sato et al. 1984). Subconfluent fibroblast cultures (passage numbers between 4 and 7, population doubling levels between 6 and 10) were trypsinized and inoculated at a seeding density of 1 × 105 cells per 33-mm dish (NUNC, Denmark) in 2 ml of DEM containing 10~o FB S. On days 7-8, after the cells had been confluent for several days, the insulin-binding assay was performed. Cell monolayers were rinsed twice in phosphate-buffered saline containing Ca 2+ and Mg 2÷ (PBS), and then incubated in PBS supplemented with 2 mM glutamine, Eagle's minimum essential medium (MEM), amino acids and MEM vitamins (Nissui Seiyaku, Tokyo), 10 mg/ml bovine serum albumin (fraction V, Sigma Chemical, St. Louis, MO), 10mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) and 10 mM N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid

231 (HEPES) at pH 7.4 or pH 8.0 in a final volume of 1.0 ml. The incubation mixture contained 0.2 ng [125I]insulin (190 Ci/g, Dinabot Radioisotope Labo., Tokyo, Japan) (Sato et al. 1984), various concentrations (0, 1, 5, 10, 20, 50, 100, 500 ng/ml) of unlabeled insulin (porcine monocomponent, Novo A/S, Denmark). Nonspecific binding was determined in the presence of a large excess of unlabeled insulin (10/~g/ml). After a 5-h incubation in room air at 15 °C the cell monolayers were washed 5 times with cold PBS and solubilized with 1.0 ml of 0.1 N NaOH. The radioactivity was assayed in a y-counter. The specific binding was determined after subtracting the nonspecific value. To examine the effect of chloroquine on cell-associated [ 125I]insulin, cell monolayers were incubated with [125I]insulin (0.2 ng/ml) in the presence or absence of 0.2 mM chloroquine for 120 min at 37 °C.

Data analysis All assays were performed in triplicate and the results were normalized to 107 cells. From the insulin competition curves the unlabeled insulin concentration required to reduce the specific binding of [125I]insulin binding (Bso) to 107 cells by 5 0 ~ were

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Fig. 1. Competitioncurvesofinsulin bindingto culturedfibroblasts.The results (mean + SD) are expressed

as specificbindingper 1 × 107 cells for (4) adult form MMD patients (0) and control adults (O), and for (B) early-onsetMMD patients (O) and control children (O).

232

obtained. As described by Scatchard (1949), the results for individual subjects were plotted as the bound-to-free insulin (B/F) ratio versus the amount of insulin bound and the affinity constant for the initial part of the c u r v e (ga) was given for the insulin receptors in the high affinity range. The low counts at high insulin concentrations preclude the reliable estimation of total receptor number, R o, and consequently the K~ (average affinity at empty sites) and the Kr (average affinity at fdled sites), described by De Meyts and Roth (1975). All data are presented as mean + SD. Statistical analysis was performed by Student's t method. RESULTS

Figure 1 shows the insulin competition curves of insulin binding at pH 7.4. The specific binding in the presence of tracer alone was not significantly different in adult form MMD and control adults (6.90 + 0.72% and 7.23 + 0.85%, respectively), or in early-onset MMD and control children (6.31 + 0.83 % and 7.02 + 0.99%, respectively). The insulin concentration required to displace 50% of the specifically bound [~25I]insulin (Bso), as a measure of binding affinity, was 13.5 + 5.3 ng/ml for the adult 0,09 0,08 0,071

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Fig. 2. Scatchard analysis of insulin binding data for (A) adult form MMD patients ( 0 ) and control adults (O), and for (B) early-onset MMD patients ( 0 ) and control children (O). Each point represents the mean + SD.

233 11

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Fig. 3. Effect o f p H on specific binding of [12sI]insulin to cultured fibroblasts. The results are represented as the mean + SD of the percent of insulin binding to 1 x 107 cells for (A) adult form M M D patients (Q) and control adults (©), and for (B) early-onset M M D patients ( Q ) and control children (O).

form of M M D and 13.0 + 5.4 ng/ml for control adults (P > 0.10). In children, the Bso values were 14.4 + 5.0 ng/ml for early-onset M M D and 13.3 + 3.5 ng/ml for controls ( e > 0.10). There were no differences in the Scatchard plots of insulin binding to cultured fibroblasts of M M D patients and controls (Fig. 2). The K a for adult form and earlyonset M M D (1.12 + 0.49 n M - ~ and 1.05 + 0.51 n M - ~, respectively) were similar to those for control adults and children (1.21 + 0 . 3 7 n M -~ and 1.13 + 0 . 2 4 n M -~, respectively). The low counts at a high insulin concentration preclude the reliable estimation of R o and consequently K~ and Kf.

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Fig. 4. Effect of chloroquine on cell-associated l~251]insulin in fibroblasts from (A) adult form M M D patients (0) and adult controls (O), and from (B) early-onset M M D patients (0) and control children (O). The results represent the mean + SD of the ratio of cell-associated [~25I]insulin in fibroblasts in the presence or absence of 0.2 mM chloroquine.

234 At pH 8.0, the specific binding in the presence of tracer alone was not significantly different in adult form M M D and control attults (9.20 + 1.43~o and 9.03 + 1.39~o, respectively), or in early-onset M M D and control children (9.21 + 1.42~o and 9.32 + 1.81 ~o, respectively) (Fig. 3). The Scatchard analysis at pH 8.0 revealed comparable curvilinear plots in M M D and controls (not shown in figures). Figure 4 shows the effect of chloroquine on cell-associated [1251]insulin in fibroblasts from MMD patients and controls. Chloroquine is a lysosomotropic agent that inhibits intracellular insulin degradation and subsequently causes the accumulation increase of cell-associated radioactivity (Marshall and Olefsky 1979; Prince et al. 1981). In the presence ofchloroquine (0.2 mM), the amount of cell-associated [ 12sI]insulin increased in both the M M D groups and controls without significant differences among them. DISCUSSION In MMD, a generalized defect in cellular membrane function had been suspected because of the involvement of a multiple organ system and various abnormalities of the erythrocyte and skeletal muscle membranes (Roses and Appel 1973,1974; Plishker et al. 1978). The process of insulin binding is one membrane function. Lain (1983) reported that insulin binding to cultured skin fibroblasts from M M D patients is decreased at pH 8.0. However, in our study insulin binding to cultured fibroblasts showed no abnormality at pH 7.4 or pH 8.0 in both adult form and early-onset MMD. It is not known why the results of two studies were different. Moreover the process of internalization of cell surface-bound insulin was also normal in cultured fibroblasts of our patients. These results of normal insulin binding and internalization in cultured fibroblasts could not explain the peripheral insulin resistance in MMD. Normal insulin binding to cultured fibroblasts and abnormal insulin binding to monocytes are also found in cases of non-insulin-dependent diabetes mellitus. In non-insulin-dependent diabetes mellitus, the reduced receptor concentration in the monocytes, erythrocytes and adipocytes is thought to be due to a reflection of secondary acquired factors because of normal insulin binding to cultured fibroblasts which express genetically determined donor characteristics (Prince et al. 1981). Similarly, abnormal insulin binding to monocytes (Festoffand Moore 1979; Tevaarwerk et al. 1979; Moxley et al. 1981; Stuart et al. 1983) and normal to cultured fibroblasts in our study may be explained by the same mechanism in M M D as in non-insulin-dependent diabetes mellitus. On the other hand, the clinical features in M M D may be ascribable to environmental factors as well as to genetic factors. For example, the existence of the gene for MMD produces the early-onset form, but the neonatal symptoms in early-onset MMD include a reflection of intrauterine environmental factors (Harper and Dyken 1972). Furthermore, an increase of deoxycholic acid (DCA) and a decrement in the ratio of cholic acid to chenodeoxycholic acid (CA/CDCA) in M M D patients may result in membrane dysfunction (Tanaka 1985). Thus, the normal binding and internalization of the insulin receptors in cultured skin fibroblasts removed from the in vivo environment of the M M D donors suggest that

235 the insulin receptors are not determined by the pathological genetic factors in MMD. Moreover the abnormal insulin binding in freshly isolated cells, reported previously, may be a reflection of environmental factors rather than a genetically determined cellular abnormality in MMD. ACKNOWLEDGEMENT

We thank Professor Kenzo Takeshita, Division of Child Neurology, Institute of Neurological Sciences, Tottori University School of Medicine, for reviewing the manuscript. REFERENCES De Meyts, P. and J. Roth (1975) Cooperativity in ligand binding: a new graphic analysis, Biochem. Biophys. Res. Commun., 66: 1118-1126. Festoff, B.W. and W.V. Moore (1979) Evaluation of the insulin receptor in myotonic dystrophy, Ann. Neurol., 6: 60-65. Harper, P.S. and P.R. Dyken (1972) Early-onset dystrophia myotonica. Evidence supporting a maternal environmental factor, Lancet, 2: 53-55. Hidaka, H., B.V. Howard, F. Ishibashi, F.C. Kosmakos, J.W. Craig, P.H. Bennett and J. Lamer (1981) Effect of pH and 3-hydroxybutyrate on insulin binding and action in cultured human fibroblasts, Diabetes, 30: 402-406. Kobayashi, M., J.C. Meek and E. Streib (1977) The insulin receptor in myotonic dystrophy, J. Clin. Endocrinol. Metab., 45: 821-824. Lam, L., A.J. Hudson, K.P. Strickland and G.J.M. Tevaarwerk (1983) Insulin binding to myotonic dystrophy fibroblasts, J. Neurol. Sci., 58: 289-295. Marshall, S. and J.M. Olefsky (1979) Effects of lysosomotropic agents on insulin interactions with adipocytes, J. Biol. Chem., 254: 10153-10160. Moxley, R. T., J. N. Livingston, D.H. Lockwood, R. C. Griggs and R. L. Hill (1981) Abnormal regulation of monocyte insulin-binding affinity after glucose ingestion in patients with myotonic dystrophy, Proc. Natl. Acad. Sci. USA, 78: 2567-2571. Ohno, K., C. Nakano, I. Eda and K. Takeshita (1984) Fibroblasts from patients with myotonic muscular dystrophy: cholesterol requirement for proliferation and sensitivity to polyene antibiotics, Brain Dev. (Tokyo), 6: 566-570. Plishker, G.A., H.J. Gitelman and S.H. Appel (1978) Myotonic muscular dystrophy: altered calcium transport in erythrocytes, Science, 200: 323-325. Prince, M.L., P. Tsai and J.M. Olefsky (1981) Insulin binding, internalization, and insulin receptor regulation in fibroblasts from type II, non-insulin-dependent diabetic subjects, Diabetes, 30: 596-600. Roses, A. D. and S.H. Appel (1973) Protein kinase activity in erythrocyte ghosts of patients with myotonic muscular dystrophy, Proc. Natl. Acad. Sci. USA, 70: 1855-1859. Roses, A.D. and S.H. Appel (1974) Muscle membrane protein kinase in myotonic muscular dystrophy, Nature, 250: 245-247. Sato, Y., M. Ono, R. Takaki and M. Kuwano (1984) Chinese hamster ovary cell variants resistant to monensin, an ionophoric antibiotic. II. Growth requirement for insulin and altered insulin-receptor activity, J. Cell PhysioL, 119: 204-210. Scatchard, G. (1949)The attraction of proteins for small molecules and ions, Ann. N.Y. Acad. Sci., 51: 660-672. Schilling, E. E., M. M. Rechler, C. Grunfeld and A. M. Rosenberg (1979) Primary defect of insulin receptors in skin fibroblasts cultured from an infant with leprechauulsm and insulin resistance, Proc. Natl. Acad. Sci. USA, 76: 5877-5881. Stuart, C.A., R.M. Armstrong, S.A. Provow and G.A. Plishker (1983) Insulin resistance in patients with myotonic dystrophy, Neurology (Cleveland), 33: 679-685. Tanaka, K. (1985) Myotonie dystrophy, Med. Hypotheses, 17: 415-425. Tevaarwerk, G.J.M. and A.J. Hudson (1977) Carbohydrate metabolism and insulin resistance in myotonia dystrophica, J. Clin. Endocrinol. Metab., 44: 491-498. Tevaarwerk, G.J.M., K.P. Strickland, C.-H. Lin and A.J. Hudson (1979) Studies on insulin resistance and insulin receptor binding in myotonia dystrophica, J. Clin. Endocrinol. Metab., 49: 216-222.