Evaluation of the Insulin Receptor in Myotonic Dystrophy B. W. Festoff, M D , and W.V. Moore, M D , P h D
Only 2 of 6 patients with myotonic dystrophy had glucose intolerance and hyperinsulinemia. All, however, had markedly reduced insulin binding to specific receptors on circulating monocytes. A significant difference in receptor affinity for insulin was observed, but t h e difference in affinity was not as marked as that in the amount o f 1251-insulinbound per cell, which was sixfold greater in control cells. No evidence for a circulating factor competing with insulin for binding sites could be demonstrated. These alterations in binding did not directly correlate with glucose intolerance or hyperinsulinemia and are consistent with a postulated generalized membrane defect. Study of the insulin receptor in this disorder may uncover regulatory mechanisms i n normal and pathological conditions. including myotonic dystrophy. Festoff BW, Moore WV: Evaluation of the insulin receptor in myotonic dystrophy. Ann Neurol 6:60-65, 1979
Myotonic dystrophy (MyD) is an autosomal dominant genetic ciisorLier with high penetrance but variable clinical presentation 181. Skeletal muscle stiffness and myotonia are the most constant clinical features of MyD and other myotonic disorders, but it is clear that MyD, at least, is a generalized systemic disease 1271 not limited t o muscle. Some patients manifest the full spectrum of skeletal, cardiac, and smooth muscle abnormalities in association with bony skeleton defects, ocular cataracts, endocrine disturbances, and intellectual dysfunction. In other patients or family members, cataracts may be the sole abnormality. Recent studies demonstrating structural 16, 22, 261 and perhaps functional disturbances [I91 in MyD erythrocyte membranes emphasize the widespread involvement of tissues besides skeletal muscle and suggest that genetically induced membrane changes may be primary in the disease. Several metabolic abnormalities of endocrine function have been investigated in MyD. Of these, disturbances of the endocrine pancreas, notably glucose intolerance (abnormal glucose tolerance test) [ 7 , 9, 17, 28, 331 and hyperinsulinism (elevated plasma insulin) 14,1 5 , 18, 25, 321, have attracted most interest. Neither reduced disappearance rates nor altered biological activity of insulin from MyD patients has been found. Many authors have noted increased peripheral resistance to insulin, while others report the lack of peripheral insulin insensitivity in MyD patients. Tevaarwerk and Hudson [ 301 argued that if peripheral insulin resistance is the cause of hyperin-
sulinism in MyD, it should be present in all affected individuals. They did find hyperinsulinism in all their patients, while 12 of 14 had glucose intolerance. All their patients had an excessive response to insulin secre tagogues (glucagon, tolbutamid e, and argi ni ne), suggesting no defect in insulin release. They concluded that impaired carbohydrate metabolism in MyD is due to primary peripheral insulin resistance in all tissues [30]. Such a universal cellular resistance to the action of insulin might reflect (1)an abnormality of insulin receptor binding sites on surface membranes, (2) disturbances of the transduction from receptor binding to initiation of enzyme activity, or (3) subsequent intermediary metabolism defects. Since the techniques for binding of iodine 125-labeled insulin to peripheral monocytes have been used for measuring the number of functional receptors on cell surfaces in several clinical states [2, 3 , 16,24,20, 311, we elected to determine the binding of '"I-insulin to mononuclear leukocytes from MyD patients. A preliminary abstract of these studies has previously appeared [ 111.
Materials and Methods All patients were recruited from the Neuromuscular Disease Clinic at the University of Kansas Medical Center. The diagnosis of MyD was made on the basis of physical findings, electromyography, and family history. Initially, four different pedigrees were evaluated. A standard oral glucose tolerance test (OGTT), 1.75 gm per kilogram of body weight, was performed o n all patients, and plasma insulin levels were obtained. The per-
60 0364-5 1111;79/O7OO6O-O(,sO1.25 @ 1978 by Barry W. Festoff
Results
cent ideal weight for each patient was determined by use of the Metropolitan Life Insurance Table. T h e normal values for OGTT and plasma insulin in the endocrine laboratory at our institution are given in Table 1. For mononuclear leukocyte preparations, 3 0 to 35 ml of heparinized blood was obtained from the antecubital vein of each patient following an overnight fast. Mononuclear leukocytes were isolated by the Ficoll-Hypaque method 151. Control cells from 15 individual volunteers were obtained from buffy coats provided by the Greater Kansas City Community Blood Center. Both heparinized and citrated blood have been used in determining normal curves, and no effect of the anticoagulant has been detected. All samples were used within 2 to 3 hours (control and patient). The cells were resuspended in buffer containing 25 mM Tris hydrochloride (pH 7.6), 120 m M sodium chloride, 1.2 mM magnesium sulfate, 2.5 mM potassium chloride, 10 mM glucose, 1 m M ethylene-diaminetetraacetic acid, and 1% albumin (“buffer A”). The number of monocytes in the mononuclear leukocyte preparations was estimated by latex particle ingestion 1341. T h e percentage of positive cells was 21.9 t 6.0% (mean t SD) in controls compared with 26.5 t 9.7% in the patients. Purified porcine monocomponent insulin for the monocyte insulin binding assay was iodinated using a modified chloramine-T method [23]. ‘“I-Insulin was separated from free and unlabeled insulin by Sephadex gel chromatography. Insulin binding to the mononuclear leukocytes was determined by a modification of a previously described method to detect thyroid stimulating hormone binding to thyroid cell membranes [ 2 3 ] . The reaction mixture consisted of 2.0 to 2.5 nmoles per milliliter of “sl-insulin (porcine monocomponent; specific activity, 20 to 5 0 pCi/pg), 0.5 to 0.6 x 106 cells in buffer A. The mixture was incubated at 15°C for 90 minutes. The cells were separated from the unbound insulin by sedimenting them (900 g for 10 minutes) through a layer of 10% sucrose containing 1% albumin and 0.05 M sodium phosphate buffer, p H 7.5, in an IEC centrifuge using swinging bucket rotors. The amount of insulin bound was determined from the percentage of 1251-insulinin the cell pellet. The insulin binding to mononuclear leukocytes from controls was determined the same way.
The maximum glucose and insulin values obtained from OGTTs for 6 MyD patients (Table 1) indicated that only 2 (Nos. 2 and 3 ) were glucose intolerant; 1 of them had accompanying hyperinsulinism (Patient 3 ) . Two of the patients were obese (>120% of ideal body weight), 1 of these having a normal OGTT. Clinical information is not available o n the 15 control subjects; however, binding to mononuclear leukocytes from 15 buffy coats of 15 separate individuals was tested to form the control curve. The amount of “51-insulin bound to the cells was determined as a function of the concentration of unlabeled insulin [16, 20, 24, 29, 311 (Fig 1). Considerable differences were found between the control curve and the curve obtained by averaging the individual binding curves of the patients. The amount of lz5Iinsulin bound to the control subjects’ monocytes at the lowest concentration of unlabeled insulin was about sixfold greater than the amount bound by the patients. The amount of ”51-insulin bound at the lowest concentration of unlabeled insulin varied from 1.6 to 27.6 (8.9 2‘4.2 SEM) pmoles per 10Ycellsfor the MyD patients compared to a mean of 61 t 5.0 (SEM) pmoles per loYcells for controls. Binding of ‘*51-insulin to the patients’ monocytes was significantly less than binding to control monocytes at any concentration of unlabeled insulin. The percentage of maximum 12sI-insulin (in the absence of unlabeled insulin) bound to cells at increasing unlabeled insulin concentration was also compared. The amounts of unlabeled insulin that produced half-maximal displacement of I2jI-insulin from the control cells was 130 ng per milliliter for controls compared to 190 ng per milliliter for the patients’ cells (Fig 2). From Scatchard plots of these binding data (Fig 3), insulin binding either occurred to multiple classes of sites or was under the influence of negative cooperativity [16, 20, 24, 311, making
Table 1 . Glucose Tolerance Tests and Body Weight of Patients with Myotonic Dystrophy a Glucose (mg/dl) Subiects
3 4 5 6
Controlb
c/c Ideal Body
30’
60’
120’ 180’ 240’ 300’ 0’
30’
60‘
120‘
180’
240’
300’
Wt/Ht (kglcm)
75
...
65 60 156 65 116 97
69 68 83 79 80 69
61 58 82 77 79 61
8 22
69 87 0 45
...
157 124 169 96 157 99
...
179 155 141
102 207 218 110 112 132
5
88
75 57 286 4.5 133 50
12 10 168 9.5 72 41
7 4 29 12.9 19 11
5 7 21 4.0 20 8
78.41187 83 80.21184 93 87.21172 141 85.51182.5 110 83.5/158 170 46.71167 87
150
134
112
86
77
79
63
28
0’
Patients 1 2
Insulin (pU/ml)
95 82 78 75
90
93
1.5 14 16
...
142 107 180 2.0 58 94
13
115
106
14
9
...
Weight
...
“Percent of ideal body weight was obtained from the Metropolitan Life Insurance Table. OGTT and insulin level determinations were performed according to standard clinical laboratory methods. bValuesobtained from healthy volunteers at the University of Kansas Medical Center Clinical Research Unit [21]; standard errors differed by less than 5%.
Festoff and Moore: Insulin Receptor Sites in Myotonic Dystrophy 61
"i1
sera impaired '"'I-insulin binding to control cells 1131. It was subsequently found that those patients' sera contained specific IgG antibodies t o their own insulin receptor sites, and it was proposed that the associated insulin resistance had the characteristics of an autoimmune process [ 121. To determine if a circulating factor (see ref 111) was present in sera o f M y D patients, similar experiments were performed; the results are shown in Table 2. In these experiments, 0.1 ml of undialyzed serum from 4 M y D patients and 4 controls were evaluated for their effect o n control monocyte '""Iinsulin binding. No statistical difference was found in insulin binding to control cells under these conditions.
1
Discussion
affinity and capacity by Scatchard plots difficult to define. However, t h e Scatchard plots indicate that the high-affinity sites are preferentially affected. T h e number of binding sites for control and M y D cells was estimated using data from Figures 1 and 2 by the formula: Sites per cell
-=
Glucose intolerance and insulin resistance have been documented in patients with M y D , although insulin insensitivity does not correlate with obesity, a known cause of insulin resistance 14, 15, 18, 301. T h e marked differences observed in the binding of "'Iinsulin t o monocytes from patients with M y D compared to those from control subjects in the present study appear to be a sixfold reduction in the number of binding sites available for insulin. Although the ratio between the '"'I-insulin bound t o M y D and control cells at any concentration of unlabeled insulin stayed fairly constant, the ratio was less at lower insulin concentrations, suggesting that the highaffinity sites might be preferentially affected. This predilection for high-affinity sites is also suggested by Scatchard plots of the binding data. These findings are identical to the characteristics of insulin binding observed in obesity [22-261. Although the affinity o f the receptor for insulin (see Fig 2 ) was significantly less in the M y D monocytes, this decrease was not as great as the decline in receptor number. T h e marked reduction in insulin binding does not appear to be
Moles of insulin bound per milliliter x 6.03 x 10"3 molecules per mole Cell concentration ~~
M y D cells have 5,107 2 -500 insulin receptor sites, while control cells have 36,542 & 3,075 receptor sites. Recently, severe insulin resistance has been observed in several unrelated patients with unusual diabetes and acanthosis nigricans [ 131. These patients were shown t o have reduced insulin binding to monocytes, which was corrected when their cells were removed from sera. In addition, the patients' No statistical difference in total rnonocytes was detected between normal and MyD blood. O u r studies were performed with total leukocytes, but nionocyres account for 85% of '251-insulin binding L20, 24, 311.
62 Annals of Neurology
Vol h
No 1 July 1979
~~~
~
~~
due to antiinsulin o r antireceptor substances 112, 131 in the serum of M y D patients since their serum does not alter insulin binding to normal monocytes (see Table 2). These results differ significantly from those of Kobayashi e t al [2I], who did not observe differences in the percentage of '"I-insulin bound t o control and M y D patients' monocytes at two different concentrations of unlabeled insulin. T h e reason for this discrepancy is not apparent. They are in accord, however, with the results o f Tevaarwerk et a1 (Tevaarwerk GJM, Strickland KA, Lin C-H, et al: Studies o n insulin resistance and insulin receptor binding in
UNLABELED INSULIN, ng/rnl
F i g 2. The percentage of maximum "Y-insulin bound at increasing concentrations of unlabeled insulin. The reaction mixture contained 2 nmoles per milliliter of 1"51-insulin.5 x I O6 sells per milliliter. and the indicated concentration of unlabeled insulin. The perrentage of total "SI-insulin bound in the absence of unlabeled hormone is plotted versus the concentration of unlabeled insulin for leukocytes from controls (circles) and patients with myotonic dystrophy (squares). Other details are as in Methods.
INSULIN BOUND, pmole/106 CELLS
Fig 3 Ssatchard plots of '"I-insulin binding t o control mononuclear leukocytes and cells from several patients with myotonir dystrophy. The ratio of the amount bound t o the concentration o j -free hormone (BE) i s plotted versus the amount bound. The control curve (open circles) is a representative plot. Scatchardplots of binding data can be rompared to OGTT by matching symbol t o patient number in Table I as follows: filled squares = Patient I ; filled circles = Patient 2; open triangles = Patient 4; open squares = Patient 6. Patient 5 is not on the scale. I
myotonic dystrophy (abstract no. 133). Presented at the IVth International Congress on Neuromuscular Diseases, Montreal, Sept 17-21, 1978), who also studied '2sI-insulin binding to monocytes of MyD and control cells. They found approximately a twofold reduction in '251-insulin bound to MyD cells. Whether the abnormality of insulin receptors in MyD represents a decrease in capacity (number) of sites or reduced affinity was not absolutely defined by their study. The mechanism of the insulin resistance and 4ecreased insulin binding in MyD is not known. Circulating factors (see ref [I]) do not appear to play a role (see Table 2). Defects in membrane function might conceivably affect latex particle ingestion, but no statistical differences in monocyte numbers were found in MyD patients and controls. In obesity, the decrease in insulin binding is suggested to be secondary to hyperinsulinism resulting from the hypercaloric state [2, 3, 14, 16, 20, 24, 291. This "downregulation" of insulin receptors in obesity may be protective [2, 3, 141. Similar conditions are not apparent to explain the insulin resistance in these patients with MyD since only 2 of the present patients studied were obese, 1 demonstrating insulin resistance by OGTT. One patient of normal weight had mild glucose intolerance with normal insulin levels. Glucose intolerance and hyperinsulinemia may vary in individual MyD patients for currently unknown reasons [4, 7, 15, 24, 30, 321. Kobayashi et al [21] found that 5 out of 7 patients were hyperinsulinemic (3 of whom were included in our study and showed normal insulin levels at that time). Since all the patients exhibited decreased insulin binding, we suggest that this quantitative abnormality may reflect a basic defect in membrane structure and function that becomes more pronounced but is not causally related to glucose intolerance or hyperinsulinism. Since data for the patients' binding were averaged, the exact
Festoff and Moore: Insulin Receptor Sites in Myotonic Dystrophy 63
Table 2. '"Y-lnsulin Birrding t o Control hffInOnlLl-lKUrLeukoi-yteJ in the Pre.renre o j Patient and Co~trrif Serum Insulin Binding"
Source of Serum
( p m o l e s i l O!' cells)
Patients 4 5 3
SD
34.4 35.8 36.8 '42.0 37.5 i 3.7
SD
28.1 27.0 32.6 43.1 32.9 i 7.2
7
Mcan
-t
Control5 1
2 3 4
Mean ~
~
&
~~~
,'Insulin binding was determined in the usual manner except that 0.1 ml o f serum per millilirer of incubation medium was added to each tube.
relationship between binding, glucose tolerance, and insulin levels is not clear. For example, Patient 5 is obese, is glucosc intolerant with increased plasma insulin at 2 hours, and has the lowest binding, which is off the scale in Figure 3. Other examples are found when Table 1 and Figure 3 are compared. Current studies are underway attempting to clarify these points. MyD is a dominantly inherited disorder in which structural o r regulatory proteins may be abnormal [101; consequently, study of the insulin receptor takes on greater importance and may uncover not only clues t o the pathogenesis of MyD, but also much-needed information regarding this critical receptor molecule in normal homeostatic mechanisms. Supported in part by the Ralph L. Smith Mental Retardation Research Center ( N I H 0 2 5 2 8 ) , N I H Grant 1 Rol Am19544-01A1, Grant R R 828 from the General Clinical Research Centers Program of the Division of Research Resources, N I H , and the Medical Research Service of the Veterans Administration. Presented in part at the 103rd Meeting of the American Neurological Association, Washington, DC. September 22, 1978. The authors wish to express their gratitude to P. Leppert for expert technical assistance and to D r Ronald Chance for providing purified porcine monocomponent insulin.
Re fete nces 1 . Appel SH, Roses AD: Membranes and myotonia, in Rowland LP (ed 1: Pathogenesis of Human Muscular Dystrophies. Amsterdam. Excerpta Medica, 1977, pp 747-758 2. Archer J A , Gorden P, Roth J : Defect in insulin binding to receptors i n obese man. Amelioration with calorie restriction. J Clin Invest 55:166-171, 1075 3. Bar RS, Gorden P. Roth J. e t al: Fluctuations in the affinity
64 Annals of N e u r o l o g y
Vol 0
No 1 July 1979
and concentration of insulin receptors o n circulating monocytes of obese patients. J Clin Invest 58:1123-1135, 1976 4. Barbosa J, Nuttall FQ, Kennedy W, e t d: Plasma insulin in patients with myotonic dystrophy and their relatives. Medicine 53:307-323, 1974 5. Boyum AA: A one-stage procedure for isolation of granulocytes and lymphocytes from human blood. Scand J Clin Lab Invest (Suppl 9 7 ) 21:51-76, 1968 6. Butterfield DA, Roses AD, Cooper ML, et al: A comparative electron spin resonance study of the erythrocyte membrane in myotonic muscular dystrophy. Biochemistry 13:5078-5082, 1974 7. Caughey JE, Brown J: Dystrophia myotonica: endocrine study. Q J Med 19:303-318, 1950 8. Caughey JE, Myrianthopoulos NC: Dystrophia Myotonica and Related Disorders. Springfield, IL, Thomas, 1963 9. Drucker W D , Rowland LP, Sterling K, et al: O n the function of the endocrine glands in myotonic muscular dystrophy. Am J Med 31941-950, 1961 10. Festoff BW: Generic alterations in surface membranes, in Rowland LP (ed): Pathogenesis of Human Muscular Dystrophies. Amsterdam, Excerpta Medica, 1977, pp 52 1-546 1 1 . Festoff BW, Moore W Jr: Decreased number of insulin receptors on monocytes of patients with rnyotonic dystrophy (abstract). Ann Neurol 4:164, 1978 12. Flier JS, Kahn CR, Jarrett DB, e t al: Characterization of antibodies to the insulin receptor. J Clin Invest 58: 1442- 1449, 1976 13. Flier JS, Kahn CR, Roth J, e t al: Antibodie$ that impair insulin receptor binding in an unusual diabetic syndrome with severe insulin resistance. Science 190f53-65. 1975 14. Gavin J R 111, Roth J, Neville D Jr, et al: Insulin-dependent regulation of insulin receptor concentrations: a direct demonstration in cell culture. Proc Natl Acad Sci USA '1:84-88, 1974 15. Gorden P, Criggs RC, Nissley SP, et al: Studies of plasma insulin in myotonic dystrophy. J Clin Endocrinol Metab 29684-690, I969 16. Harrison LC, Martin FIR, Melick RA: Correlation between insulin receptor binding in isolated fat cells and insulin sensitivity in obese human subjects.J Clin Invest 5 8 : 1435-1441, 1976 17. Huff TA, Horton ES, Lebovitz HE: Abnormal insulin secretion in myotonic dystrophy. N Engl J Med 277:837-841, 1967 18. Huff TA, Lebovitz HE: Dynamics of insulin secretion in myotonic dystrophy. J Clin Endocrinol Metab 28:992-998, I968 10. Hull KL, Roses AD: Stoichiometry of socliurn and potassium transport in erythrocytes from patients with myotonic muscular dystrophy. J Physiol (Lond) 254: 169- 181, 19'6 20. Kahn CR, Neville D M Jr, Roth J: Insulin receptor interaction in the obese-hyperglycemic mouse. J Biol Chem 248:244250, 1973 21. Kobayashi M, Meek J Streib E: The insulin receptor in myotonic dystrophy. J lin Endocrinol Metab 45:821-823, 1977 22. Miller SE, Roses AD, Appel SH: Scanning electron microscopy studies in muscular dystrophy. Arch Neurol 33: 172174, 1976 23. Moore W, Wolff J: Thyroid-stimulating hormone binding t o beef thyroid membranes. J Biol Chem 249:0255-6263, 1074 24. Olefsky JM: Decreased insulin binding to adipocytes and circulating monocytes from obese subjects. J Clin Invest 57:1165-1172, 1976 25. Olson N D : Plasmaglucose and free fatty acid response to l o w dose insulin infusion as a tool for the assessment o f peripheral
26
2-
26 29
30
insulin resistance in myotonic dystrophy. Diabetes (Suppl 1) 25:379a, 1976 Roses AD, Appel SH: Protein kinase activity in erythrocyte ghosts of' patients with myotonic muscular dystrophy. Proc Natl Acad Sci USA 70:1855-1859, 1973 Rowland LP: Pathogenesis of muscular dystrophies. Arch Neurol 33:315-321, 1976 Simon KA: Diabetes and lens changes in myotonic dystrophy. Arch Ophthalmol 67:312-315, 1962 Sol1 A H , Goldfine ID, Roth J, e t al: Thymic lymphocytes in obese (ob-ob) mice. A mirror of the insulin receptor defect in liver and fat. J Biol Chem 249:4127-4131, 1974 Tevaamerk GJ, Hudson AJ: Carbohydrate metabolism and
3 1.
32.
33.
34.
insulin resistance in myotonia dystrophica. J Clin Endocrinol Metab 44:491-498, 1977 Thorsson AV, Hintz RL: Insulin receptors in the newborn: an increase in affinity and number. N Engl J Med 297908-912, 1977 Walsh JC, Turtle JR, Miller S, e t al: Abnormalities of insulin secretion in dystrophia myotonia. Brain 93:73 1-742, 1970 Ziegler DK, Rogoff J: Rare variant of myotonia atrophicaclinical and electromyographic study of a family. Brain 791349-357, 1056 Zucker-Franklin D : The percentage of monocytes among "mononuclear" cell fractions obtained from normal human blood. J Immunol 112:234-240, 1974
Festoff and Moore: Insulin Receptor Sites in Myotonic Dystrophy
65
Only 2 of 6 patients with myotonic dystrophy had glucose intolerance and hyperinsulinemia. All, however, had markedly reduced insulin binding to specific receptors on circulating monocytes. A significant difference in receptor affinity for insulin was observed, but t h e difference in affinity was not as marked as that in the amount o f 1251-insulinbound per cell, which was sixfold greater in control cells. No evidence for a circulating factor competing with insulin for binding sites could be demonstrated. These alterations in binding did not directly correlate with glucose intolerance or hyperinsulinemia and are consistent with a postulated generalized membrane defect. Study of the insulin receptor in this disorder may uncover regulatory mechanisms i n normal and pathological conditions. including myotonic dystrophy. Festoff BW, Moore WV: Evaluation of the insulin receptor in myotonic dystrophy. Ann Neurol 6:60-65, 1979
Myotonic dystrophy (MyD) is an autosomal dominant genetic ciisorLier with high penetrance but variable clinical presentation 181. Skeletal muscle stiffness and myotonia are the most constant clinical features of MyD and other myotonic disorders, but it is clear that MyD, at least, is a generalized systemic disease 1271 not limited t o muscle. Some patients manifest the full spectrum of skeletal, cardiac, and smooth muscle abnormalities in association with bony skeleton defects, ocular cataracts, endocrine disturbances, and intellectual dysfunction. In other patients or family members, cataracts may be the sole abnormality. Recent studies demonstrating structural 16, 22, 261 and perhaps functional disturbances [I91 in MyD erythrocyte membranes emphasize the widespread involvement of tissues besides skeletal muscle and suggest that genetically induced membrane changes may be primary in the disease. Several metabolic abnormalities of endocrine function have been investigated in MyD. Of these, disturbances of the endocrine pancreas, notably glucose intolerance (abnormal glucose tolerance test) [ 7 , 9, 17, 28, 331 and hyperinsulinism (elevated plasma insulin) 14,1 5 , 18, 25, 321, have attracted most interest. Neither reduced disappearance rates nor altered biological activity of insulin from MyD patients has been found. Many authors have noted increased peripheral resistance to insulin, while others report the lack of peripheral insulin insensitivity in MyD patients. Tevaarwerk and Hudson [ 301 argued that if peripheral insulin resistance is the cause of hyperin-
sulinism in MyD, it should be present in all affected individuals. They did find hyperinsulinism in all their patients, while 12 of 14 had glucose intolerance. All their patients had an excessive response to insulin secre tagogues (glucagon, tolbutamid e, and argi ni ne), suggesting no defect in insulin release. They concluded that impaired carbohydrate metabolism in MyD is due to primary peripheral insulin resistance in all tissues [30]. Such a universal cellular resistance to the action of insulin might reflect (1)an abnormality of insulin receptor binding sites on surface membranes, (2) disturbances of the transduction from receptor binding to initiation of enzyme activity, or (3) subsequent intermediary metabolism defects. Since the techniques for binding of iodine 125-labeled insulin to peripheral monocytes have been used for measuring the number of functional receptors on cell surfaces in several clinical states [2, 3 , 16,24,20, 311, we elected to determine the binding of '"I-insulin to mononuclear leukocytes from MyD patients. A preliminary abstract of these studies has previously appeared [ 111.
Materials and Methods All patients were recruited from the Neuromuscular Disease Clinic at the University of Kansas Medical Center. The diagnosis of MyD was made on the basis of physical findings, electromyography, and family history. Initially, four different pedigrees were evaluated. A standard oral glucose tolerance test (OGTT), 1.75 gm per kilogram of body weight, was performed o n all patients, and plasma insulin levels were obtained. The per-
60 0364-5 1111;79/O7OO6O-O(,sO1.25 @ 1978 by Barry W. Festoff
Results
cent ideal weight for each patient was determined by use of the Metropolitan Life Insurance Table. T h e normal values for OGTT and plasma insulin in the endocrine laboratory at our institution are given in Table 1. For mononuclear leukocyte preparations, 3 0 to 35 ml of heparinized blood was obtained from the antecubital vein of each patient following an overnight fast. Mononuclear leukocytes were isolated by the Ficoll-Hypaque method 151. Control cells from 15 individual volunteers were obtained from buffy coats provided by the Greater Kansas City Community Blood Center. Both heparinized and citrated blood have been used in determining normal curves, and no effect of the anticoagulant has been detected. All samples were used within 2 to 3 hours (control and patient). The cells were resuspended in buffer containing 25 mM Tris hydrochloride (pH 7.6), 120 m M sodium chloride, 1.2 mM magnesium sulfate, 2.5 mM potassium chloride, 10 mM glucose, 1 m M ethylene-diaminetetraacetic acid, and 1% albumin (“buffer A”). The number of monocytes in the mononuclear leukocyte preparations was estimated by latex particle ingestion 1341. T h e percentage of positive cells was 21.9 t 6.0% (mean t SD) in controls compared with 26.5 t 9.7% in the patients. Purified porcine monocomponent insulin for the monocyte insulin binding assay was iodinated using a modified chloramine-T method [23]. ‘“I-Insulin was separated from free and unlabeled insulin by Sephadex gel chromatography. Insulin binding to the mononuclear leukocytes was determined by a modification of a previously described method to detect thyroid stimulating hormone binding to thyroid cell membranes [ 2 3 ] . The reaction mixture consisted of 2.0 to 2.5 nmoles per milliliter of “sl-insulin (porcine monocomponent; specific activity, 20 to 5 0 pCi/pg), 0.5 to 0.6 x 106 cells in buffer A. The mixture was incubated at 15°C for 90 minutes. The cells were separated from the unbound insulin by sedimenting them (900 g for 10 minutes) through a layer of 10% sucrose containing 1% albumin and 0.05 M sodium phosphate buffer, p H 7.5, in an IEC centrifuge using swinging bucket rotors. The amount of insulin bound was determined from the percentage of 1251-insulinin the cell pellet. The insulin binding to mononuclear leukocytes from controls was determined the same way.
The maximum glucose and insulin values obtained from OGTTs for 6 MyD patients (Table 1) indicated that only 2 (Nos. 2 and 3 ) were glucose intolerant; 1 of them had accompanying hyperinsulinism (Patient 3 ) . Two of the patients were obese (>120% of ideal body weight), 1 of these having a normal OGTT. Clinical information is not available o n the 15 control subjects; however, binding to mononuclear leukocytes from 15 buffy coats of 15 separate individuals was tested to form the control curve. The amount of “51-insulin bound to the cells was determined as a function of the concentration of unlabeled insulin [16, 20, 24, 29, 311 (Fig 1). Considerable differences were found between the control curve and the curve obtained by averaging the individual binding curves of the patients. The amount of lz5Iinsulin bound to the control subjects’ monocytes at the lowest concentration of unlabeled insulin was about sixfold greater than the amount bound by the patients. The amount of ”51-insulin bound at the lowest concentration of unlabeled insulin varied from 1.6 to 27.6 (8.9 2‘4.2 SEM) pmoles per 10Ycellsfor the MyD patients compared to a mean of 61 t 5.0 (SEM) pmoles per loYcells for controls. Binding of ‘*51-insulin to the patients’ monocytes was significantly less than binding to control monocytes at any concentration of unlabeled insulin. The percentage of maximum 12sI-insulin (in the absence of unlabeled insulin) bound to cells at increasing unlabeled insulin concentration was also compared. The amounts of unlabeled insulin that produced half-maximal displacement of I2jI-insulin from the control cells was 130 ng per milliliter for controls compared to 190 ng per milliliter for the patients’ cells (Fig 2). From Scatchard plots of these binding data (Fig 3), insulin binding either occurred to multiple classes of sites or was under the influence of negative cooperativity [16, 20, 24, 311, making
Table 1 . Glucose Tolerance Tests and Body Weight of Patients with Myotonic Dystrophy a Glucose (mg/dl) Subiects
3 4 5 6
Controlb
c/c Ideal Body
30’
60’
120’ 180’ 240’ 300’ 0’
30’
60‘
120‘
180’
240’
300’
Wt/Ht (kglcm)
75
...
65 60 156 65 116 97
69 68 83 79 80 69
61 58 82 77 79 61
8 22
69 87 0 45
...
157 124 169 96 157 99
...
179 155 141
102 207 218 110 112 132
5
88
75 57 286 4.5 133 50
12 10 168 9.5 72 41
7 4 29 12.9 19 11
5 7 21 4.0 20 8
78.41187 83 80.21184 93 87.21172 141 85.51182.5 110 83.5/158 170 46.71167 87
150
134
112
86
77
79
63
28
0’
Patients 1 2
Insulin (pU/ml)
95 82 78 75
90
93
1.5 14 16
...
142 107 180 2.0 58 94
13
115
106
14
9
...
Weight
...
“Percent of ideal body weight was obtained from the Metropolitan Life Insurance Table. OGTT and insulin level determinations were performed according to standard clinical laboratory methods. bValuesobtained from healthy volunteers at the University of Kansas Medical Center Clinical Research Unit [21]; standard errors differed by less than 5%.
Festoff and Moore: Insulin Receptor Sites in Myotonic Dystrophy 61
"i1
sera impaired '"'I-insulin binding to control cells 1131. It was subsequently found that those patients' sera contained specific IgG antibodies t o their own insulin receptor sites, and it was proposed that the associated insulin resistance had the characteristics of an autoimmune process [ 121. To determine if a circulating factor (see ref 111) was present in sera o f M y D patients, similar experiments were performed; the results are shown in Table 2. In these experiments, 0.1 ml of undialyzed serum from 4 M y D patients and 4 controls were evaluated for their effect o n control monocyte '""Iinsulin binding. No statistical difference was found in insulin binding to control cells under these conditions.
1
Discussion
affinity and capacity by Scatchard plots difficult to define. However, t h e Scatchard plots indicate that the high-affinity sites are preferentially affected. T h e number of binding sites for control and M y D cells was estimated using data from Figures 1 and 2 by the formula: Sites per cell
-=
Glucose intolerance and insulin resistance have been documented in patients with M y D , although insulin insensitivity does not correlate with obesity, a known cause of insulin resistance 14, 15, 18, 301. T h e marked differences observed in the binding of "'Iinsulin t o monocytes from patients with M y D compared to those from control subjects in the present study appear to be a sixfold reduction in the number of binding sites available for insulin. Although the ratio between the '"'I-insulin bound t o M y D and control cells at any concentration of unlabeled insulin stayed fairly constant, the ratio was less at lower insulin concentrations, suggesting that the highaffinity sites might be preferentially affected. This predilection for high-affinity sites is also suggested by Scatchard plots of the binding data. These findings are identical to the characteristics of insulin binding observed in obesity [22-261. Although the affinity o f the receptor for insulin (see Fig 2 ) was significantly less in the M y D monocytes, this decrease was not as great as the decline in receptor number. T h e marked reduction in insulin binding does not appear to be
Moles of insulin bound per milliliter x 6.03 x 10"3 molecules per mole Cell concentration ~~
M y D cells have 5,107 2 -500 insulin receptor sites, while control cells have 36,542 & 3,075 receptor sites. Recently, severe insulin resistance has been observed in several unrelated patients with unusual diabetes and acanthosis nigricans [ 131. These patients were shown t o have reduced insulin binding to monocytes, which was corrected when their cells were removed from sera. In addition, the patients' No statistical difference in total rnonocytes was detected between normal and MyD blood. O u r studies were performed with total leukocytes, but nionocyres account for 85% of '251-insulin binding L20, 24, 311.
62 Annals of Neurology
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~~~
~
~~
due to antiinsulin o r antireceptor substances 112, 131 in the serum of M y D patients since their serum does not alter insulin binding to normal monocytes (see Table 2). These results differ significantly from those of Kobayashi e t al [2I], who did not observe differences in the percentage of '"I-insulin bound t o control and M y D patients' monocytes at two different concentrations of unlabeled insulin. T h e reason for this discrepancy is not apparent. They are in accord, however, with the results o f Tevaarwerk et a1 (Tevaarwerk GJM, Strickland KA, Lin C-H, et al: Studies o n insulin resistance and insulin receptor binding in
UNLABELED INSULIN, ng/rnl
F i g 2. The percentage of maximum "Y-insulin bound at increasing concentrations of unlabeled insulin. The reaction mixture contained 2 nmoles per milliliter of 1"51-insulin.5 x I O6 sells per milliliter. and the indicated concentration of unlabeled insulin. The perrentage of total "SI-insulin bound in the absence of unlabeled hormone is plotted versus the concentration of unlabeled insulin for leukocytes from controls (circles) and patients with myotonic dystrophy (squares). Other details are as in Methods.
INSULIN BOUND, pmole/106 CELLS
Fig 3 Ssatchard plots of '"I-insulin binding t o control mononuclear leukocytes and cells from several patients with myotonir dystrophy. The ratio of the amount bound t o the concentration o j -free hormone (BE) i s plotted versus the amount bound. The control curve (open circles) is a representative plot. Scatchardplots of binding data can be rompared to OGTT by matching symbol t o patient number in Table I as follows: filled squares = Patient I ; filled circles = Patient 2; open triangles = Patient 4; open squares = Patient 6. Patient 5 is not on the scale. I
myotonic dystrophy (abstract no. 133). Presented at the IVth International Congress on Neuromuscular Diseases, Montreal, Sept 17-21, 1978), who also studied '2sI-insulin binding to monocytes of MyD and control cells. They found approximately a twofold reduction in '251-insulin bound to MyD cells. Whether the abnormality of insulin receptors in MyD represents a decrease in capacity (number) of sites or reduced affinity was not absolutely defined by their study. The mechanism of the insulin resistance and 4ecreased insulin binding in MyD is not known. Circulating factors (see ref [I]) do not appear to play a role (see Table 2). Defects in membrane function might conceivably affect latex particle ingestion, but no statistical differences in monocyte numbers were found in MyD patients and controls. In obesity, the decrease in insulin binding is suggested to be secondary to hyperinsulinism resulting from the hypercaloric state [2, 3, 14, 16, 20, 24, 291. This "downregulation" of insulin receptors in obesity may be protective [2, 3, 141. Similar conditions are not apparent to explain the insulin resistance in these patients with MyD since only 2 of the present patients studied were obese, 1 demonstrating insulin resistance by OGTT. One patient of normal weight had mild glucose intolerance with normal insulin levels. Glucose intolerance and hyperinsulinemia may vary in individual MyD patients for currently unknown reasons [4, 7, 15, 24, 30, 321. Kobayashi et al [21] found that 5 out of 7 patients were hyperinsulinemic (3 of whom were included in our study and showed normal insulin levels at that time). Since all the patients exhibited decreased insulin binding, we suggest that this quantitative abnormality may reflect a basic defect in membrane structure and function that becomes more pronounced but is not causally related to glucose intolerance or hyperinsulinism. Since data for the patients' binding were averaged, the exact
Festoff and Moore: Insulin Receptor Sites in Myotonic Dystrophy 63
Table 2. '"Y-lnsulin Birrding t o Control hffInOnlLl-lKUrLeukoi-yteJ in the Pre.renre o j Patient and Co~trrif Serum Insulin Binding"
Source of Serum
( p m o l e s i l O!' cells)
Patients 4 5 3
SD
34.4 35.8 36.8 '42.0 37.5 i 3.7
SD
28.1 27.0 32.6 43.1 32.9 i 7.2
7
Mcan
-t
Control5 1
2 3 4
Mean ~
~
&
~~~
,'Insulin binding was determined in the usual manner except that 0.1 ml o f serum per millilirer of incubation medium was added to each tube.
relationship between binding, glucose tolerance, and insulin levels is not clear. For example, Patient 5 is obese, is glucosc intolerant with increased plasma insulin at 2 hours, and has the lowest binding, which is off the scale in Figure 3. Other examples are found when Table 1 and Figure 3 are compared. Current studies are underway attempting to clarify these points. MyD is a dominantly inherited disorder in which structural o r regulatory proteins may be abnormal [101; consequently, study of the insulin receptor takes on greater importance and may uncover not only clues t o the pathogenesis of MyD, but also much-needed information regarding this critical receptor molecule in normal homeostatic mechanisms. Supported in part by the Ralph L. Smith Mental Retardation Research Center ( N I H 0 2 5 2 8 ) , N I H Grant 1 Rol Am19544-01A1, Grant R R 828 from the General Clinical Research Centers Program of the Division of Research Resources, N I H , and the Medical Research Service of the Veterans Administration. Presented in part at the 103rd Meeting of the American Neurological Association, Washington, DC. September 22, 1978. The authors wish to express their gratitude to P. Leppert for expert technical assistance and to D r Ronald Chance for providing purified porcine monocomponent insulin.
Re fete nces 1 . Appel SH, Roses AD: Membranes and myotonia, in Rowland LP (ed 1: Pathogenesis of Human Muscular Dystrophies. Amsterdam. Excerpta Medica, 1977, pp 747-758 2. Archer J A , Gorden P, Roth J : Defect in insulin binding to receptors i n obese man. Amelioration with calorie restriction. J Clin Invest 55:166-171, 1075 3. Bar RS, Gorden P. Roth J. e t al: Fluctuations in the affinity
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and concentration of insulin receptors o n circulating monocytes of obese patients. J Clin Invest 58:1123-1135, 1976 4. Barbosa J, Nuttall FQ, Kennedy W, e t d: Plasma insulin in patients with myotonic dystrophy and their relatives. Medicine 53:307-323, 1974 5. Boyum AA: A one-stage procedure for isolation of granulocytes and lymphocytes from human blood. Scand J Clin Lab Invest (Suppl 9 7 ) 21:51-76, 1968 6. Butterfield DA, Roses AD, Cooper ML, et al: A comparative electron spin resonance study of the erythrocyte membrane in myotonic muscular dystrophy. Biochemistry 13:5078-5082, 1974 7. Caughey JE, Brown J: Dystrophia myotonica: endocrine study. Q J Med 19:303-318, 1950 8. Caughey JE, Myrianthopoulos NC: Dystrophia Myotonica and Related Disorders. Springfield, IL, Thomas, 1963 9. Drucker W D , Rowland LP, Sterling K, et al: O n the function of the endocrine glands in myotonic muscular dystrophy. Am J Med 31941-950, 1961 10. Festoff BW: Generic alterations in surface membranes, in Rowland LP (ed): Pathogenesis of Human Muscular Dystrophies. Amsterdam, Excerpta Medica, 1977, pp 52 1-546 1 1 . Festoff BW, Moore W Jr: Decreased number of insulin receptors on monocytes of patients with rnyotonic dystrophy (abstract). Ann Neurol 4:164, 1978 12. Flier JS, Kahn CR, Jarrett DB, e t al: Characterization of antibodies to the insulin receptor. J Clin Invest 58: 1442- 1449, 1976 13. Flier JS, Kahn CR, Roth J, e t al: Antibodie$ that impair insulin receptor binding in an unusual diabetic syndrome with severe insulin resistance. Science 190f53-65. 1975 14. Gavin J R 111, Roth J, Neville D Jr, et al: Insulin-dependent regulation of insulin receptor concentrations: a direct demonstration in cell culture. Proc Natl Acad Sci USA '1:84-88, 1974 15. Gorden P, Criggs RC, Nissley SP, et al: Studies of plasma insulin in myotonic dystrophy. J Clin Endocrinol Metab 29684-690, I969 16. Harrison LC, Martin FIR, Melick RA: Correlation between insulin receptor binding in isolated fat cells and insulin sensitivity in obese human subjects.J Clin Invest 5 8 : 1435-1441, 1976 17. Huff TA, Horton ES, Lebovitz HE: Abnormal insulin secretion in myotonic dystrophy. N Engl J Med 277:837-841, 1967 18. Huff TA, Lebovitz HE: Dynamics of insulin secretion in myotonic dystrophy. J Clin Endocrinol Metab 28:992-998, I968 10. Hull KL, Roses AD: Stoichiometry of socliurn and potassium transport in erythrocytes from patients with myotonic muscular dystrophy. J Physiol (Lond) 254: 169- 181, 19'6 20. Kahn CR, Neville D M Jr, Roth J: Insulin receptor interaction in the obese-hyperglycemic mouse. J Biol Chem 248:244250, 1973 21. Kobayashi M, Meek J Streib E: The insulin receptor in myotonic dystrophy. J lin Endocrinol Metab 45:821-823, 1977 22. Miller SE, Roses AD, Appel SH: Scanning electron microscopy studies in muscular dystrophy. Arch Neurol 33: 172174, 1976 23. Moore W, Wolff J: Thyroid-stimulating hormone binding t o beef thyroid membranes. J Biol Chem 249:0255-6263, 1074 24. Olefsky JM: Decreased insulin binding to adipocytes and circulating monocytes from obese subjects. J Clin Invest 57:1165-1172, 1976 25. Olson N D : Plasmaglucose and free fatty acid response to l o w dose insulin infusion as a tool for the assessment o f peripheral
26
2-
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30
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