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Environmental influence on altered receptor function in a genetic disease: Insulin and glucose affect insulin receptors in myotonic dystrophy
Journal of the Neurological Sciences, 1989, 89:15-25
15
Elsevier JNS 03098
Environmental influence on altered receptor function in a genetic disease: insulin and glucose affect insulin receptors in myotonic dystrophy Osvaldo H. Perurena* and Barry W. Festoff Neurobiology Research Laboratory, Kansas City, Missouri Veterans Administration Medical Center, and Department of Neurology, University of Kansas Medical Center, Kansas City, KS (U.S.A.)
(Received27 January, 1988) (Revised, received9 September, 1988) (Accepted 9 September, 1988)
SUMMARY Insulin action in vivo and insulin binding to monocytes in vitro were correlated in patients with myotonic dystrophy (MyD) and compared with healthy controls. Confh,-ming our previous studies and those of others, the present results show that the glucose infusion rate (DR), an estimate of in vivo insulin sensitivity, was significantly diminished in MyD. At the same per cent of ideal body weight DR in MyD patients was considerably less than controls suggesting that obesity could not solely account for decreased insulin sensitivity in MyD. The relative capacity (RC), and relative affinity (EDso) of the insulin receptor in monocytes was significantly less in patients. The relative affmity (EDso) was improved by changing environmental insulin levels while receptor numbers (RC) were not. Insulin sensitivity and RC showed a trend toward a positive correlation although this did not reach statistical siotmificance. Our data suggest that the alteration of the insulin receptor in MyD is different from obesity and from other disorders of the motor unit such as amyotrophic lateral sclerosis, where insulin sensitivity and RC are reduced but EDso is unchanged. Thus, in MyD the receptor may be one of the loci where the resistance occurs.
Key words: Myotonic dystrophy; Insulin receptor; Insulin resistance; Molecular genetics; Chromosome; eDNA; Euglycemic insulin clamp; Monocyte
* Present address: NeurologyService(151), Colmery-O'NeilVAMC,Topeka, KS 66622, U.S.A. Correspondence to." Barry W. Festoff,M.D., NeurobiologyResearch Laboratory(151)VA Medical
Center, 4801 LinwoodBlvd., Kansas City, MO 64128, U.S.A. 0022-510X/89/$03.50 © 1989Elsevier Science Publishers B,V.(BiomedicalDivision)
16 INTRODUCTION
Myotonic dystrophy (MyD) is an autosomal dominant genetic disorder manifested commonly by myopathy and myotonia and accompanied frequently by a vast array of bony and endocrine abnormalities, cataract and mental retardation (Harper 1979). Recent studies suggest that genetically induced-membrane changes may be primary in the disease (Roses et al. 1980). A marked decrease in capacity of high affinity monocyte receptors for insulin in patients with MyD has frequently been found (Festoff and Moore 1979; Tevaarwerk et al. 1979; Stuart et al. 1983). To determine if altered insulin receptors correlate with peripheral insulin resistance, we have utilized the euglycemic clamp method (DeFronzo et al. 1979) to quantify the composite glucose uptake by the three main insulin targets: liver, fat, and muscle. Simultaneously, we evaluated insulin receptors on circulating monocytes of MyD patients and controls. Portions of this work have been previously reported (Festoffet al. 1981; Perurena et al. 1981). METHODS
Subjects Informed consent was obtained for monocyte insulin binding and glucose clamping in both MyD patients and controls. Patients were recruited from the Neuromuscular Disease Clinics of the University of Kansas and Kansas City Veterans Administration Medical Centers. The diagnosis of MyD was made on the basis of physical findings, electromyography, muscle biopsy and family history. All were males aged 20-55 years with various degrees of functional impairment. Seven female and 8 male controls, aged 21-34 years, were healthy with normal oral glucose tolerance tests (OGTT) and displayed a similar moderate amount of physical activity. Patients and controls fasted for 12 h before all studies. Insulin binding of [ ~25I]insulin by peripheral monocytes was determined in MyD patients immediately before and after the euglycemic clamp, while binding in controls was determined only before the clamp. Iodination of insulin, preparation of cells and binding studies to monocytes were as described by Perurena and Festoff (1987).
Euglycemic clamp One indwelling catheter was inserted in a forearm vein for insulin and glucose infusion. A double lumen catheter allowing an admixture of subject's blood with heparin, was the input to the Biostator Controller (Miles Laboratories, Elkhart, IN) for blood glucose monitoring at 60-s intervals. Insulin (Eli Lilly, Indianapolis, IN) was infused by a Harvard pump (Harvard Apparatus, Millis, MA): a priming initial dose of 2.4 mU. kg- i. rain - t was given during the fn'st 10 rain and a subsequent dose of 1.2 mU. kg - 1. rain - 1 for the remaining 110 rain. Serum was obtained for estimation of insulin levels during the infusion and exceeded 85/~U/ml in all subjects.
17 Euglycemic insulin clamp parameters were calculated according to the preprogrammed algorithm for the Biostator Controller. Glucose infusion was administered using the following equation: DR (mg. kg- 1. min- 1) = RD. (BD - G/25
+ 1) 4
where DR is the glucose infusion rate, RD is the glucose infusion rate at the pre-selected basal glucose level, BD is the preselected basal glucose, and G is the last one-minute average level recorded in the computer. In some subjects, it was necessary to supplement the glucose infusion by the Controller via a separate line in order to maintain blood glucose at the preseleeted basal levels. In these cases RD was higher than the maximum RD of the machine.
RESULTS
Subjects The mean percent of ideal body weight in the 6 MyD patients was 122.5 + 9.82 (Table 1). Two of them (J. C. and JaC) were brothers and had abnormal oral glucose tolerance tests (OGTTs). The mean basal insulin level was 24.8 #U/ml. In 3 patients they were above 25/~U/ml. Patient R.B. had a family history and biopsy compatible with the disease, but no muscle weakness and minimal myotonia. Patients D.E. and M.G. had the complete syndrome, but were ambulatory with only mild weakness. TABLE 1 S U M M A R Y O F IN VIVO G L U C O S E A N D I N S U L I N M E T A B O L I S M A N D M O N O C Y T E I N S U L I N B I N D I N G IN M y D PATIENTS A N D N O R M A L C O N T R O L S MyD
% Ideal body weight (IBW) a (n = 6)
Abnormal OGTT
Fasting insulin b (/~U/rnl)
Glucose infusion rate (DR) (mg. k g - 1. m i n - i)
J.C. JaC R.B. D.E. M.G. R.J. Mean + SEM
136 ! 39 113 128 78 141 122.5 9.8
Yes Yes No No No No
26 54 8 27 16 18 24.6 15.9
3.73 1.16 5.01 2.54 4.63 2.0 3.17 0.62
No
14.3 1.28
8.21 0.83
Controls (n = 15) Mean _+ SEM
97.2 3.85
P < 0.01 a IBW was determined using the tables of the Metropolitan Life Insurance Co. b For insulin levels determinations see Methods section.
18 14
\ "~ ~"~1-12 " ~ ~
• CONTROLS a MyD
Z
~4 ::3
°
I I0 INSULIN
I00 I000 I0,000 CONCENTRATION ( n g / m l )
Fig. l. Percent [~2Sl]insulinbindingto 10 7 monocytes/mlat increasingconcentrationsof unlabeled insulin. The percent of [~2sI]insulinbound is plotted versus the quantity (ng/ml)of unlabeledinsulin for leukocytes from 15 controls (closed circles) and 6 patients with MyD (squares).
Patients J.C. and JaC were more severely impaired, but still ambulatory using a cane. Patient R.J. was intermittently in a wheelchair. The 15 controls for the clamp studies (Table 1) were 97.2 + 3.85% of ideal body weight and only 1 control subject had basal insulin levels above 20 #U/ml (mean 14.3 #U/ml). All had a similar moderate amount of physical activity.
Insulin binding to monocytes The percent of [~25I]insulin bound to the cells at 15 °C was determined as a function of the concentration of unlabeled insulin. The amount of [~25I]insulin bound to the control subjects' monocytes at the lowest concentration of unlabeled insulin was 2-fold greater than the amount hound by the patients (Fig. 1). The mean percent specific binding to 10 7 cells was 6.36 + 0.84% in MyD patients. The mean percent specific binding for control cells was 13.6 + 1.9% per 107 cells. This difference between both means is statistically significant (P < 0.05). The amount of unlabeled insulin producing half maximal displacement of [~25I]insulin was determined from logit-log plots (Rodbard et al. 1968). The EDso determined in this way was significantly lower in controls (20.0 + 2.1 x 10-9 M) than in MyD (52.0 + 10.0 × 10 - 9 M; P < 0.02) implying a greater affinity for insulin in these subjects' cells. Scatchard plots of these data were curvilinear as is typical for insulin binding (not shown).
Euglycemic clamp The glucose disposal rate (DR) for controls was 8.21 + 0.83 and for MyD patients was 3.17 + 0.62 mg" kg- 1. min (P = 0.001: Table 1). Insulin levels during the clamp were similar for both groups (Table 1, Fig. 2, remaining relatively constant during the euglycemic clamp). DR showed a significant inverse correlation with percent of ideal body weight (Fig. 3; r = - 0.67; P < 0.01). It should be noted, however, that at the same
19
tlJ
8
160
o
=80 o
E
J (9
~4o 0
Controls
0
MyD
Controls MyD
Fig. 2. Mean ( + SEM) glucosedisposal rates (A) and steady state plasma insulin concentrations(B) in 15 normal and 6 MyD patients in euglycemicglucose clamps studies performedwith an insulin infusion rate of 1.2 mU. kg- ~' min - ~.
percent of ideal body weight glucose disposal in MyD patients was considerably less than controls (Fig. 3; open circles) suggesting that obesity could not solely account for decreased insulin sensitivity in MyD. Correlation of percent specific binding and glucose disposal rate in both groups (compared in 10 control subjects) showed a trend towards a positive correlation although this was not statistically significant (r = 0.44).
Effect of euglycemic clamp on insulin binding and EDso in monocytes In order to determine whether the alteration in insulin receptors in MyD monocytes could be acutely modified we determined relative capacity and affinity (EDso) preand post-clamp. There were no differences in the pre- and post-clamp averaged receptor capacity binding curves in MyD patients. However, a significant improvement in EDso post-clamp was seen in 4 MyD patients (JaC., JoC, M . G . and R.B.) as shown in Table 2 (pre-clamp EDso 52.0 + 10, post-clamp EDso 24.56 + 3.6; P = 0.01).
w
14
,
,
~
'
,
r =-0.67 p
/
oT= 8 N
" 6
8 (.9
o
,I
'
0
% IDEAL
'
80 160 BODY WEIGHT
Fig. 3. Linear regression showingthe correlationbetween % of ideal body weight and DR in MyD (open circles) and controls (closed circles); r = -0.67, P < 0.01.
2O TABLE 2 COMPARISON OF ~o INSULIN BOUND AND EDs0 (LOGIT LOG) OF MyD MONOCYTES PREAND POST-EUGLYCEMIC CLAMPS Patient
Pre-clamp a
Post-clamp b
~o Boundc
EDsoa
~ Bound
EDso
D.E. JaC M.G. R.J. JoC R.B.
7.97 7.99 7.6 6.0 3.6 6.37
8.0 32.0 45.0 18.0 120.0 90.0
7.8 5.4 7.2 2.6 3.0 10.4
16.5 12.5 14.0 15.0 49.4 40.0
Mean _+ SEM
6~58 0.42
52.0 10.0
6.06 0.74
24.56 3.63
EDso, pre- and post-clamp: P = 0.01. ~o Bound, pre- and post-clamp: P = 0.5. a, b Euglycemic clamp performed as described in Methods section. Pre-elamp cells were obtained at time of catheter insertion. Post-clamp cells were obtained immediately aRer infusion. c,d Relative capacity and EDso calculated from data of insulin binding of [125I]-insulin to monocytes as described in Methods section.
DISCUSSION
MyD patients have a 4-fold increase in the risk of diabetes (Harper 1979). Abnormal OGTTs have been found in about 16~ of MyD cases and insulin hyperresponsiveness during OGTT in about 62 ~ (Harper 1979). Kobayashi et al. (1977)did not observe differences in insulin receptor number of MyD patients' mononuclear cells. However, only 2 unlabelled insulin concentrations were used in that study. Subsequently, Festoff and Moore (1979), utilizing a complete displacement curve, found a markedly decreased receptor number preferentially affecting the high affinity binding sites. Tevaarwerk et al. (1979) and Stuart et al. (1983) independently showed modest to significant decrease in insulin binding accompanied by substantial decreases in receptor affinity. These studies suggest that the decrease in affinity is the main reason for the decrease in insulin binding in MyD. However, Fratino et al. (1982) reported only a decrease in receptor numbers. Whether the fundamental abnormality consists of a general decrease in affinity or a small reduction of receptors of fixed high affinity has, consequently, not been clearly defined by any study. More recently, Vialettes et al. (1986) could only show loss of down-regulation in MyD erythrocytes, however, no effort was made to study "young" red ceils. In addition, although binding of insulin to Duchenne muscular dystrophy erythrocytes was reduced, no abnormality in carbohydrate tolerance is known in this genetic muscle disease (DePirro 1982). Hudson and colleagues (1987) have studied this problem in cultured fibroblasts from MyD patients. In an environment which was insulin-free, MyD cells had decreased receptor numbers with normal affinity. An intrinsic cellular defect was suggested to explain this situation.
21 Insulin resistance is defined, in the endocrine literature, as a state in which greater than normal amounts of insulin are required to elicit a quantitatively normal response (Berson and Yalow 1970). Such an assessment in vivo has been attempted in MyD using several methods. Early studies suggested alterations in insulin secretion (Huff et al. 1967; Huffand Lebovitz 1968). The results of insulin sensitivity using intravenous insulin tolerance tests (IITT)have been contradictory: Tevaarwerk and Hudson (1977), Barbosa et al. (1974) and Stuart et al. (1983) reported significantly higher mean nadir IITT values. However, Gorden et al. (1969), Bird and Tzgournis (1970), Nuttal et al. (1974), and Marshall (1959) found responses similar to normal controls. Moxley et al. (1980) showed that there was resistance to the effect of physiologic concentrations of insulin on glucose uptake in forearm skeletal muscle, but not of skin and fat in MyD patients. They interpreted these results as showing restricted insulin resistance in skeletal muscle in MyD. Furthermore, higher concentrations of insulin did not correct this apparent resistance. The glucose clamp technique (DeFronzo et al. 1979) averages the glucose removal from blood by all insulin sensitive tissues, but does not discriminate the effect on the three main targets: muscle, fat and liver. However, muscle accounts for more than 85~o of glucose disposal (Jones et al. 1985). The present study, using this sensitive technique demonstrates total body resistance in MyD and confLrms our own (Perurena et al. 1981) and the results of others (Moxley et al. 1984; Vialettes et al. 1986). The inverse relationship found between ideal body weight and insulin sensitivity for MyD patients and controls groups is in agreement with the findings of Olefsky and Kolterman (1981) and Rizza et al, (1981), using similar methods. Since 2 of our MyD patients were less sensitive than controls at the same ideal body weight, this suggests that a factor other than obesity is involved in determining glucose disposal rate in MyD. We acknowledge that ideal true body weight may not fully reflect body codposition in MyD. It probably underestimates the amount of fat and does not take into consideration the disproportionate effect on Type ! more than Type II muscle fibers that occurs in muscle in MyD patients early in the disease (Harper 1979). However, the probable greater amount of fat in MyD patients should, in fact, compensate for muscle insulin resistance if fat tissue was normally sensitive as suggested (Moxley et al. 1978, 1980). The trend towards direct correlation between relative capacity and insulin sensitivity in our study indicates that the receptor is the regulator where the resistance occurs. The affinity in 4 MyD patients improved after an acute sustained delivery of glucose and insulin, and this may be analogous to the findings in normals after an OGTT as described by Muggeo et al. (1977). This finding suggests potential environmental modification of the receptor characteristics in this disease. Furthermore, the post-prandial relative affmity was markedly diminished in our patients, a fact that clearly differentiates them from obese subjects and amyotrophic lateral sclerosis (ALS) patients where alTmity is normal (Bar et al. 1979; Perurena and Festoff 1987). Our results clearly show that the reduced receptor affinity of MyD monocyte insulin receptors can be improved by supraphysiological infusion of insulin during the clamp. Although an earlier study by Moxley et al. (1981) reported no increase in insulin receptor affinity of MyD monocytes after an OGTT, their results might be explained
22 by an increased fasting receptor affinity in MyD patients compared to controls. Our data suggest that receptor affinity alteration is modifiable by changing environmental insulin levels while receptor numbers are not. This is similar to the results of Hudson et al. (1987) with MyD fibroblasts in tissue culture. However, the direction of the change is different. In culture, absence of insulin resulted in normal receptor affinity. Since oxidative muscle fibers are the most dependent on insulin action (James et al. 1985) the disproportionate amount of Type I atrophy may be the early pathological locus of the insulin resistance found with the clamp. The results of other authors utilizing the euglycemic clamp technique with dose-response curves were compatible with a failure at the receptor (Moxley et al. 1984) or at the post-receptor level (Vialettes et al. 1986). This disparity may be more apparent than real since the co-existence of binding and post-binding defects leading to decreased insulin action is found in other insulin resistance conditions (Truglia et al. 1985). It has recently been reported that MyD patients lack normal enhancement of insulin sensitivity after an OGTT (Moxley et al. 1987). Such results may depend on an intrinsic defect in adaptation of the receptor in target tissues. The abnormality of insulin action, however, is extended to the regulation of serum amino acid levels (Moxley et al. 1986) and protein anabolism (Griggs et al. 1980). Of great interest are the recent results from molecular biology and genetics. The genes for MyD and the insulin receptor are both on chromosome 19, but in different regions (Shaw et al. 1985; Yang-Feng et al. 1985; Pericak-Vance et al. 1986; Bartlett et al. 1987; Friedrich et al. 1987). Linkage studies suggest that MyD is at or close to the centromere (Pericak-Vance et al. 1986; Bartlett et al. 1987), i.e., the 19cen-q13.2 region (Schepens et al. 1987) or 19p13-19q13 (Shaw et al. 1985). The cDNA for the human insulin receptor has been cloned (Ebina et al. 1985; Ullrich et al. 1985) which has allowed the insulin receptor gene to be mapped to the short arm of chromosome 19 (Strauss et al. 1985; Yang-Feng et al. 1985). The gene for the human insulin receptor has not yet been cloned but recent studies, using genomic cloning and DNA restriction length analysis, have provided a partial map of the gene's structure (Milller-Wieland et al. 1988). The gene is quite large with a minimum estimated size of 150 kb, approximately 96 kb corresponding to the alpha and 60 kb to the beta subunits (Mtlller-Wieland et al. 1988). In MyD the alteration in insulin receptor function is retained in cultured cells, suggesting the involvement of a genetic mechanism (Hudson et al. 1987). The deficit in MyD of a regulatory protease which normally acts on the insulin proreceptor could, theoretically, lead to conformational changes modifying its binding and affinity. Furthermore, as in two other multisystem disorders with insulin resistance (type A insulin resistance and leprechaunism), a specific point mutation of the insulin receptor gene could also be associated with MyD producing a defect in the expression of the insulin gene (Grigorescu et al. 1987; Yoshimasa et al. 1987; Kadowaki et al. 1988). Further understanding of the molecular genetics of the insulin resistance in MyD will require cloning and sequencing of genomic insulin receptor fragments or cDNA, as well as studying the protein chemistry of the insulin receptor, utilizing available affinity labeling, affinity purification and specific immunoprecipitation techniques (Czech 1985).
23 ACKNOWLEDGEMENTS
The authors express their gratitude to Drs. W.V. Moore, Jr. and R. Guthrie in the inception of these studies as well as to R. Jorgensen, R.N. who participated in the Biostator studies. Support for these studies came, in part, from the Muscular Dystrophy Association, BRSG S07 RR05373 (awarded by the Biomedical Research Support Grant Program, Division of Research Resources, NIH), and the Medical Research Service of the Veterans Administration. Grateful acknowledgment is given to Joyce Capps for preparation of the manuscript and to Dr. R. Kahn and associates for advance viewing of their unpublished manuscript.
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25 Ullrich, A., J. R. Bell, E.Y. Chen, R. Herrera, L.M. Petruzelli, T.J. Dull, A. Gray, L. Coussens, Y.-C. Liao, M. Tsubokawa, A. Mason, P.H. Seeburg, C. GTunfeld, O.M. Rosen and J. Ramachandran (1985) Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature, 313: 756-761. Vialettes, B., J. Pouget, R. Viard, J.P. Moulin, G. Serratrice and P. Vague (1986) Mechanism and significance of insulin resistance in myotonic dystrophy. Horm. Metab. Res., 18: 395-399. Yang-Feng, T., U. Francke and A. Ullrich (1985) Gene for human insulin receptor: localization to site on chromosome 19 involved in pre-B-cell leukemia. Science, 228: 728-730. Yoshimasa, Y., S. Seino, J. Whittaker, T. Kakehi, A. Kosaki, H. Kuzuya, H. Imura, G.I. Bell and D.F. Steiner (1987) Insulin-resistant diabetes due to a point mutation that prevents insulin proreceptor processing. Science, 240: 784-787.
15
Elsevier JNS 03098
Environmental influence on altered receptor function in a genetic disease: insulin and glucose affect insulin receptors in myotonic dystrophy Osvaldo H. Perurena* and Barry W. Festoff Neurobiology Research Laboratory, Kansas City, Missouri Veterans Administration Medical Center, and Department of Neurology, University of Kansas Medical Center, Kansas City, KS (U.S.A.)
(Received27 January, 1988) (Revised, received9 September, 1988) (Accepted 9 September, 1988)
SUMMARY Insulin action in vivo and insulin binding to monocytes in vitro were correlated in patients with myotonic dystrophy (MyD) and compared with healthy controls. Confh,-ming our previous studies and those of others, the present results show that the glucose infusion rate (DR), an estimate of in vivo insulin sensitivity, was significantly diminished in MyD. At the same per cent of ideal body weight DR in MyD patients was considerably less than controls suggesting that obesity could not solely account for decreased insulin sensitivity in MyD. The relative capacity (RC), and relative affinity (EDso) of the insulin receptor in monocytes was significantly less in patients. The relative affmity (EDso) was improved by changing environmental insulin levels while receptor numbers (RC) were not. Insulin sensitivity and RC showed a trend toward a positive correlation although this did not reach statistical siotmificance. Our data suggest that the alteration of the insulin receptor in MyD is different from obesity and from other disorders of the motor unit such as amyotrophic lateral sclerosis, where insulin sensitivity and RC are reduced but EDso is unchanged. Thus, in MyD the receptor may be one of the loci where the resistance occurs.
Key words: Myotonic dystrophy; Insulin receptor; Insulin resistance; Molecular genetics; Chromosome; eDNA; Euglycemic insulin clamp; Monocyte
* Present address: NeurologyService(151), Colmery-O'NeilVAMC,Topeka, KS 66622, U.S.A. Correspondence to." Barry W. Festoff,M.D., NeurobiologyResearch Laboratory(151)VA Medical
Center, 4801 LinwoodBlvd., Kansas City, MO 64128, U.S.A. 0022-510X/89/$03.50 © 1989Elsevier Science Publishers B,V.(BiomedicalDivision)
16 INTRODUCTION
Myotonic dystrophy (MyD) is an autosomal dominant genetic disorder manifested commonly by myopathy and myotonia and accompanied frequently by a vast array of bony and endocrine abnormalities, cataract and mental retardation (Harper 1979). Recent studies suggest that genetically induced-membrane changes may be primary in the disease (Roses et al. 1980). A marked decrease in capacity of high affinity monocyte receptors for insulin in patients with MyD has frequently been found (Festoff and Moore 1979; Tevaarwerk et al. 1979; Stuart et al. 1983). To determine if altered insulin receptors correlate with peripheral insulin resistance, we have utilized the euglycemic clamp method (DeFronzo et al. 1979) to quantify the composite glucose uptake by the three main insulin targets: liver, fat, and muscle. Simultaneously, we evaluated insulin receptors on circulating monocytes of MyD patients and controls. Portions of this work have been previously reported (Festoffet al. 1981; Perurena et al. 1981). METHODS
Subjects Informed consent was obtained for monocyte insulin binding and glucose clamping in both MyD patients and controls. Patients were recruited from the Neuromuscular Disease Clinics of the University of Kansas and Kansas City Veterans Administration Medical Centers. The diagnosis of MyD was made on the basis of physical findings, electromyography, muscle biopsy and family history. All were males aged 20-55 years with various degrees of functional impairment. Seven female and 8 male controls, aged 21-34 years, were healthy with normal oral glucose tolerance tests (OGTT) and displayed a similar moderate amount of physical activity. Patients and controls fasted for 12 h before all studies. Insulin binding of [ ~25I]insulin by peripheral monocytes was determined in MyD patients immediately before and after the euglycemic clamp, while binding in controls was determined only before the clamp. Iodination of insulin, preparation of cells and binding studies to monocytes were as described by Perurena and Festoff (1987).
Euglycemic clamp One indwelling catheter was inserted in a forearm vein for insulin and glucose infusion. A double lumen catheter allowing an admixture of subject's blood with heparin, was the input to the Biostator Controller (Miles Laboratories, Elkhart, IN) for blood glucose monitoring at 60-s intervals. Insulin (Eli Lilly, Indianapolis, IN) was infused by a Harvard pump (Harvard Apparatus, Millis, MA): a priming initial dose of 2.4 mU. kg- i. rain - t was given during the fn'st 10 rain and a subsequent dose of 1.2 mU. kg - 1. rain - 1 for the remaining 110 rain. Serum was obtained for estimation of insulin levels during the infusion and exceeded 85/~U/ml in all subjects.
17 Euglycemic insulin clamp parameters were calculated according to the preprogrammed algorithm for the Biostator Controller. Glucose infusion was administered using the following equation: DR (mg. kg- 1. min- 1) = RD. (BD - G/25
+ 1) 4
where DR is the glucose infusion rate, RD is the glucose infusion rate at the pre-selected basal glucose level, BD is the preselected basal glucose, and G is the last one-minute average level recorded in the computer. In some subjects, it was necessary to supplement the glucose infusion by the Controller via a separate line in order to maintain blood glucose at the preseleeted basal levels. In these cases RD was higher than the maximum RD of the machine.
RESULTS
Subjects The mean percent of ideal body weight in the 6 MyD patients was 122.5 + 9.82 (Table 1). Two of them (J. C. and JaC) were brothers and had abnormal oral glucose tolerance tests (OGTTs). The mean basal insulin level was 24.8 #U/ml. In 3 patients they were above 25/~U/ml. Patient R.B. had a family history and biopsy compatible with the disease, but no muscle weakness and minimal myotonia. Patients D.E. and M.G. had the complete syndrome, but were ambulatory with only mild weakness. TABLE 1 S U M M A R Y O F IN VIVO G L U C O S E A N D I N S U L I N M E T A B O L I S M A N D M O N O C Y T E I N S U L I N B I N D I N G IN M y D PATIENTS A N D N O R M A L C O N T R O L S MyD
% Ideal body weight (IBW) a (n = 6)
Abnormal OGTT
Fasting insulin b (/~U/rnl)
Glucose infusion rate (DR) (mg. k g - 1. m i n - i)
J.C. JaC R.B. D.E. M.G. R.J. Mean + SEM
136 ! 39 113 128 78 141 122.5 9.8
Yes Yes No No No No
26 54 8 27 16 18 24.6 15.9
3.73 1.16 5.01 2.54 4.63 2.0 3.17 0.62
No
14.3 1.28
8.21 0.83
Controls (n = 15) Mean _+ SEM
97.2 3.85
P < 0.01 a IBW was determined using the tables of the Metropolitan Life Insurance Co. b For insulin levels determinations see Methods section.
18 14
\ "~ ~"~1-12 " ~ ~
• CONTROLS a MyD
Z
~4 ::3
°
I I0 INSULIN
I00 I000 I0,000 CONCENTRATION ( n g / m l )
Fig. l. Percent [~2Sl]insulinbindingto 10 7 monocytes/mlat increasingconcentrationsof unlabeled insulin. The percent of [~2sI]insulinbound is plotted versus the quantity (ng/ml)of unlabeledinsulin for leukocytes from 15 controls (closed circles) and 6 patients with MyD (squares).
Patients J.C. and JaC were more severely impaired, but still ambulatory using a cane. Patient R.J. was intermittently in a wheelchair. The 15 controls for the clamp studies (Table 1) were 97.2 + 3.85% of ideal body weight and only 1 control subject had basal insulin levels above 20 #U/ml (mean 14.3 #U/ml). All had a similar moderate amount of physical activity.
Insulin binding to monocytes The percent of [~25I]insulin bound to the cells at 15 °C was determined as a function of the concentration of unlabeled insulin. The amount of [~25I]insulin bound to the control subjects' monocytes at the lowest concentration of unlabeled insulin was 2-fold greater than the amount hound by the patients (Fig. 1). The mean percent specific binding to 10 7 cells was 6.36 + 0.84% in MyD patients. The mean percent specific binding for control cells was 13.6 + 1.9% per 107 cells. This difference between both means is statistically significant (P < 0.05). The amount of unlabeled insulin producing half maximal displacement of [~25I]insulin was determined from logit-log plots (Rodbard et al. 1968). The EDso determined in this way was significantly lower in controls (20.0 + 2.1 x 10-9 M) than in MyD (52.0 + 10.0 × 10 - 9 M; P < 0.02) implying a greater affinity for insulin in these subjects' cells. Scatchard plots of these data were curvilinear as is typical for insulin binding (not shown).
Euglycemic clamp The glucose disposal rate (DR) for controls was 8.21 + 0.83 and for MyD patients was 3.17 + 0.62 mg" kg- 1. min (P = 0.001: Table 1). Insulin levels during the clamp were similar for both groups (Table 1, Fig. 2, remaining relatively constant during the euglycemic clamp). DR showed a significant inverse correlation with percent of ideal body weight (Fig. 3; r = - 0.67; P < 0.01). It should be noted, however, that at the same
19
tlJ
8
160
o
=80 o
E
J (9
~4o 0
Controls
0
MyD
Controls MyD
Fig. 2. Mean ( + SEM) glucosedisposal rates (A) and steady state plasma insulin concentrations(B) in 15 normal and 6 MyD patients in euglycemicglucose clamps studies performedwith an insulin infusion rate of 1.2 mU. kg- ~' min - ~.
percent of ideal body weight glucose disposal in MyD patients was considerably less than controls (Fig. 3; open circles) suggesting that obesity could not solely account for decreased insulin sensitivity in MyD. Correlation of percent specific binding and glucose disposal rate in both groups (compared in 10 control subjects) showed a trend towards a positive correlation although this was not statistically significant (r = 0.44).
Effect of euglycemic clamp on insulin binding and EDso in monocytes In order to determine whether the alteration in insulin receptors in MyD monocytes could be acutely modified we determined relative capacity and affinity (EDso) preand post-clamp. There were no differences in the pre- and post-clamp averaged receptor capacity binding curves in MyD patients. However, a significant improvement in EDso post-clamp was seen in 4 MyD patients (JaC., JoC, M . G . and R.B.) as shown in Table 2 (pre-clamp EDso 52.0 + 10, post-clamp EDso 24.56 + 3.6; P = 0.01).
w
14
,
,
~
'
,
r =-0.67 p
/
oT= 8 N
" 6
8 (.9
o
,I
'
0
% IDEAL
'
80 160 BODY WEIGHT
Fig. 3. Linear regression showingthe correlationbetween % of ideal body weight and DR in MyD (open circles) and controls (closed circles); r = -0.67, P < 0.01.
2O TABLE 2 COMPARISON OF ~o INSULIN BOUND AND EDs0 (LOGIT LOG) OF MyD MONOCYTES PREAND POST-EUGLYCEMIC CLAMPS Patient
Pre-clamp a
Post-clamp b
~o Boundc
EDsoa
~ Bound
EDso
D.E. JaC M.G. R.J. JoC R.B.
7.97 7.99 7.6 6.0 3.6 6.37
8.0 32.0 45.0 18.0 120.0 90.0
7.8 5.4 7.2 2.6 3.0 10.4
16.5 12.5 14.0 15.0 49.4 40.0
Mean _+ SEM
6~58 0.42
52.0 10.0
6.06 0.74
24.56 3.63
EDso, pre- and post-clamp: P = 0.01. ~o Bound, pre- and post-clamp: P = 0.5. a, b Euglycemic clamp performed as described in Methods section. Pre-elamp cells were obtained at time of catheter insertion. Post-clamp cells were obtained immediately aRer infusion. c,d Relative capacity and EDso calculated from data of insulin binding of [125I]-insulin to monocytes as described in Methods section.
DISCUSSION
MyD patients have a 4-fold increase in the risk of diabetes (Harper 1979). Abnormal OGTTs have been found in about 16~ of MyD cases and insulin hyperresponsiveness during OGTT in about 62 ~ (Harper 1979). Kobayashi et al. (1977)did not observe differences in insulin receptor number of MyD patients' mononuclear cells. However, only 2 unlabelled insulin concentrations were used in that study. Subsequently, Festoff and Moore (1979), utilizing a complete displacement curve, found a markedly decreased receptor number preferentially affecting the high affinity binding sites. Tevaarwerk et al. (1979) and Stuart et al. (1983) independently showed modest to significant decrease in insulin binding accompanied by substantial decreases in receptor affinity. These studies suggest that the decrease in affinity is the main reason for the decrease in insulin binding in MyD. However, Fratino et al. (1982) reported only a decrease in receptor numbers. Whether the fundamental abnormality consists of a general decrease in affinity or a small reduction of receptors of fixed high affinity has, consequently, not been clearly defined by any study. More recently, Vialettes et al. (1986) could only show loss of down-regulation in MyD erythrocytes, however, no effort was made to study "young" red ceils. In addition, although binding of insulin to Duchenne muscular dystrophy erythrocytes was reduced, no abnormality in carbohydrate tolerance is known in this genetic muscle disease (DePirro 1982). Hudson and colleagues (1987) have studied this problem in cultured fibroblasts from MyD patients. In an environment which was insulin-free, MyD cells had decreased receptor numbers with normal affinity. An intrinsic cellular defect was suggested to explain this situation.
21 Insulin resistance is defined, in the endocrine literature, as a state in which greater than normal amounts of insulin are required to elicit a quantitatively normal response (Berson and Yalow 1970). Such an assessment in vivo has been attempted in MyD using several methods. Early studies suggested alterations in insulin secretion (Huff et al. 1967; Huffand Lebovitz 1968). The results of insulin sensitivity using intravenous insulin tolerance tests (IITT)have been contradictory: Tevaarwerk and Hudson (1977), Barbosa et al. (1974) and Stuart et al. (1983) reported significantly higher mean nadir IITT values. However, Gorden et al. (1969), Bird and Tzgournis (1970), Nuttal et al. (1974), and Marshall (1959) found responses similar to normal controls. Moxley et al. (1980) showed that there was resistance to the effect of physiologic concentrations of insulin on glucose uptake in forearm skeletal muscle, but not of skin and fat in MyD patients. They interpreted these results as showing restricted insulin resistance in skeletal muscle in MyD. Furthermore, higher concentrations of insulin did not correct this apparent resistance. The glucose clamp technique (DeFronzo et al. 1979) averages the glucose removal from blood by all insulin sensitive tissues, but does not discriminate the effect on the three main targets: muscle, fat and liver. However, muscle accounts for more than 85~o of glucose disposal (Jones et al. 1985). The present study, using this sensitive technique demonstrates total body resistance in MyD and confLrms our own (Perurena et al. 1981) and the results of others (Moxley et al. 1984; Vialettes et al. 1986). The inverse relationship found between ideal body weight and insulin sensitivity for MyD patients and controls groups is in agreement with the findings of Olefsky and Kolterman (1981) and Rizza et al, (1981), using similar methods. Since 2 of our MyD patients were less sensitive than controls at the same ideal body weight, this suggests that a factor other than obesity is involved in determining glucose disposal rate in MyD. We acknowledge that ideal true body weight may not fully reflect body codposition in MyD. It probably underestimates the amount of fat and does not take into consideration the disproportionate effect on Type ! more than Type II muscle fibers that occurs in muscle in MyD patients early in the disease (Harper 1979). However, the probable greater amount of fat in MyD patients should, in fact, compensate for muscle insulin resistance if fat tissue was normally sensitive as suggested (Moxley et al. 1978, 1980). The trend towards direct correlation between relative capacity and insulin sensitivity in our study indicates that the receptor is the regulator where the resistance occurs. The affinity in 4 MyD patients improved after an acute sustained delivery of glucose and insulin, and this may be analogous to the findings in normals after an OGTT as described by Muggeo et al. (1977). This finding suggests potential environmental modification of the receptor characteristics in this disease. Furthermore, the post-prandial relative affmity was markedly diminished in our patients, a fact that clearly differentiates them from obese subjects and amyotrophic lateral sclerosis (ALS) patients where alTmity is normal (Bar et al. 1979; Perurena and Festoff 1987). Our results clearly show that the reduced receptor affinity of MyD monocyte insulin receptors can be improved by supraphysiological infusion of insulin during the clamp. Although an earlier study by Moxley et al. (1981) reported no increase in insulin receptor affinity of MyD monocytes after an OGTT, their results might be explained
22 by an increased fasting receptor affinity in MyD patients compared to controls. Our data suggest that receptor affinity alteration is modifiable by changing environmental insulin levels while receptor numbers are not. This is similar to the results of Hudson et al. (1987) with MyD fibroblasts in tissue culture. However, the direction of the change is different. In culture, absence of insulin resulted in normal receptor affinity. Since oxidative muscle fibers are the most dependent on insulin action (James et al. 1985) the disproportionate amount of Type I atrophy may be the early pathological locus of the insulin resistance found with the clamp. The results of other authors utilizing the euglycemic clamp technique with dose-response curves were compatible with a failure at the receptor (Moxley et al. 1984) or at the post-receptor level (Vialettes et al. 1986). This disparity may be more apparent than real since the co-existence of binding and post-binding defects leading to decreased insulin action is found in other insulin resistance conditions (Truglia et al. 1985). It has recently been reported that MyD patients lack normal enhancement of insulin sensitivity after an OGTT (Moxley et al. 1987). Such results may depend on an intrinsic defect in adaptation of the receptor in target tissues. The abnormality of insulin action, however, is extended to the regulation of serum amino acid levels (Moxley et al. 1986) and protein anabolism (Griggs et al. 1980). Of great interest are the recent results from molecular biology and genetics. The genes for MyD and the insulin receptor are both on chromosome 19, but in different regions (Shaw et al. 1985; Yang-Feng et al. 1985; Pericak-Vance et al. 1986; Bartlett et al. 1987; Friedrich et al. 1987). Linkage studies suggest that MyD is at or close to the centromere (Pericak-Vance et al. 1986; Bartlett et al. 1987), i.e., the 19cen-q13.2 region (Schepens et al. 1987) or 19p13-19q13 (Shaw et al. 1985). The cDNA for the human insulin receptor has been cloned (Ebina et al. 1985; Ullrich et al. 1985) which has allowed the insulin receptor gene to be mapped to the short arm of chromosome 19 (Strauss et al. 1985; Yang-Feng et al. 1985). The gene for the human insulin receptor has not yet been cloned but recent studies, using genomic cloning and DNA restriction length analysis, have provided a partial map of the gene's structure (Milller-Wieland et al. 1988). The gene is quite large with a minimum estimated size of 150 kb, approximately 96 kb corresponding to the alpha and 60 kb to the beta subunits (Mtlller-Wieland et al. 1988). In MyD the alteration in insulin receptor function is retained in cultured cells, suggesting the involvement of a genetic mechanism (Hudson et al. 1987). The deficit in MyD of a regulatory protease which normally acts on the insulin proreceptor could, theoretically, lead to conformational changes modifying its binding and affinity. Furthermore, as in two other multisystem disorders with insulin resistance (type A insulin resistance and leprechaunism), a specific point mutation of the insulin receptor gene could also be associated with MyD producing a defect in the expression of the insulin gene (Grigorescu et al. 1987; Yoshimasa et al. 1987; Kadowaki et al. 1988). Further understanding of the molecular genetics of the insulin resistance in MyD will require cloning and sequencing of genomic insulin receptor fragments or cDNA, as well as studying the protein chemistry of the insulin receptor, utilizing available affinity labeling, affinity purification and specific immunoprecipitation techniques (Czech 1985).
23 ACKNOWLEDGEMENTS
The authors express their gratitude to Drs. W.V. Moore, Jr. and R. Guthrie in the inception of these studies as well as to R. Jorgensen, R.N. who participated in the Biostator studies. Support for these studies came, in part, from the Muscular Dystrophy Association, BRSG S07 RR05373 (awarded by the Biomedical Research Support Grant Program, Division of Research Resources, NIH), and the Medical Research Service of the Veterans Administration. Grateful acknowledgment is given to Joyce Capps for preparation of the manuscript and to Dr. R. Kahn and associates for advance viewing of their unpublished manuscript.
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