Somatomedin in Insulin-Dependent Diabetes Mellitus MARGO P. COHEN, KRISHNA JASTI, AND DIANA L. RYE Department of Medicine, Wayne State University of School of Medicine, Detroit, Michigan ABSTRACT. Somatomedin activity in sera from twelve insulin-dependent diabetics was measured by the chick embryo cartilage assay system. All patients required insulin for control of hyperglycemia, and had been continuously treated with exogenous insulin for 3 to 25 years. Mean fasting somatomedin activity was elevated in this group of
S
EVERAL studies have suggested the existence of a causal relationship between growth hormone and the development of diabetic vascular disease. One approach to this problem considers the role of somatomedins, which stimulate anabolic processes in sensitive tissues (1,2). The prototype of these substances was originally identified by Salmon and Daughaday as a factor present in serum which enhanced the uptake of radioactive sulfate into cartilage from hypophysectomized rats (3). Subsequent work revealed additional important effects, including the ability to stimulate the incorporation of labeled thymidine into DNA, uridine into RNA, and leucine into protein (4). Poor growth in acute streptozotocin diabetes is associated with diminished serum somatomedin activity in rats (5), and somatomedin has been reported to be in the low normal range in untreated juvenile diabetics (6) and in long-term diabetics with severe diabetic angiopathy (7). However, information regarding duration, glucose levels, treatment and type of diabetes in the latter series was not provided. We therefore considered it of interest to determine levels of somatomedin activity in a group of insulin-dependent diabetics in whom these
Received October 20, 1976. Supported in part by Research Grants from the Michigan Department of Public Health, the American Diabetes Association (Michigan Affiliate), and the Detroit General Hospital Research Corporation. Correspondence to: Margo P. Cohen, M.D., Wayne State University School of Medicine, 540 East Canfield, Detroit, Michigan 48201.
diabetics, and activity did not correlate with the simultaneous blood glucose concentrations. No significant differences were demonstrable between levels in diabetics with and without retinopathy or in patients with and without proteinuria. (J Clin Endocrinol Metab 45: 236, 1977)
factors were defined, and to explore the relationship between somatomedin and the presence of retinopathy and/or proteinuria. Materials and Methods Patients Twelve patients with a diagnosis of diabetes mellitus being followed at Detroit General Hospital (Wayne State University Medical Center) were studied. All subjects were classified as insulin-dependent diabetics on the basis of body mass (< 115% of ideal body weight) and documented ketonuria upon insulin withdrawal. Age of onset of diabetes was less than 45 years in every patient, and serum creatinine was normal at the time of study except in patients 9 and 12 (2.1 and 2.8 mg/dl, respectively). Duration of diabetes and of exogenous insulin therapy ranged from 3 to 25 years. Pertinent clinical findings are summarized in Table 1. Informed consent was obtained from all patients, to whom the purpose of the student and amount of blood sampling had been explained. Serum somatomedin assays Sera used for these assays were obtained in the fasting state, prior to morning insulin injections, and frozen at - 2 0 C until use. Somatomedin (sulfation factor) activity was measured by a modification of the method of Hall (8,9) using pelvic rudiments from 11-day old chick embryos incubated in 2 ml of Eagle's basal medium made 0.21 inM in glutamine. Samples were preincubated for 1 h, and then pulsed for 4 h with 35Ssulfate. Following incubation, samples were successively washed with unlabeled 0.2M sodium sulfate and distilled water, blotted dry, and weighed. After overnight digestion with papain,
236
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237
SOMATOMEDIN IN DIABETES TABLE 1. Clinical data in diabetic patients
Patient 1 2 3 4 5 6 7 8 9 10 11 12
Sex M F F F M M M F M M M F
Age 31 26 19 34 34 40 28 27 54 48 56 55
Duration of diabetes (yrs) 3 3 4 5 6 9 12 12 12 15 17 25
MeanFBS* (mg/dl)
Urinary protein Retinopathyf _ — + — — _ + + + + + +
278 283 160 310 291 234 213 200 250 219 130 216
(g/24 h) 0.04 0.13 0.01 0.43 0.02 0.55 7.05 0.02 3.50 0.24 4.54 5.60
* Mean fasting blood sugar taken from review of record throughout patients clinic course; 3-10 fasting blood glucose determinations per year of clinic attendance available for computation. t Retinopathy defined as the presence of microaneurysms and/or exudates on routine funduscopic exam. protein was precipitated with 5% trichloroacetic acid, and the glycosaminoglycans in the supernatant fraction were dialyzed against distilled water. Aliquots were removed for determination of radioactivity by liquid scintillation in 10 ml of Bray's solution (10) and for uronic acid measurement by the carbazole method (11). A symmetrical 4-point design was used in all incubations, with two concentrations each of reference serum (10 and 20%) and of the test serum; pooled normal human serum (NHS) was used as the reference material. Only assays in which the response to test serum was parallel to the reference curve were considered valid; if necessary, assays were repeated with appropriate alterations in the serum concentrations to meet these criteria. The somatomedin activity of NHS was arbitrarily defined as 1.00, and the activity of the test sera was compared to and expressed in terms of this standard according to parallel line analysis (12). The mean index of precision in this assay was .30 ± .08. Results
Serum somatomedin activity and simultaneous blood sugars were determined in four separate samples from each patient. These results are shown in Table 2. The range of normal for somatomedin is 0.8 to 1.2 in our laboratory, determined with triplicate or quadruplicate assays in about 20 nondiabetic patients with a variety of other illnesses that were not of a debilitating nature.
Assay of three different acromegalic sera gave relative potency values of 1.53, 1.95 and 2.51, and those of sera from two hypopituitary patients were 0.63 and 0.71. Mean somatomedin levels were therefore significantly increased in this group of diabetic patients, although basal growth hormone levels in the diabetic samples used for glucose and somatomedin activity were not different from non-diabetic levels. This TABLE 2. Somatomedin levels in diabetic patients Patient
Glucose (mg/dl)*
Somatomedin f
1 2 3 4 5 6 7 8 9 10 11 12
275 213 222 143 176 248 237 158 343 219 122 216
1.60 ± 0.17 0.96 ± 0.15 1.55 ± 0.06 2.09 d: 0.06 0.83 dt0.05 1.52 Ht0.08 2.20 dt0.29 0.89 dt0.08 1.10 2t0.04 1.82 -t0.16 1.36: t0.09 3.21:tO.22
Mean
214
1.60 ± 0.14
* Blood glucose levels are mean of 4 determinations performed on 4 separate samples used for somatomedin assays. f Somatomedin potency of patients' sera expressed in relation to potency of pooled NHS, which was arbitrarily defined as 1.00. Results given as the mean ± SE of 4 separate assays performed in each subject.
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238
JCE & M • 1977 Vol 45 • No 2
COHEN, JASTI AND RYE
finding was confirmed by separately assaying sera from six non-diabetic individuals and calculating potency ratios compared to pooled normal human serum. Mean somatomedin activity was 1.05 ± .10 in this group of healthy house officers and laboratory personnel, while mean serum somatomedin activity in the diabetic group was 1.60 ± .14 (F < .01 normal versus diabetic). There was no correlation between simultaneous blood glucose concentrations and levels of somatomedin activity. No significant differences were demonstrable between somatomedin activity in diabetics with and without retinopathy or in patients with and without proteinuria. Discussion Hyperglycemia in untreated human (6) and experimental diabetes (5) is associated with lowered somatomedin activity. Normalization of blood glucose with insulin in acutely streptozotocin-diabetic rats restores somatomedin activity to control values, and activity is inversely correlated to the blood sugar in treated animals (16). The increased mean somatomedin activity and absence of correlation with the simultaneous blood glucose concentrations found in the present study suggest that the acute and chronic effects of hyperglycemia differ in respect to their influence on somatomedin generation and/or potency. While mean somatomedin activity was low in the group of 22 long-term diabetics with retinopathy studied by Yde (7), pertinent data regarding factors which might affect somatomedin are not given. Detailed information concerning renal function, nutritional status, average blood glucose, and duration, type and treatment of diabetes is not provided for the individual patients in that study. Differences in such clinical factors, as well as in assay techniques (chick embryo versus hypophysectomized rat cartilage) may account for these conflicting results. Evidence that growth hormone secretion or metabolism is altered in juvenile diabetes
is convincing. For example, diurnal and mean 24 h growth hormone levels are higher in diabetics than in control subjects (13,14), and an increased growth hormone response to exercise occurs in diabetics (15). While the role of growth hormone in the pathogenesis of chronic complications of diabetes remains speculative, the present study extends the significance of previously reported abnormalities in growth hormone by estab lishing the existence of changes in the growth-hormone dependent somatomedin in long-term, treated, insulin-dependent diabetes. Acknowledgment The skillful secretarial support of Ms. Joan Quinones is acknowledged.
References 1. Daughaday, W. H., K. Hall, M. S. Raben, W. D, Salmon, J. L. Van den Brande, and J. J. Van Wyk. Somatomedin: Proposed designation for sulfation factor, Nature 235: 107, 1972. 2. Van Wyk, J. J., L. E. Underwood, R. C. Lister, and R. N. Marshall, The somatomedins. A new class oi growth regulating hormones?, AmJ Dis Child 126: 705, 1973. 3. Salmon, W. D., and W. H. Daughaday, A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro, J Lab Clin Med 49: 825, 1957. 4. Salmon, W. D., and M. R. DuVall, A serum fraction with "sulfation factor activity" which stimulates in vitro incorporation of leucine and sulfate into protein-polysaccharide complexes, uridine into RNA, and thymidine into DNA of costal cartilage from hypophysectomized rats, Endocrinology 86: 721, 1970. 5. Phillips, L. S., and H. S. Young: Nutrition and somatomedin. II. Serum somatomedin activity and cartilage growth in streptozotocin-diabetic rats, Diabetes 25: 516, 1976. 6. Van den Brande, J. L., and M. V. L. Du Caju. Plasma somatomedin activity in children with growth disturbances, Adv. Growth Hormone Res., DHEW Publ. no. (NIH) 74-612, U.S. Government Printing Office, 1974, p. 98. 7. Yde, H., The growth hormone dependent sulfation factor in serum from patients with various types of diabetes, Ada Med Scand 186: 293, 1969 8. Hall, K., Quantitative determination of the sulphation factor activity in human sera, Ada Endocrinol (Kbh) 63: 338, 1970.
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SOMATOMEDIN IN DIABETES 9. Audhya, T. K., and K. D. Gibson, Serum inorganic sulfate and apparent somatomedin activity in an assay using chick embryo cartilage, Endocrinology 95: 1614, 1974. 10. Bray, G. A., A simple and efficient liquid scintillator for counting aqueous solutions in a liquid scintillator counter, Anal Biochem 1: 279, 1960. 11. Bitter, T., and H. Muir, A modified uronic acid carbazole reaction, Anal Biochem 4: 330, 1962. 12. Phillips, L. S., A. C. Herington, and W. H. Daughaday, Steroid hormone effects on somatomedin. I Somatomedin action in vitro, Endocrinology 97: 780, 1975. 13. Molnar, G. D., W. F. Taylor, A. Langworthy, and V. Fatourechi, Diurnal growth hormone and glu-
239
cose abnormalities in unstable diabetics: Studies of ambulatory-fed subjects during continuous blood glucose analysis, J Clin Endocrinol Metab 34: 837, 1972. 14. Hansen, A. P., and K. Johansen, Diurnal patterns of blood glucose, serum free fatty acids, insulin, glucagon and growth hormone in normal and juvenile diabetics, Diabetologia 6: 27, 1970. 15. Hansen, A. P., Abnormal serum growth hormone response to exercise in juvenile diabetes, J Clin Invest 49: 1467, 1970 16. Phillips, L. S., and A. T. Orawski, Diabetic control, somatomedin and growth in rats. Diabetes (Suppl 1) 25: 335, 1976 (Abstract).
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S
EVERAL studies have suggested the existence of a causal relationship between growth hormone and the development of diabetic vascular disease. One approach to this problem considers the role of somatomedins, which stimulate anabolic processes in sensitive tissues (1,2). The prototype of these substances was originally identified by Salmon and Daughaday as a factor present in serum which enhanced the uptake of radioactive sulfate into cartilage from hypophysectomized rats (3). Subsequent work revealed additional important effects, including the ability to stimulate the incorporation of labeled thymidine into DNA, uridine into RNA, and leucine into protein (4). Poor growth in acute streptozotocin diabetes is associated with diminished serum somatomedin activity in rats (5), and somatomedin has been reported to be in the low normal range in untreated juvenile diabetics (6) and in long-term diabetics with severe diabetic angiopathy (7). However, information regarding duration, glucose levels, treatment and type of diabetes in the latter series was not provided. We therefore considered it of interest to determine levels of somatomedin activity in a group of insulin-dependent diabetics in whom these
Received October 20, 1976. Supported in part by Research Grants from the Michigan Department of Public Health, the American Diabetes Association (Michigan Affiliate), and the Detroit General Hospital Research Corporation. Correspondence to: Margo P. Cohen, M.D., Wayne State University School of Medicine, 540 East Canfield, Detroit, Michigan 48201.
diabetics, and activity did not correlate with the simultaneous blood glucose concentrations. No significant differences were demonstrable between levels in diabetics with and without retinopathy or in patients with and without proteinuria. (J Clin Endocrinol Metab 45: 236, 1977)
factors were defined, and to explore the relationship between somatomedin and the presence of retinopathy and/or proteinuria. Materials and Methods Patients Twelve patients with a diagnosis of diabetes mellitus being followed at Detroit General Hospital (Wayne State University Medical Center) were studied. All subjects were classified as insulin-dependent diabetics on the basis of body mass (< 115% of ideal body weight) and documented ketonuria upon insulin withdrawal. Age of onset of diabetes was less than 45 years in every patient, and serum creatinine was normal at the time of study except in patients 9 and 12 (2.1 and 2.8 mg/dl, respectively). Duration of diabetes and of exogenous insulin therapy ranged from 3 to 25 years. Pertinent clinical findings are summarized in Table 1. Informed consent was obtained from all patients, to whom the purpose of the student and amount of blood sampling had been explained. Serum somatomedin assays Sera used for these assays were obtained in the fasting state, prior to morning insulin injections, and frozen at - 2 0 C until use. Somatomedin (sulfation factor) activity was measured by a modification of the method of Hall (8,9) using pelvic rudiments from 11-day old chick embryos incubated in 2 ml of Eagle's basal medium made 0.21 inM in glutamine. Samples were preincubated for 1 h, and then pulsed for 4 h with 35Ssulfate. Following incubation, samples were successively washed with unlabeled 0.2M sodium sulfate and distilled water, blotted dry, and weighed. After overnight digestion with papain,
236
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237
SOMATOMEDIN IN DIABETES TABLE 1. Clinical data in diabetic patients
Patient 1 2 3 4 5 6 7 8 9 10 11 12
Sex M F F F M M M F M M M F
Age 31 26 19 34 34 40 28 27 54 48 56 55
Duration of diabetes (yrs) 3 3 4 5 6 9 12 12 12 15 17 25
MeanFBS* (mg/dl)
Urinary protein Retinopathyf _ — + — — _ + + + + + +
278 283 160 310 291 234 213 200 250 219 130 216
(g/24 h) 0.04 0.13 0.01 0.43 0.02 0.55 7.05 0.02 3.50 0.24 4.54 5.60
* Mean fasting blood sugar taken from review of record throughout patients clinic course; 3-10 fasting blood glucose determinations per year of clinic attendance available for computation. t Retinopathy defined as the presence of microaneurysms and/or exudates on routine funduscopic exam. protein was precipitated with 5% trichloroacetic acid, and the glycosaminoglycans in the supernatant fraction were dialyzed against distilled water. Aliquots were removed for determination of radioactivity by liquid scintillation in 10 ml of Bray's solution (10) and for uronic acid measurement by the carbazole method (11). A symmetrical 4-point design was used in all incubations, with two concentrations each of reference serum (10 and 20%) and of the test serum; pooled normal human serum (NHS) was used as the reference material. Only assays in which the response to test serum was parallel to the reference curve were considered valid; if necessary, assays were repeated with appropriate alterations in the serum concentrations to meet these criteria. The somatomedin activity of NHS was arbitrarily defined as 1.00, and the activity of the test sera was compared to and expressed in terms of this standard according to parallel line analysis (12). The mean index of precision in this assay was .30 ± .08. Results
Serum somatomedin activity and simultaneous blood sugars were determined in four separate samples from each patient. These results are shown in Table 2. The range of normal for somatomedin is 0.8 to 1.2 in our laboratory, determined with triplicate or quadruplicate assays in about 20 nondiabetic patients with a variety of other illnesses that were not of a debilitating nature.
Assay of three different acromegalic sera gave relative potency values of 1.53, 1.95 and 2.51, and those of sera from two hypopituitary patients were 0.63 and 0.71. Mean somatomedin levels were therefore significantly increased in this group of diabetic patients, although basal growth hormone levels in the diabetic samples used for glucose and somatomedin activity were not different from non-diabetic levels. This TABLE 2. Somatomedin levels in diabetic patients Patient
Glucose (mg/dl)*
Somatomedin f
1 2 3 4 5 6 7 8 9 10 11 12
275 213 222 143 176 248 237 158 343 219 122 216
1.60 ± 0.17 0.96 ± 0.15 1.55 ± 0.06 2.09 d: 0.06 0.83 dt0.05 1.52 Ht0.08 2.20 dt0.29 0.89 dt0.08 1.10 2t0.04 1.82 -t0.16 1.36: t0.09 3.21:tO.22
Mean
214
1.60 ± 0.14
* Blood glucose levels are mean of 4 determinations performed on 4 separate samples used for somatomedin assays. f Somatomedin potency of patients' sera expressed in relation to potency of pooled NHS, which was arbitrarily defined as 1.00. Results given as the mean ± SE of 4 separate assays performed in each subject.
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238
JCE & M • 1977 Vol 45 • No 2
COHEN, JASTI AND RYE
finding was confirmed by separately assaying sera from six non-diabetic individuals and calculating potency ratios compared to pooled normal human serum. Mean somatomedin activity was 1.05 ± .10 in this group of healthy house officers and laboratory personnel, while mean serum somatomedin activity in the diabetic group was 1.60 ± .14 (F < .01 normal versus diabetic). There was no correlation between simultaneous blood glucose concentrations and levels of somatomedin activity. No significant differences were demonstrable between somatomedin activity in diabetics with and without retinopathy or in patients with and without proteinuria. Discussion Hyperglycemia in untreated human (6) and experimental diabetes (5) is associated with lowered somatomedin activity. Normalization of blood glucose with insulin in acutely streptozotocin-diabetic rats restores somatomedin activity to control values, and activity is inversely correlated to the blood sugar in treated animals (16). The increased mean somatomedin activity and absence of correlation with the simultaneous blood glucose concentrations found in the present study suggest that the acute and chronic effects of hyperglycemia differ in respect to their influence on somatomedin generation and/or potency. While mean somatomedin activity was low in the group of 22 long-term diabetics with retinopathy studied by Yde (7), pertinent data regarding factors which might affect somatomedin are not given. Detailed information concerning renal function, nutritional status, average blood glucose, and duration, type and treatment of diabetes is not provided for the individual patients in that study. Differences in such clinical factors, as well as in assay techniques (chick embryo versus hypophysectomized rat cartilage) may account for these conflicting results. Evidence that growth hormone secretion or metabolism is altered in juvenile diabetes
is convincing. For example, diurnal and mean 24 h growth hormone levels are higher in diabetics than in control subjects (13,14), and an increased growth hormone response to exercise occurs in diabetics (15). While the role of growth hormone in the pathogenesis of chronic complications of diabetes remains speculative, the present study extends the significance of previously reported abnormalities in growth hormone by estab lishing the existence of changes in the growth-hormone dependent somatomedin in long-term, treated, insulin-dependent diabetes. Acknowledgment The skillful secretarial support of Ms. Joan Quinones is acknowledged.
References 1. Daughaday, W. H., K. Hall, M. S. Raben, W. D, Salmon, J. L. Van den Brande, and J. J. Van Wyk. Somatomedin: Proposed designation for sulfation factor, Nature 235: 107, 1972. 2. Van Wyk, J. J., L. E. Underwood, R. C. Lister, and R. N. Marshall, The somatomedins. A new class oi growth regulating hormones?, AmJ Dis Child 126: 705, 1973. 3. Salmon, W. D., and W. H. Daughaday, A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro, J Lab Clin Med 49: 825, 1957. 4. Salmon, W. D., and M. R. DuVall, A serum fraction with "sulfation factor activity" which stimulates in vitro incorporation of leucine and sulfate into protein-polysaccharide complexes, uridine into RNA, and thymidine into DNA of costal cartilage from hypophysectomized rats, Endocrinology 86: 721, 1970. 5. Phillips, L. S., and H. S. Young: Nutrition and somatomedin. II. Serum somatomedin activity and cartilage growth in streptozotocin-diabetic rats, Diabetes 25: 516, 1976. 6. Van den Brande, J. L., and M. V. L. Du Caju. Plasma somatomedin activity in children with growth disturbances, Adv. Growth Hormone Res., DHEW Publ. no. (NIH) 74-612, U.S. Government Printing Office, 1974, p. 98. 7. Yde, H., The growth hormone dependent sulfation factor in serum from patients with various types of diabetes, Ada Med Scand 186: 293, 1969 8. Hall, K., Quantitative determination of the sulphation factor activity in human sera, Ada Endocrinol (Kbh) 63: 338, 1970.
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SOMATOMEDIN IN DIABETES 9. Audhya, T. K., and K. D. Gibson, Serum inorganic sulfate and apparent somatomedin activity in an assay using chick embryo cartilage, Endocrinology 95: 1614, 1974. 10. Bray, G. A., A simple and efficient liquid scintillator for counting aqueous solutions in a liquid scintillator counter, Anal Biochem 1: 279, 1960. 11. Bitter, T., and H. Muir, A modified uronic acid carbazole reaction, Anal Biochem 4: 330, 1962. 12. Phillips, L. S., A. C. Herington, and W. H. Daughaday, Steroid hormone effects on somatomedin. I Somatomedin action in vitro, Endocrinology 97: 780, 1975. 13. Molnar, G. D., W. F. Taylor, A. Langworthy, and V. Fatourechi, Diurnal growth hormone and glu-
239
cose abnormalities in unstable diabetics: Studies of ambulatory-fed subjects during continuous blood glucose analysis, J Clin Endocrinol Metab 34: 837, 1972. 14. Hansen, A. P., and K. Johansen, Diurnal patterns of blood glucose, serum free fatty acids, insulin, glucagon and growth hormone in normal and juvenile diabetics, Diabetologia 6: 27, 1970. 15. Hansen, A. P., Abnormal serum growth hormone response to exercise in juvenile diabetes, J Clin Invest 49: 1467, 1970 16. Phillips, L. S., and A. T. Orawski, Diabetic control, somatomedin and growth in rats. Diabetes (Suppl 1) 25: 335, 1976 (Abstract).
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