- Email: [email protected]
Glucose, insulin, HGH, and cortisol plasma levels in granuloma annulare patients during glucose tolerance and cortisone glucose tolerance test
Clin. Biochem. 7, 150-160 (1974) GLUCOSE, I N S U L I N , HGH, AND. CORTISOL P L A S M A L E V E L S I N G R A N U L O M A A N N U L A R E P A T I E N T S D U R I N G GLUCOSE T O L E R A N C E A N D C O R T I S O N E GLUCOSE T O L E R A N C E T E S T A. CASTRO, R. HAMMOND and J. FOTTS Papanieolaou Cancer Reseat, oh Institute, Miami, Florida and Departments of Pediatrics and Dermatology, University of Oregon Medical School, Portland, Oregon (Accepted December 18, 197~)
CLBIA, 7 (2): 150-160 (1974) Clin. Biochem. Castro, A., Hammond, R., and Potts, J. Papanicolaou Cancer Research Institute, Miami, Florida and Depa]rtments of Pediatrics and Dermatology, University of Oregon Medical School, Portland, Or6gon, USA GLUCOSE, INSULIN, HGH, AND CORTISOL PLASMA LEVELS IN GRANULOMA ANNULARE PATIENTS DURING GLUCOSE TOLERANCE AND CORTISONE GLUCOSE TOLERANCE TEST Glucose, insulin, human growth hormone and cortisol levels are reported during both a standard glucose tolerance test and a cortisone-glucose tolerance test in eleven Granuloma Annulare patients and five healthy subjects. Although little difference seemed to exist between these two groups during the standard GTT they differed significantly (p~0.01) in their insulin response to glucose during the cortisone GTT. The GA patients also showed a significantly lower HGH level during both the standard GTT and the cortisone GTT. These "high risk" GA patients show a blunted growth hormone profile similar to that found in mildly glucose intolerant subjects reported by other investigators. The significance of these HGH levels in glucose metabolism is discussed. This data indicates that routine study of plasma growth hormone during challenge tests can be extremely useful in evaluating possible glucose intolerant patients.
IN A PREVIOUS REPORT we n o t e d t h a t a h i g h incidence of glucose i n t o l e r a n c e can be d e m o n s t r a t e d in G r a n u l o m a A n n u l a r e ( G A ) p a t i e n t s b y u s i n g m o r e sensitive m e a n s of testing1. W e e m p l o y e d a s t a n d a r d glucose tolerance t e s t as well as a cortisone-glucose t o l e r a n c e test, a n d d e t e r m i n e d both insulin a n d glucose values a n d t h e i r r e l a t i o n s h i p as e x p r e s s e d in t h e r a tio insulin/glucose. I t h a s been r e p o r t e d b y D a n o w s k i et al 2 t h a t u n t r e a t e d diabetics a n d obese individuals d i f f e r f r o m n o n d i a b e t i c n o n o b e s e p e r s o n s in t h a t t h e l a t t e r f r e q u e n t l y develop m e a s u r a b l e i n c r e a s e s in p l a s m a g r o w t h h o r m o n e d u r i n g a glucose load. N u m e r o u s o t h e r r e p o r t s also h a v e s h o w n t h e s e d i f f e r e n c e s 3.4,5,6,7,s. B e c a u s e of t h e possible i m p o r t a n c e of GA as a n indication of c a r b o h y d r a t e i n t o l e r a n c e a n d t h e value of an e a r l y r e c o g n i t i o n of tfiis imbalance, we h a v e i n v e s t i g a t e d cortisol a n d h u m a n g r o w t h h o r m o n e levels in GA patients. A l t h o u g h t h e diabetogenic p r o p e r Correspondence: Dr. Albert Castro, Papanicolaou Cancer Research Institute, 1425 N.W. 10th Ave., Miami, Florida 33136, USA
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ties of both cortico-steroids and growth hormone have been studied previously ~,9,1o they have been related mainly to the treatment of hypoglycemia. Levels of HGH and cortisol during oral glucose tolerance tests in both diabetics and normals have been reported; however, the relationship of these hormone levels in possible glucose intolerant patients with GA are not available. Also, we are not aware of any previous reports rela~ing HGH to cortisal levels in either normals, diabetics or GA patients during a cortisone stimulated glucose tolerance test. Several reports are available which deal with the cortisol-HGH-insulin-glucose relationships during Metyrapone treatment. The effect which Metyrapone has on cortisol levels and subsequently insulin, glucose, and growth hormone levels during a standard glucose tolerance test has stimulated further investigation into the relationship of these four diabetogenic parameters in both normal controis and possible glucose intolerant patients 11, 9. By studying the cortisolHGH-insulin-glucose relationships of GA patients who are high risk for carbohydrate intolerance and five normal control subjects of comparable age during both the standard GTT and contisone GTT, we hope to better establish the relationship of HGH and cortisol before and after steroid glucose tolerance in normal subjects and mildly glucose intolerant patients.
~METHODS Subjects: All 5 subjects in the normal group were healthy, ambulatory, with no family history of diabetes mellitus. Body weight ranged from 100-102% of ideal weight (Metropolitan Life Insurance Table) (mean 101%). Their mean age was 30 years, and the r a n g e was 22 to 54 years. The 11 patients with GA were ambulatory and ranged f r o m 91 to 129% of ideal body weight (mean 103.5%). Only one patient was 1297/o his ideal body weight, with the remaining ten all under 110%. Their mean age was 33 years r a n g i n g from 17 to 62 years. In addition to GA, 8 of the 11 patients were considered "high risk" f o r diabetes due to other reasons, either overweight o r a family history of diabetes; however, none had been previously diagnosed as diabetic or carbohydrate intolerant (Table 1). Clinical features: The group of GA patients consisted of 7 women and 4 men. The duration on the disease ranged from 1 month to 14 years. Of the 11 patients, 10 had lesions on the extremities and 1 had lesions on both t r u n k and extremities. Most of the lesions were flesh-colored or erythmatous papules in an annular arrangement. The diagnosis was verified by biopsy in all cases. The glucose dose was 100 gm in each test. The two challenge tests were performed in intervals exceeding two weeks to assure t h a t each test was not affected by the preceding one. The oral glucose tolerance test and the steroid glucose tolerance test were done in the early afternoon 12. 13. Considerable care was taken to employ rigorously standardized conditions including: (a) pretest preparation with a normal caloric diet containing more than 300 gm carbohydrate for 3 days preceding each test, (b) prohibition of
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any medication for one month preceding the test, (c) at least a 12 hour fasting period before each test during which smoking was prohibited, (d) administration of 50 mg of cortisone acetate orally 8V2 and 2 hours before glucose ingestion for the cortisone glucose tolerance test, (e) at least 1 hour of rest in a recumbent position previous to each test, (f) ingestion of glucose during a 5 minute period in the semi-recumbent position with the subject confined to bed TM in the recumbent position for the remainder of the test, (g) constant load volume, e.g., 500 ml iced water containing the glucose and 3 gm citric acid for flavoring, (h) sampling periods of 15 to 30 minutes with an intravenous cannula to minimize trauma. Blood was drawn before glucose ingestion and 15, 30, 45, 60, 90, 120, 150, and 180 minutes thereafter. Serum sodium and potassium concentrations and packed cell volume were determined on all subjects at the end of each test and were within normal limits. Assay methods: Venous blood was collected in a tube containing 10 U of heparin per ml of blood. Plasma was immediately separated and divided into 2 portions, 1 of which was frozen until insulin, growth hormone, and cortisol assays could be performed. The other was analyzed promptly for glucose. Glucose was determined with the Technicon Analyzer by a ferricyanide reduction method. A sample of plasma glucose pool, which was kept frozen in small aliquots, was incorporated into each experimental run as a check on the variability of the glucose estimations; the results of the plasma pool did not vary by more than 2 mg per 100 ml during the study. Plasma insulin and human growth hormone concentration were assayed using the simultaneous assay technique of Morgan and Lazarow 15. The 1-131-bovine insulin and 1-125 human growth hormone were obtained from Abbott Laboratories, and its purity was verified before use by radioactive scanning of paper strips after electrophoresis of samples in veronal buffer and chromatography in an ethanol-water system. As a check on the variability of the radioimmunoassay, a standard plasma pool sample was assayed with each run o~. unknown plasma insulin, growth hormone samples. Constant values were obtained throughout the study (32 uU/ml, S.D. ~ 3.0 uU/ml for insulin and 3.8 mug/rag, S.D. ---- ----- 0.8 mug/ml for HGH). The human insulin standards were kindly donated by Dr. Mary Root, Eli Lilly & Co. The competitive protein-binding radioassay of B. E. Murphy TM was used in determining plasma cortisol levels. Again a plasma pool was incorporated into each run and constant values were obtair~ed throughout the study (8.5 ug%, S. D. ± 0.9 ug% cortisol). Statistical interpretations of data: We employed the correlation coefficient and the t-test for paired comparisons in analyzing the different responses of the high risk GA patients and the healthy subjects with respect to their glucose, insulin, cortisol and human growth hormone levels during both the standard GTT and the cortisone-GTT.
RESULTS In observing insulin/glucose ratios during the standard oral GTT we f o u n d no s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e h i g h r i s k GA p a t i e n t s a n d the healthy subjects. However, the cortisone GTT results showed a s i g n i f i c a n t l y l o w e r insulin r e s p o n s e to glucose levels ( p < 0 . 0 1 ) in G A p a t i e n t s as c o m p a r e d t o t h e h e a l t h y p a t i e n t s . I n s u l i n r e s p o n s e s t o g l u c o s e levels a r e s h o w n in F i g . 1. A v e r a g e g l u c o s e , i n s u l i n , g r o w t h h o r m o n e , a n d c o r t i s o l l evel s f o r b o t h g r o u p s a r e g i v e n i n T a b l e 2. U s i n g a t - t e s t f o r p a i r e d c o m p a r i s o n s w e compared the two groups with respect to the above four variables at each
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time of sampling. During the GTT the GA patients gave significantly higher (p<0.001) glucose values than the healthy patients. However, these elevated glucose levels were accompanied by significantly higher (p<0.05) insulin levels in the GA high risk group. The glucose levels of the high risk group were again greatly exaggerated (p<0.01) in comparison to the healthy subjects during the cortisone GTT. But during this challenge the insulin was not enhanced significantly to overcome the added stress. When the two challenge tests in each of these groups were considered separately we found a significant rise in insulin levels in the control subjects during the C-GTT as compared to the GTT with no significant rise in their glucose values. The high risk GA patients showed significantly higher glucose (p<.01) levels during cortisone GTT and no significant rise in insulin responses. During the standard GTT the high risk GA patients had higher (p< 0.001) cortisol levels and lower HGH values than the healthy patients. After cortisone acetate ingestion and another glucose challenge the high risk GA patients continued to demonstrate lower HGH levels (p<0.05) than the healthy patients and also exhibited lower circulating plasma cortisol levels (p<0.02) after the same amount of cortisone acetate had been ingested (Fig. 2). When correlating the four parameters - - glucose, insulin, HGH, cortisol - - we found significant correlation between glucose and insulin values during both the GTT and C-GTT in the GA patients as well as the normals. HGH and cortisol levels were significantly correlated only during the C-GTT in the normal controls. During the standard GTT glucose vs cortisol levels and the insulin vs HGH levels were not correlated in the normals but were correlated in the GA patient group. DISCUSSION There has long been an interest regarding the relationship between glucose, insulin, cortisol and human growth hormone in diabetics as well as in mildly glucose intolerant subjects. Sonksen et al s recently reported the relationship between these variables to be quite different in normal control patients than in diabetic patients during slow continuous infusions of monocomponent human insulin. While in the controls serum HGH and cortisol rose in direct proportion to the degree of hypoglycemia achieved, the diabetic patients showed a striking rise in HGH and cortisol within 30 minutes of the start of the insulin infusion: i.e. the HGH and cortisol responses did not seem to be related to a fall in plasma glucose but to be directly related to the administration of insulin. Fatourechi et al lr through constant monitoring revealed in insulin treated diabetics that circulating levels of growth hormone can increase without a fall in the
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blood sugar. Sabeh et al7 reported a blunted g r o w t h h o r m o n e response following an oral glucose load in both patients with mild carbohydrate intolerance and obese patients displaying no carbohydrate intolerance. They found that the combination of obesity and mild glucose intolerance was not associated with decreases in growth hormone beyond those observed with either alone. Our data also shows a blunted growth hormone profile similar to that found in mild glucose intolerant subjects, in our granuloma annulare patients when compared to our normal control subjects. The normal control subjects responded with greater terminal growth hormone levels during the cortisone GTT; however, terminal differences in HGH levels during the standard GTT between the GA patients and controls were not apparent. Hunter et al25 postulated that the terminal rise in HGH secretion following glucose loading is related not so much to the magnitude or rapidity of the reduction in glycemia but to the value to which it has fallen, relating to the concept of tissue glucose assimilation threshold values. In our normal controls the higher terminal HGH values during the C-GTT may be related to the increased insulin responses observed in relation to glucose levels. If insulin is the link promoting the entry of glucose into tissues our results are in agreement with Hunter's hypothesis. Baird et al, in studying the relationship between human growth hormone and the development of clinical diabetes, found different responses in this hormone level seemingly associated with the severity of the disease 1s. They reported as others n. ~, 2 low HGH responses in the obese and mildly glucose intolerant subjects during a standard GTT. However, in diabetics ketotic at the time of the study they reported high HGH levels. These high HGH levels were also present in ~liabetic patients receiving t r e a t m e n t with high protein diet, oral hypoglycemic agent, or exogenous insulin. From this information it seems that HGH regulated the utilizational intracellular glucose and by this means alters the sensitivity to insulin. Our high risk GA patients seem to be representative of the mildly glucose intolerant patients reported by both Baird et al and Hales et a/1 s. 5 It is currently believed that HGH is secreted in normal adults when there is a metabolic need for mobilization of stored fat which is consistent with the hypothesis that a stimulus to this secretion is intracellular glucose deprivation as in the severe, possibly ketotic diabetic. However, in the mildly glucose intolerant patient the pancreatic overreaction to the glucose load is still successful in avoiding any intracellular glucose deprivation and mobilization of stored fat is not necessary; therefore, growth hormone levels remain low. De Moor and Heyns ~9 reported diabetics to have a higher mean cortisol binding capacity (CBC) of plasma transcortin than normal control subjects. We did not measure CBC levels in our patients, however, these levels might prove to be very interesting during the C-GTT.
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The relationship of cortisol levels and diabetes is not well established; it has, however, been shown that by diminishing endogenous production and exogenous administration of cortisol at the proper time, it is possible to revert an abnormal GTT response to normal 12. It has been hypothesized that resulting increased insulin production is actually secondary to an improvement of the beta cells of the pancreas following a decrease in cortisol levels 2°, 21. It has also been reported" that a decrease in blood cortisol levels indirectly decrease ACTH production, which in turn initiates a rise in blood insulin levels normalizing diabetic GTT responses. In the eleven GA patients as well as the normal controls there was an increase in terminal HGH levels during the cortisone GTT as compared to the standard GTT, possibly accentuated by an increased insulin response to glucose caused by the cortisone acetate. However, this increase was much more pronounced in the normal subjects than in the controls and seemed to relate well to the prolonged cortisol levels in these subjects. The normal control subjects had definitely higher cortisol levels at the beginning of the C-GTT and a decrease in levels to about 2/3 of the original level. This decrease in cortisol in the normals began at about one hour and seemed to relate well with the increase in HGH response. This relationship was not as pronounced in the GA patients (Fig. 2). The initial decrease in HGH upon ingesting the glucose load in all subjects studied is in keeping with the generally recognized reduction of the normally high fasting human growth hormone levels. In interpreting our findings we kept in mind the variety of stimuli such as prolonged starvation, stress, exercise, etc., that can produce increases in plasma growth hormone in man. We require the subjects to rest one hour before beginning each test, and to remain recumbent during the test in order to avoid unreliable results. Our data indicates that routine study of plasma growth hormone association with both a glucose tolerance test and a cortisone glucose tolerance test can be used to f u r t h e r accentuate the differences between normal control subjects and possible glucose intolerant patients. Our findings are in agreement with the general hypothesis that the plasma growth hormone response to "relative" hypoglycemia may be blunted in mildly glucose intolerant patients (GA) or diabetics. ACKNOWLEDGEMENTS
Research support was provided by grants from the Oregon Heart Association, The Diabetic Research Foundation of Good Samaritan Hospital, Portland, Oregon and by a PHS training g r a n t 5T1 AM-5300 from the National Institute of Arthritis and Metabolic Diseases, U.S. Department
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of Health, Education and Welfare. The authors wish to thank Mrs. Kirby Dyess and S. Bradley for their technical assistance. We also wish to thank the National Pituitary Agency for supplying the material for the radioimmuno-assay of HGH.
REFERENCES 1. Hammond, R., Dyess, K. and Castro, A. (1972). Brit. J. D e n . 87, 540-547. 2. Danowski, T. S., Mintz, D. H., Finster, J., Sabeh, G. and Morgan, C. R. (1968). Ann. N . Y . Acad. Sci. 148, 796-807. 3. Hunter, W. M., Clarke, B. F. and Duncan, J. P. (1966). Metabolism 15, 596-607. 4. Mitchell, M. L., Raben, M. S. and Ernest, M. (1970). Diabetes 19, 196-199. 5. Hales, C. N., Greenwood, F. C., Mitchell, F. L. and Strauss, W. T. (1968). Diabetologia 4, 73-82. 6. Sabeh, G., et al (1968). Metabolism 18, 73-~2. 7. Sabeh, G., Corredor, D. G., Mendelsohn, L. V., Morgan, C. R., Sieracki, J. C., Sunder, J. H., Wingert, J. P. and Danowski, T. S. (1969). Metabolism 13, 741-747. 8. Sanksen, P. H., Tompkins, C. V., Srivastava, M. D. and Nabarro, J. D. N. (1972). Lancet 155-159, July 22. 9. Bruno, 0. D., Leclercq, R., Virasoro, E. and Copinschi, G. (1971). J. Clin. Endocr. 32, 260-265. 10. Meek, J. C., Reitz, R. P. and Bolinger, R . . (1970). Proc. Soc. Exp. Biol. Med. 135, 123-126. 11. Grinberg, Rauland, Mazar and Stephen (1970). N.Y. State J. Med. 70, 2341-2343. 12. Robert, J. D. (1964). J. Am. Ge~iat. Soe. 12, 423-434. 13. Toyota, T. and Goto, Y. (1969). Tohokii J. Exp. Med. 9, 309-313. 14. Castro, A., Scott, J., Grettie, D., MacFarlane, D. and Bailey, R. (1970). Diabetes 19, 843-851. 15. Morgan, C. and Lazarow, A. (1962). Proc. Soe. Exp. Biol. Med. 110, 29-32. 16. Murphy, B. E. P. (1967). J. Clin. Endoer. 27, 973.090. 17. Fataureehi, V., Molnar, G. D., Service, F. J., Ackerman, E., Rosevear, J. W., Moxness, K. E. and Taylor, W. F. (1969). J. Clin. Endocr. 29, 319-328. 18. Baird, J. D., Hunter, W. M. and Smith, A. W. (1969). J. Endocr. 43, 13-14. 19. De Moor, P., and Heyns, W. (1967). J. Clin. Endocr. 27, 706-708. 20. Barr, H. S. and Wolff, O. H. (1957). Lancet 1, 812-813. 21. Carona, F. A. and Liebow, A. A. (1957). N e w Eng. J. Med. 257, 690-701.
CLBIA, 7 (2): 150-160 (1974) Clin. Biochem. Castro, A., Hammond, R., and Potts, J. Papanicolaou Cancer Research Institute, Miami, Florida and Depa]rtments of Pediatrics and Dermatology, University of Oregon Medical School, Portland, Or6gon, USA GLUCOSE, INSULIN, HGH, AND CORTISOL PLASMA LEVELS IN GRANULOMA ANNULARE PATIENTS DURING GLUCOSE TOLERANCE AND CORTISONE GLUCOSE TOLERANCE TEST Glucose, insulin, human growth hormone and cortisol levels are reported during both a standard glucose tolerance test and a cortisone-glucose tolerance test in eleven Granuloma Annulare patients and five healthy subjects. Although little difference seemed to exist between these two groups during the standard GTT they differed significantly (p~0.01) in their insulin response to glucose during the cortisone GTT. The GA patients also showed a significantly lower HGH level during both the standard GTT and the cortisone GTT. These "high risk" GA patients show a blunted growth hormone profile similar to that found in mildly glucose intolerant subjects reported by other investigators. The significance of these HGH levels in glucose metabolism is discussed. This data indicates that routine study of plasma growth hormone during challenge tests can be extremely useful in evaluating possible glucose intolerant patients.
IN A PREVIOUS REPORT we n o t e d t h a t a h i g h incidence of glucose i n t o l e r a n c e can be d e m o n s t r a t e d in G r a n u l o m a A n n u l a r e ( G A ) p a t i e n t s b y u s i n g m o r e sensitive m e a n s of testing1. W e e m p l o y e d a s t a n d a r d glucose tolerance t e s t as well as a cortisone-glucose t o l e r a n c e test, a n d d e t e r m i n e d both insulin a n d glucose values a n d t h e i r r e l a t i o n s h i p as e x p r e s s e d in t h e r a tio insulin/glucose. I t h a s been r e p o r t e d b y D a n o w s k i et al 2 t h a t u n t r e a t e d diabetics a n d obese individuals d i f f e r f r o m n o n d i a b e t i c n o n o b e s e p e r s o n s in t h a t t h e l a t t e r f r e q u e n t l y develop m e a s u r a b l e i n c r e a s e s in p l a s m a g r o w t h h o r m o n e d u r i n g a glucose load. N u m e r o u s o t h e r r e p o r t s also h a v e s h o w n t h e s e d i f f e r e n c e s 3.4,5,6,7,s. B e c a u s e of t h e possible i m p o r t a n c e of GA as a n indication of c a r b o h y d r a t e i n t o l e r a n c e a n d t h e value of an e a r l y r e c o g n i t i o n of tfiis imbalance, we h a v e i n v e s t i g a t e d cortisol a n d h u m a n g r o w t h h o r m o n e levels in GA patients. A l t h o u g h t h e diabetogenic p r o p e r Correspondence: Dr. Albert Castro, Papanicolaou Cancer Research Institute, 1425 N.W. 10th Ave., Miami, Florida 33136, USA
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ties of both cortico-steroids and growth hormone have been studied previously ~,9,1o they have been related mainly to the treatment of hypoglycemia. Levels of HGH and cortisol during oral glucose tolerance tests in both diabetics and normals have been reported; however, the relationship of these hormone levels in possible glucose intolerant patients with GA are not available. Also, we are not aware of any previous reports rela~ing HGH to cortisal levels in either normals, diabetics or GA patients during a cortisone stimulated glucose tolerance test. Several reports are available which deal with the cortisol-HGH-insulin-glucose relationships during Metyrapone treatment. The effect which Metyrapone has on cortisol levels and subsequently insulin, glucose, and growth hormone levels during a standard glucose tolerance test has stimulated further investigation into the relationship of these four diabetogenic parameters in both normal controis and possible glucose intolerant patients 11, 9. By studying the cortisolHGH-insulin-glucose relationships of GA patients who are high risk for carbohydrate intolerance and five normal control subjects of comparable age during both the standard GTT and contisone GTT, we hope to better establish the relationship of HGH and cortisol before and after steroid glucose tolerance in normal subjects and mildly glucose intolerant patients.
~METHODS Subjects: All 5 subjects in the normal group were healthy, ambulatory, with no family history of diabetes mellitus. Body weight ranged from 100-102% of ideal weight (Metropolitan Life Insurance Table) (mean 101%). Their mean age was 30 years, and the r a n g e was 22 to 54 years. The 11 patients with GA were ambulatory and ranged f r o m 91 to 129% of ideal body weight (mean 103.5%). Only one patient was 1297/o his ideal body weight, with the remaining ten all under 110%. Their mean age was 33 years r a n g i n g from 17 to 62 years. In addition to GA, 8 of the 11 patients were considered "high risk" f o r diabetes due to other reasons, either overweight o r a family history of diabetes; however, none had been previously diagnosed as diabetic or carbohydrate intolerant (Table 1). Clinical features: The group of GA patients consisted of 7 women and 4 men. The duration on the disease ranged from 1 month to 14 years. Of the 11 patients, 10 had lesions on the extremities and 1 had lesions on both t r u n k and extremities. Most of the lesions were flesh-colored or erythmatous papules in an annular arrangement. The diagnosis was verified by biopsy in all cases. The glucose dose was 100 gm in each test. The two challenge tests were performed in intervals exceeding two weeks to assure t h a t each test was not affected by the preceding one. The oral glucose tolerance test and the steroid glucose tolerance test were done in the early afternoon 12. 13. Considerable care was taken to employ rigorously standardized conditions including: (a) pretest preparation with a normal caloric diet containing more than 300 gm carbohydrate for 3 days preceding each test, (b) prohibition of
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any medication for one month preceding the test, (c) at least a 12 hour fasting period before each test during which smoking was prohibited, (d) administration of 50 mg of cortisone acetate orally 8V2 and 2 hours before glucose ingestion for the cortisone glucose tolerance test, (e) at least 1 hour of rest in a recumbent position previous to each test, (f) ingestion of glucose during a 5 minute period in the semi-recumbent position with the subject confined to bed TM in the recumbent position for the remainder of the test, (g) constant load volume, e.g., 500 ml iced water containing the glucose and 3 gm citric acid for flavoring, (h) sampling periods of 15 to 30 minutes with an intravenous cannula to minimize trauma. Blood was drawn before glucose ingestion and 15, 30, 45, 60, 90, 120, 150, and 180 minutes thereafter. Serum sodium and potassium concentrations and packed cell volume were determined on all subjects at the end of each test and were within normal limits. Assay methods: Venous blood was collected in a tube containing 10 U of heparin per ml of blood. Plasma was immediately separated and divided into 2 portions, 1 of which was frozen until insulin, growth hormone, and cortisol assays could be performed. The other was analyzed promptly for glucose. Glucose was determined with the Technicon Analyzer by a ferricyanide reduction method. A sample of plasma glucose pool, which was kept frozen in small aliquots, was incorporated into each experimental run as a check on the variability of the glucose estimations; the results of the plasma pool did not vary by more than 2 mg per 100 ml during the study. Plasma insulin and human growth hormone concentration were assayed using the simultaneous assay technique of Morgan and Lazarow 15. The 1-131-bovine insulin and 1-125 human growth hormone were obtained from Abbott Laboratories, and its purity was verified before use by radioactive scanning of paper strips after electrophoresis of samples in veronal buffer and chromatography in an ethanol-water system. As a check on the variability of the radioimmunoassay, a standard plasma pool sample was assayed with each run o~. unknown plasma insulin, growth hormone samples. Constant values were obtained throughout the study (32 uU/ml, S.D. ~ 3.0 uU/ml for insulin and 3.8 mug/rag, S.D. ---- ----- 0.8 mug/ml for HGH). The human insulin standards were kindly donated by Dr. Mary Root, Eli Lilly & Co. The competitive protein-binding radioassay of B. E. Murphy TM was used in determining plasma cortisol levels. Again a plasma pool was incorporated into each run and constant values were obtair~ed throughout the study (8.5 ug%, S. D. ± 0.9 ug% cortisol). Statistical interpretations of data: We employed the correlation coefficient and the t-test for paired comparisons in analyzing the different responses of the high risk GA patients and the healthy subjects with respect to their glucose, insulin, cortisol and human growth hormone levels during both the standard GTT and the cortisone-GTT.
RESULTS In observing insulin/glucose ratios during the standard oral GTT we f o u n d no s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e h i g h r i s k GA p a t i e n t s a n d the healthy subjects. However, the cortisone GTT results showed a s i g n i f i c a n t l y l o w e r insulin r e s p o n s e to glucose levels ( p < 0 . 0 1 ) in G A p a t i e n t s as c o m p a r e d t o t h e h e a l t h y p a t i e n t s . I n s u l i n r e s p o n s e s t o g l u c o s e levels a r e s h o w n in F i g . 1. A v e r a g e g l u c o s e , i n s u l i n , g r o w t h h o r m o n e , a n d c o r t i s o l l evel s f o r b o t h g r o u p s a r e g i v e n i n T a b l e 2. U s i n g a t - t e s t f o r p a i r e d c o m p a r i s o n s w e compared the two groups with respect to the above four variables at each
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time of sampling. During the GTT the GA patients gave significantly higher (p<0.001) glucose values than the healthy patients. However, these elevated glucose levels were accompanied by significantly higher (p<0.05) insulin levels in the GA high risk group. The glucose levels of the high risk group were again greatly exaggerated (p<0.01) in comparison to the healthy subjects during the cortisone GTT. But during this challenge the insulin was not enhanced significantly to overcome the added stress. When the two challenge tests in each of these groups were considered separately we found a significant rise in insulin levels in the control subjects during the C-GTT as compared to the GTT with no significant rise in their glucose values. The high risk GA patients showed significantly higher glucose (p<.01) levels during cortisone GTT and no significant rise in insulin responses. During the standard GTT the high risk GA patients had higher (p< 0.001) cortisol levels and lower HGH values than the healthy patients. After cortisone acetate ingestion and another glucose challenge the high risk GA patients continued to demonstrate lower HGH levels (p<0.05) than the healthy patients and also exhibited lower circulating plasma cortisol levels (p<0.02) after the same amount of cortisone acetate had been ingested (Fig. 2). When correlating the four parameters - - glucose, insulin, HGH, cortisol - - we found significant correlation between glucose and insulin values during both the GTT and C-GTT in the GA patients as well as the normals. HGH and cortisol levels were significantly correlated only during the C-GTT in the normal controls. During the standard GTT glucose vs cortisol levels and the insulin vs HGH levels were not correlated in the normals but were correlated in the GA patient group. DISCUSSION There has long been an interest regarding the relationship between glucose, insulin, cortisol and human growth hormone in diabetics as well as in mildly glucose intolerant subjects. Sonksen et al s recently reported the relationship between these variables to be quite different in normal control patients than in diabetic patients during slow continuous infusions of monocomponent human insulin. While in the controls serum HGH and cortisol rose in direct proportion to the degree of hypoglycemia achieved, the diabetic patients showed a striking rise in HGH and cortisol within 30 minutes of the start of the insulin infusion: i.e. the HGH and cortisol responses did not seem to be related to a fall in plasma glucose but to be directly related to the administration of insulin. Fatourechi et al lr through constant monitoring revealed in insulin treated diabetics that circulating levels of growth hormone can increase without a fall in the
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blood sugar. Sabeh et al7 reported a blunted g r o w t h h o r m o n e response following an oral glucose load in both patients with mild carbohydrate intolerance and obese patients displaying no carbohydrate intolerance. They found that the combination of obesity and mild glucose intolerance was not associated with decreases in growth hormone beyond those observed with either alone. Our data also shows a blunted growth hormone profile similar to that found in mild glucose intolerant subjects, in our granuloma annulare patients when compared to our normal control subjects. The normal control subjects responded with greater terminal growth hormone levels during the cortisone GTT; however, terminal differences in HGH levels during the standard GTT between the GA patients and controls were not apparent. Hunter et al25 postulated that the terminal rise in HGH secretion following glucose loading is related not so much to the magnitude or rapidity of the reduction in glycemia but to the value to which it has fallen, relating to the concept of tissue glucose assimilation threshold values. In our normal controls the higher terminal HGH values during the C-GTT may be related to the increased insulin responses observed in relation to glucose levels. If insulin is the link promoting the entry of glucose into tissues our results are in agreement with Hunter's hypothesis. Baird et al, in studying the relationship between human growth hormone and the development of clinical diabetes, found different responses in this hormone level seemingly associated with the severity of the disease 1s. They reported as others n. ~, 2 low HGH responses in the obese and mildly glucose intolerant subjects during a standard GTT. However, in diabetics ketotic at the time of the study they reported high HGH levels. These high HGH levels were also present in ~liabetic patients receiving t r e a t m e n t with high protein diet, oral hypoglycemic agent, or exogenous insulin. From this information it seems that HGH regulated the utilizational intracellular glucose and by this means alters the sensitivity to insulin. Our high risk GA patients seem to be representative of the mildly glucose intolerant patients reported by both Baird et al and Hales et a/1 s. 5 It is currently believed that HGH is secreted in normal adults when there is a metabolic need for mobilization of stored fat which is consistent with the hypothesis that a stimulus to this secretion is intracellular glucose deprivation as in the severe, possibly ketotic diabetic. However, in the mildly glucose intolerant patient the pancreatic overreaction to the glucose load is still successful in avoiding any intracellular glucose deprivation and mobilization of stored fat is not necessary; therefore, growth hormone levels remain low. De Moor and Heyns ~9 reported diabetics to have a higher mean cortisol binding capacity (CBC) of plasma transcortin than normal control subjects. We did not measure CBC levels in our patients, however, these levels might prove to be very interesting during the C-GTT.
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159
The relationship of cortisol levels and diabetes is not well established; it has, however, been shown that by diminishing endogenous production and exogenous administration of cortisol at the proper time, it is possible to revert an abnormal GTT response to normal 12. It has been hypothesized that resulting increased insulin production is actually secondary to an improvement of the beta cells of the pancreas following a decrease in cortisol levels 2°, 21. It has also been reported" that a decrease in blood cortisol levels indirectly decrease ACTH production, which in turn initiates a rise in blood insulin levels normalizing diabetic GTT responses. In the eleven GA patients as well as the normal controls there was an increase in terminal HGH levels during the cortisone GTT as compared to the standard GTT, possibly accentuated by an increased insulin response to glucose caused by the cortisone acetate. However, this increase was much more pronounced in the normal subjects than in the controls and seemed to relate well to the prolonged cortisol levels in these subjects. The normal control subjects had definitely higher cortisol levels at the beginning of the C-GTT and a decrease in levels to about 2/3 of the original level. This decrease in cortisol in the normals began at about one hour and seemed to relate well with the increase in HGH response. This relationship was not as pronounced in the GA patients (Fig. 2). The initial decrease in HGH upon ingesting the glucose load in all subjects studied is in keeping with the generally recognized reduction of the normally high fasting human growth hormone levels. In interpreting our findings we kept in mind the variety of stimuli such as prolonged starvation, stress, exercise, etc., that can produce increases in plasma growth hormone in man. We require the subjects to rest one hour before beginning each test, and to remain recumbent during the test in order to avoid unreliable results. Our data indicates that routine study of plasma growth hormone association with both a glucose tolerance test and a cortisone glucose tolerance test can be used to f u r t h e r accentuate the differences between normal control subjects and possible glucose intolerant patients. Our findings are in agreement with the general hypothesis that the plasma growth hormone response to "relative" hypoglycemia may be blunted in mildly glucose intolerant patients (GA) or diabetics. ACKNOWLEDGEMENTS
Research support was provided by grants from the Oregon Heart Association, The Diabetic Research Foundation of Good Samaritan Hospital, Portland, Oregon and by a PHS training g r a n t 5T1 AM-5300 from the National Institute of Arthritis and Metabolic Diseases, U.S. Department
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of Health, Education and Welfare. The authors wish to thank Mrs. Kirby Dyess and S. Bradley for their technical assistance. We also wish to thank the National Pituitary Agency for supplying the material for the radioimmuno-assay of HGH.
REFERENCES 1. Hammond, R., Dyess, K. and Castro, A. (1972). Brit. J. D e n . 87, 540-547. 2. Danowski, T. S., Mintz, D. H., Finster, J., Sabeh, G. and Morgan, C. R. (1968). Ann. N . Y . Acad. Sci. 148, 796-807. 3. Hunter, W. M., Clarke, B. F. and Duncan, J. P. (1966). Metabolism 15, 596-607. 4. Mitchell, M. L., Raben, M. S. and Ernest, M. (1970). Diabetes 19, 196-199. 5. Hales, C. N., Greenwood, F. C., Mitchell, F. L. and Strauss, W. T. (1968). Diabetologia 4, 73-82. 6. Sabeh, G., et al (1968). Metabolism 18, 73-~2. 7. Sabeh, G., Corredor, D. G., Mendelsohn, L. V., Morgan, C. R., Sieracki, J. C., Sunder, J. H., Wingert, J. P. and Danowski, T. S. (1969). Metabolism 13, 741-747. 8. Sanksen, P. H., Tompkins, C. V., Srivastava, M. D. and Nabarro, J. D. N. (1972). Lancet 155-159, July 22. 9. Bruno, 0. D., Leclercq, R., Virasoro, E. and Copinschi, G. (1971). J. Clin. Endocr. 32, 260-265. 10. Meek, J. C., Reitz, R. P. and Bolinger, R . . (1970). Proc. Soc. Exp. Biol. Med. 135, 123-126. 11. Grinberg, Rauland, Mazar and Stephen (1970). N.Y. State J. Med. 70, 2341-2343. 12. Robert, J. D. (1964). J. Am. Ge~iat. Soe. 12, 423-434. 13. Toyota, T. and Goto, Y. (1969). Tohokii J. Exp. Med. 9, 309-313. 14. Castro, A., Scott, J., Grettie, D., MacFarlane, D. and Bailey, R. (1970). Diabetes 19, 843-851. 15. Morgan, C. and Lazarow, A. (1962). Proc. Soe. Exp. Biol. Med. 110, 29-32. 16. Murphy, B. E. P. (1967). J. Clin. Endoer. 27, 973.090. 17. Fataureehi, V., Molnar, G. D., Service, F. J., Ackerman, E., Rosevear, J. W., Moxness, K. E. and Taylor, W. F. (1969). J. Clin. Endocr. 29, 319-328. 18. Baird, J. D., Hunter, W. M. and Smith, A. W. (1969). J. Endocr. 43, 13-14. 19. De Moor, P., and Heyns, W. (1967). J. Clin. Endocr. 27, 706-708. 20. Barr, H. S. and Wolff, O. H. (1957). Lancet 1, 812-813. 21. Carona, F. A. and Liebow, A. A. (1957). N e w Eng. J. Med. 257, 690-701.