Effect of 12 months of recombinant human growth hormone replacement therapy on insulin sensitivity in GH-deficient adults as determined by different methods.D Micic, G Cvijovic, M Doknic, A Kendereski, V Popovic
Institute of Endocrinology, Diabetes and Diseases of Metabolism, Clinical Center of Serbia, Dr Subotica 13, 11000 Beograd, Yugoslavia. , Yugoslavia
Correspondence Address: Source of Support: None, Conflict of Interest: None PMID: 12571379
Source of Support: None, Conflict of Interest: None
BACKGROUND: Controversial results have been obtained in measuring insulin sensitivity (S(I)) during recombinant human growth hormone (rhGH) treatment in adult growth hormone deficient (GH-deficient) patients. AIMS: The aim of our study was to estimate S(I) before and during treatment using three different methods for quantifying insulin sensitivity in GH-deficient adults treated with rhGH. SETTINGS AND DESIGN: Twenty-one GH-deficient adults were treated with rhGH during 12 months. S(I) was estimated using Minimal model analysis, Homeostatic Model of Assessment (HOMA) and Quantitative Insulin Sensitivity Check Index (QUICKI) before and after 3, 6, 9 and 12 months of rhGH therapy. MATERIAL AND METHODS: Oral Glucose Tolerance Test (OGTT) and Frequently Sampled Intravenous Glucose Tolerance Test (FSIGT) were performed in each patient at respective time intervals. QUICKI and HOMA were calculated using basal values of glucose and insulin from FSIGT. Minimal model computer analysis was calculated from glucose and insulin data obtained during FSIGT. STATISTICAL ANALYSIS: Area under the curve for glucose, insulin and C-peptide were calculated using trapezoidal rule from OGTT data. Differences and correlations were tested using ANOVA for repeated measures, Wilcoxon's matched-paired test, paired t-test, Pearson's correlation and Bland Altman plot. RESULTS: There were no significant changes in S(I) using Minimal model analysis and QUICKI during rhGH treatment. On the contrary, HOMA analysis indicated significant deterioration in S(I) after 12 months of therapy. CONCLUSION: Our study did not demonstrate any changes in S(I) using Minimal model and QUICKI analysis, while there was significant increase in insulin resistance using HOMA model. We suggest that the choice of method for the determination of S(I) may influence the interpretation of results concerning the effect of rhGH therapy on S(I) in GH-deficient adults.
Keywords: Adult, Comparative Study, Female, Glucose Tolerance Test, Growth Disorders, drug therapy,physiopathology,Growth Hormone, pharmacology,Human, Insulin Resistance, physiology,Male, Radioimmunoassay, Support, Non-U.S. Gov′t,
There are contrasting reports concerning the effect of recombinant human growth hormone (rhGH) replacement therapy on glucose metabolism. Some studies have reported obvious deterioration of insulin sensitivity (SI) with concomitant elevation of basal and stimulated glucose and insulin levels., Changes in the kinetics of insulin and C-peptide have also been noted. There are others who have not found any significant change in SI in comparison with pretreatment values.,  Different methods used for the estimation of SI and variable pretreatment characteristics of GH-deficient patients that were included in the studies could account for these differences.
Euglycaemic hyper-insulinaemic clamp is considered as the “gold standard” for quantifying SI in vivo, but this method has procedure-related practical limitations in clinical investigations. Introduction of Minimal model analysis, Homeostatic Model of Assessment (HOMA) and Quantitative Insulin Sensitivity Check Index (QUICKI) for determination of SI and/or insulin resistance permitted the investigation of larger number of patients. Relatively simple investigational procedure is the chief attribute of these methods. It was recently shown that measures of SI is inversely related to resistance and it was suggested that correlations should be examined among sensitivities or among resistance measurements. The most straightforward comparison between a different index of SI and the HOMA index, which is a measure of insulin resistance, is obtained by first inverting it so that it is also expressed as a sensitivity.
Therefore, we decided to evaluate the effect of rhGH replacement therapy on peripheral SI and/or insulin resistance during the twelve months course of rhGH therapy in a group of adult patients with hypopituitarism using three different methods for its determination - Minimal model analysis (MINMOD), QUICKI and HOMA.
Twenty-one GH-deficient patients participated in 12 months rhGH (Norditropin, Novo Nordisk A/S) replacement therapy study. GH-deficiency was defined as GH peak level being less than 3 ?g/l after insulin-induced hypoglycaemia. All study subjects were adequately treated with hydrocortisone, testosterone and/or L-thyroxin, if necessary, for at least one year before initiation of rhGH therapy. The local Ethics committee approved the study, and all patients were informed about the possible side effects and their consent was obtained prior to enrolment. The dose titration for rhGH was adjusted in order to achieve the mean value for normal IGF-I range (normal: 15 – 35 nmol/l). Mean rhGH dose was 0.75 IU/day for men and 1.25 IU/day for women; mean IGF-I during the therapy was 26.8±3.2 nmol/l. In two female patients the dose of rhGH was reduced to 1 IU/day due to the side effects of therapy. A control group consisting of 13 healthy subjects (9 men and 4 women; age: 30.73±2.88 years; BMI: 24.35±1.61 kg/m2) was also studied.
Oral glucose tolerance test (OGTT) and frequently sampled intravenous glucose tolerance test (FSIGT) were performed before and after 3, 6, 9 and 12 months of rhGH therapy in GH-deficient adults and in controls at baseline. The tests were done after overnight (12 hours) fasting. Baseline blood samples were drawn at -20, -10 and 0 min from an indwelling cannula, which was inserted in the antecubital vein.
OGTT was performed after oral ingestion of 75-g glucose monohydrate diluted in 300 ml of water. Venous blood samples were drawn at 0, 30, 60, 90 and 120 minutes and analysed for glucose, insulin and C-peptide levels.
FSIGT was performed after glucose injection (0.3 g/kg body weight) at 0 min. After 20 min, a bolus of 0.05 IU/kg body weight of insulin (Actrapid HM, Novo Nordisk) was injected intravenously. Venous blood samples were drawn at 2, 4, 8, 19, 22, 25, 30, 40, 50, 70, 90 and 180 minutes and analysed for glucose and insulin levels.8
Assays: Blood glucose levels (mmol/l) were measured by glucose-oxidase method (Randox, UK) using autoanalyser (Beckman, Austria). Serum GH levels were measured using a time-resolved fluoroimmunoassay (Delfia, Wallac Oy, Turky, Finland; ?g/l) with a sensitivity of 0.018 ?g/l, and the intra-assay coefficient of variation (CV) was 5.0, 2.1 and 4.1% for concentrations of 0.2±0.01, 11.36±0.23 and 29.7±1.22 ?g/l, respectively, and for inter-assay was 6.3, 5.4 and 4.2% for concentrations of 0.26±0.01, 13.9±0.75 and 28.9±1.26 ?g/l, respectively. IGF-I was performed using polyclonal RIA (INEP, Zemun; nmol/l) after acid ethanol extraction. Coefficient of variation for intra-assay ranged from 3.0-6.0 % and for inter-assay was less than 12%. Plasma insulin was measured by radioimmunoassay (RIA INEP, Zemun; mIU/l). Coefficient of variation for intra-assay was 20.4, 7.6 and 6.1% for concentrations of 9.4±1.9, 23.7±1.8 and 181.6±11.0 mIU/l, and for inter-assay was 22.1, 10.6 and 16.8% for concentrations of 8.6±1.9, 33.8±3.6 and 153.9±25.8 mIU/l, respectively. C-peptide levels were measured using commercial radioimmunoassay kit (RIA, INEP, Zemun, nmol/l), coefficient of variations for intra-assay were 15.3, 7.2 and 9.8% for concentrations of 0.047±0.007, 0.199±0.014 and 1.225±0.12 nmol/l, respectively.
Calculations: Insulin sensitivity (SI) was estimated using the MINMOD computer program,8 Quantitative Insulin Sensitivity Check Index (QUICKI) and Homeostatic Model of Assessment (HOMA). QUICKI was calculated by following formula: QUICKI= 1 / [log (insulin0) + log (glucose0)], HOMA IR was calculated as fasting insulin (IU/l) x fasting glucose (mmol/l) / 22.5 and HOMA SI was calculated as 1/HOMA IR.7, 9, 10 Fasting glucose and insulin concentrations were calculated as the mean values from -20, -10 and 0 min from Minimal model analysis. Glucose effectiveness (SG) was calculated using the MINMOD computer program.8 Acute insulin response to glucose (AIRG) was calculated as area under the curve (AUC) for insulin, 0-8 min after glucose bolus injection, using the trapezoidal rule.11 Disposition index (DI) was calculated as product of SI and AIRG.11 The changes of glucose, insulin and C-peptide during OGTT were analysed as AUC using the trapezoidal rule.
Statistical analysis: Data are expressed as mean±SEM. ANOVA for repeated measures, paired t-test, Wilcoxon’s matched-paired test, Pearson’s correlation and Bland Altman plot were used for statistical analysis. Results were considered significant at P <0.05.
In the twenty-one patients (10 men and 11 women; age: 36.6±2.3 years; BMI: 26.45±4.40 kg/m2) the underlying pathology was: nonfunctional pituitary adenoma - 8, prolactinoma - 6, craniopharyngioma - 3, Cushing - 1, histiocytosis - 1, astrocytoma - 1 and idiopathic GH-deficiency - 1. There was no statistically significant difference in SI (3.06±0.37 vs. 2.73±0.54, P >0.05) using MINMOD analysis, as well as in AIRG (603.66±157.18 vs. 688.71±155.51, P > 0.05) before and after therapy. There was no significant difference in SG before and after the therapy (2.67±0.34 vs. 2.30±0.32, P > 0.05). Also, there was no change in DI [Table - 1]; [Figure - 1]. SI and AIRG had shown a hyperbolic relation before, during and after rhGH therapy. In comparison with the controls, there was no difference in SI at baseline (3.06±0.37 vs. 4.00±0.86, P > 0.05).
There was no difference in SI before and after 12 months of rhGH therapy using QUICKI formula (0.362±0.013 vs. 0.334±0.005, p = 0.079) [Figure - 1]. There was no difference in QUICKI between GH-deficient adults before therapy versus controls (0.362±0.013 vs. 0.361±0.06, P > 0.05).
HOMA index changed significantly after 12 months of therapy ( HOMA IR : 1.71±0.32 vs. 2.82±0.42, P > 0.05; HOMA SI : 0.94±0.19 vs. 0.42±0.04, P > 0.05) [Figure - 1].
Compared to controls, there was no significant difference at baseline in SI (3.06±0.37 vs. 4.00±0.86, P > 0.05), but after 12 months of rhGH therapy, the difference was significant (2.73±0.54 vs. 4.00±0.86, P > 0.05). Contrary to Minimal model analysis, there was no difference in QUICKI between GH-deficient adults and controls before (0.362±0.013 vs. 0.361±0.06, P > 0.05) and after (0.339±0.05 vs. 0.361±0.06, P > 0.05) rhGH therapy.
There was a significant correlation between: MINMOD and QUICKI (r = 0.46; P < 0.01), MINMOD and HOMA (MINMOD and HOMA IR: r = -0.37, P < 0.01; MINMOD and HOMA SI: r = 0.47, P < 0.01), and QUICKI and HOMA (QUICKI and HOMA IR: r = -0.82, P < 0.01; QUICKI and HOMA SI: r = 0.95; P < 0.01) [Figure - 2]. The results obtained by Bland Altman plot suggest comparability between MINMOD and HOMA as methods for quantification of insulin sensitivity, so the differences obtained using these tests are probably due to a small sample size [Figure - 3].
There was a significant increase in AUC under the glucose curve during OGTT before and after rhGH therapy (619.57±22.38 vs. 698.16±40.77, P < 0.05) as well as in the AUC under insulin curve during OGTT (5956.78±779.49 vs. 7803.05±970.27, P < 0.05). There was no significant difference in AUC for C-peptide during OGTT before and after therapy (178.93±25.63 vs. 147.03±21.62, P < 0.05). Only one patient developed impaired glucose tolerance during 12 months of rhGH therapy.
There was no difference in BMI of patients before and after therapy (26.45±0.96 vs. 26.65±1.03 kg/m2, P < 0.05).
Our data suggests that rhGH therapy in GH-deficient (GHD) adults decrease SI in this group of patients. HOMA analysis, before and after therapy, clearly indicates significant decrease of SI. On the contrary, MINMOD and QUICKI analysis demonstrated non-significant decrease in SI. This is most likely due to a small sample size. Changes in AUC for glucose and insulin before and after therapy, suggests an increase in insulin resistance, as confirmed by the HOMA index. One patient without familial predisposition for diabetes mellitus developed impaired glucose tolerance (IGT) after therapy in absence of change in the BMI. Similar results were obtained during other studies, with exceptions in the percentage of patients who developed IGT. Others authors did not find a change in glucose tolerance after 6 months of rhGH therapy. It is postulated that the observed difference amongst studies may be due to a difference in number of patients enrolled and/or differences in pretreatment characteristics of patients, particularly those who are susceptible to develop IGT or diabetes. It was also suggested that only children susceptible to develop diabetes mellitus, before initiation of rhGH therapy, are at a greater risk to develop IGT or diabetes during therapy course.
The existence of insulin resistance and its consequences in GHD adults before the initiation of the rhGH therapy was observed in several studies., It was shown that this group of patients had normal fasting levels of glucose, insulin, C-peptide and free fatty acids (FFA).
It was suggested that insulin resistance in GHD patients is generated due to severe inhibition of insulin-stimulated glycogen synthase (GlySyn) activity, accompanied by a reduced baseline glycogen content, low-to-normal glucose 6-phosphate (G6P) levels and high total intracellular glucose concentrations in the presence of persisting euglycaemia. These abnormalities are not present in other insulin resistant states associated with reduced insulin-stimulated GlySyn activity (i.e. obesity, diabetes mellitus type 2).
Controversial results in measuring SI during rhGH treatment were obtained in a number of studies.
An initial deterioration of SI during the course of rhGH therapy was observed in most studies,,, as we observed in our study. However, both glucose and insulin levels have a tendency to return to pre-treatment values during continuation of rhGH treatment. A persistent deterioration of SI during and after rhGH therapy was demonstrated using euglycaemic hyper-insulinaemic clamp as a method for assessment of SI. 17 Index of insulin resistance after 12-months of rhGH therapy remains unchanged using HOMA.
In two long–term controlled studies, there was no significant difference in SI before and after rhGH therapy in GHD adults, or between rhGH treated and non-treated GHD adults, as well as between rhGH-treated GHD adults and healthy controls., Our study also did not demonstrate any significant difference in insulin sensitivity (SI) using MINMOD and QUICKI index.
Recently, Katz et al suggested QUICKI as the most relevant substitution for hyper-insulinaemic euglycaemic clamp in estimation of SI. HOMA represents a structural computer model of the glucose/insulin feedback system for the estimation of insulin resistance. Both QUICKI and HOMA methods use only basal concentrations of glucose and insulin for estimation of SI. Increased glomerular filtration rate and renal blood flow during rhGH therapy may alter clearance of insulin and C-peptide. Therefore, actual concentrations of insulin and C-peptide underestimate the increase in insulin secretion during rhGH treatment, limiting their role in calculation of index of SI. MINMOD is more convenient procedure than the euglycaemic hyper-insulinaemic clamp and it appears that MINMOD may be the most convenient method for estimation of SI in GHD adults on the rhGH replacement therapy. Concerning the correlations between the results obtained by different methods, it was suggested that correlation between SI indices obtained from QUICKI and HOMA analysis and SI index of euglycaemic hyper-insulinaemic clamp is better than correlation between SI indices obtained from clamp and MINMOD.
The use of different methods for determination of SI could contribute to the observed polarisation in SI results and may influence the degree of significance of deterioration of SI during rhGH replacement therapy. Discrepant results concerning SI in different studies may reflect differential rhGH dose titration, preexisting SI, the age of studied patients, and according to our experience in this study, different methods used for the assessment of SI.
In conclusion, our results indicate that the choice of method used for quantification of SI could influence the conclusion concerning the effect of rhGH on SI. MINMOD and QUICKI index did not demonstrate any significant difference in SI in adult growth hormone deficient patients before and after the 12 month therapy with rhGH, while HOMA index indicated a significant increase in insulin resistance. This difference could be accounted for due to a small sample size. Observed differences in the results between the different methodologies may limit the comparisons between groups that used different method for quantification of SI and could contribute to observed controversial results of the effect of rhGH therapy on SI.
Novo Nordisk A/S Regional office Belgrade kindly supported our study, providing us the rhGH. Study was supported from Republic Ministry of Science of Serbia, project No: 1973.
[Figure - 1], [Figure - 2], [Figure - 3][Table - 1]