Inaccuracy of insulin-like growth factor (IGF) binding protein (IGFBP)-3 assessment in the diagnosis of growth hormone (GH) deficiency from childhood to young adulthood: association to low GH dependency of IGF-II and presence of circulating IGFBP-3 18-kilodalton fragment

CONTEXT: Poor sensitivity of IGF binding protein (IGFBP)-3 assessment in the work-up of GH deficiency (GHD) has been ascribed to the equal affinity of IGFBP-3 for IGF-I and IGF-II and to IGFBP-3 proteolysis. OBJECTIVE: The objective of this study was to determine the IGF-II GH dependency and IGFBP-3 proteolysis in patients with GHD from childhood to young adulthood. DESIGN: This study was cross-sectional. SETTING: This was a national multicenter study performed in university hospitals. PATIENTS: One hundred thirty-one subjects (chronological age, 1.3-25 yr), 72 patients with GHD and 59 subjects with idiopathic short stature, were studied. INTERVENTIONS: IGF-I, IGF-II, and IGFBP-3 serum concentrations were measured by immunoradiometric assay. IGFBP-3 circulating forms were assessed by Western immunoblot (WIB) analysis. MAIN OUTCOME MEASURES: Main outcome measures were sensitivity and specificity of IGF-I, IGF-II, and IGFBP-3 measurements. RESULTS: Sensitivity and specificity of IGFBP-3 measurement were 27 and 100%, respectively. IGFBP-3 sensitivity was 46% in young adulthood. Sensitivity and specificity of IGF-I were 69 and 81%, respectively. Sensitivity and specificity of IGF-II assessment were 23 and 97%, respectively. IGFBP-3 WIB revealed the presence of the intact form and the major 29-kDa fragment in both GHD and subjects with idiopathic short stature. In patients with GHD, WIB showed the presence of an additional smaller IGFBP-3 fragment migrating at approximately 18 kDa. CONCLUSIONS: Our results suggest that in children and young adults with GHD, the low GH dependency of IGF-II together with IGFBP-3 proteolytic activity yielding the 18-kDa fragment concur to reduce the sensitivity of IGFBP-3 assessment, ultimately making it too inaccurate as a screening test in the work-up of GHD.     

The study of spontaneous GH secretion after 36-h fasting distinguishes between GH-deficient and normal adults

OBJECTIVE: Within an appropriate clinical context, GH deficiency (GHD) in adults can only be diagnosed biochemically by provocative testing. The evaluation of IGF-I, IGFBP-3 and even of spontaneous GH secretion do not establish the diagnosis of adult GHD. In fact, remarkable overlaps between normal and GHD adults have been reported for all these parameters. On the other hand, it is well known that even short-term fasting stimulates GH secretion in normal subjects. The aim of our study was to determine the effects of 36 h fasting on 8-h diurnal GH, insulin and glucose levels as well as on basal IGF-I, IGFBP-3, acid-labile subunit (ALS), IGFBP-1, GHBP and free fatty acid (FFA) levels. SUBJECTS: We studied 9 GHD adults (GHD, 8 males, 1 female; age, mean +/- SEM: 37.6 +/- 2.3 years, body mass index (BMI): 24.5 +/- 1.0 kg/m2) and 20 age-matched normal subjects (NS) as controls (13 males, 7 females; age: 28.9 +/- 0.6 years, BMI: 21.6 +/- 0.4 kg/m2). STUDY DESIGN: In all subjects we studied the effects of 36 h fasting on 8-h daytime GH, insulin and glucose levels (assay every 30 min from 0800 h to 1600 h) as well as on basal IGF-I, IGFBP-3, ALS, IGFBP-1, GHBP and FFA levels. RESULTS: Before fasting, basal mean IGF-I, IGFBP-3 and ALS levels in GHD were lower (P < 0. 0001) than in NS. IGFBP-1, GHBP and FFA levels were similar in both groups. Before fasting mean GH concentration (mGHc) in GHD was lower (P < 0.05) than in NS (0.4 +/- 0.2 vs. 2.2 +/- 0.6 mu/l) but with a clear overlap between the 2 groups (range 0.4-0.8 vs. 0.4-6.8 mu/l). After fasting, both in GHD and NS basal IGF-I, IGFBP-3, ALS and GHBP levels did not change significantly. On the other hand, in both GHD and in NS IGFBP-1 was increased (P < 0.0001) to a similar extent, while FFA increased in NS more (P < 0.01) than in GHD. Fasting significantly increased mGHc in NS (12.0 +/- 1.2 mu/l, P < 0.0001) but not in GHD (0.6 +/- 0.2 mu/l). After fasting, no overlap was present between GHD and NS (0.4-1.6 vs. 2.4-20.8 mu/l, respectively). Mean glucose and insulin concentrations over 8 h in GHD and NS in basal conditions were similar and were reduced to the same extent in both groups. CONCLUSIONS: Our findings demonstrate that after short-term fasting, the study of spontaneous GH secretion distinguishes between GH-deficient adults and normal subjects; this phenomenon occurs before significant changes in IGF-I and IGFBP-3 levels. These results suggest that the assessment of spontaneous GH secretion could be useful for the diagnosis of adult GH deficiency only after short-term fasting.     

Effect of severe growth hormone (GH) deficiency due to a mutation in the GH-releasing hormone receptor on insulin-like growth factors (IGFs), IGF-binding proteins, and ternary complex formation throughout life

Measurement of the insulin-like growth factors (IGFs) and their binding proteins has become commonplace in the indirect assessment of the integrity of the GH axis. However, the relative effect of GH deficiency (GHD) on each component of the IGF axis and the merit of any one parameter as a diagnostic test have not been defined in a homogeneous population across all ages. We therefore measured IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), IGFBP-2, IGFBP-3, and acid labile subunit (ALS) in 27 GHD subjects (aged 5-82 yr) from an extended kindred in Northeast Brazil with an identical GHRH receptor mutation and in 55 indigenous controls (aged 5-80 yr). The effect of GHD on the theoretical distribution of IGFs between the IGFBPs and the ternary complex was also examined. All components of the IGF axis, measured and theoretical, showed complete separation between GHD and control subjects, except IGFBP-1 and IGFBP-2 concentrations, which did not differ. The most profound effects of GHD were on total IGF-I, IGF-I in the ternary complex, and ALS. The proportion of IGF-I associated with IGFBP-3 remained constant throughout life, but was significantly lower in GHD due to an increase in IGF-I/IGFBP-2 complexes. IGF-I in the ternary complex was determined principally by concentrations of ALS in GHD and IGFBP-3 in controls, implying that ALS has greater GH dependency. In the controls, IGF-II was associated primarily with IGFBP-3 and to a lesser extent with IGFBP-2, whereas in GHD the reverse was found. There was also a dramatic decline in the proportion of free ALS in GHD adults that was not evident in controls. As diagnostic tests, IGF-I in the ternary complex and total IGF-I provided the greatest separation between GHD and controls in childhood. Similarly, in older adults the best separation was achieved with IGF-I in the ternary complex, with free ALS being optimal in younger adults. Severe GHD not only reduces the amounts of IGFs, IGFBP-3, and ALS, but also modifies the distribution of the IGFs bound to each IGFBP. Diagnostic tests used in the investigation of GHD should be tailored to the age of the individual. In particular, measurement of IGF-I in the ternary complex may prove useful in the diagnosis of GHD in children and older adults, whereas free ALS may be more relevant to younger adults.     

Differential effects of GH replacement on the components of the leptin system in GH-deficient individuals

GH therapy is associated with a reduction in fat mass and an increase in lean mass in subjects with GH deficiency (GHD). Leptin, like GH, plays an important role in the regulation of body composition. GH treatment has been shown to reduce serum leptin; however, the physiological interactions between the leptin system (free leptin, bound leptin, and soluble leptin receptor) and the GH/IGF-I system largely remain unknown. Twenty-five patients with childhood (n = 10) and adult-onset (n = 15) GHD were studied. GH status had previously been determined using an insulin tolerance test and/or an arginine stimulation test. The following parameters were recorded at baseline (V1) and then after 3 months (V2) and 6 months (V3) on GH treatment: fat mass, body mass index (BMI), and waist/hip ratio (WHR); blood samples were taken after an overnight fast for free leptin, bound leptin, soluble leptin receptor, insulin, and IGF-I. At V2 and V3, respectively, a fall in free leptin (P < 0.001 for each), and at V3 a fall in in percent fat mass (P < 0.001) were observed. There were no significant changes in BMI or WHR. Simultaneously, there was a rise in insulin (P = 0.068 and P < 0.001), IGF-I (P < 0.001 and P < 0.001), bound leptin (P = 0.005 and P < 0.001), and soluble leptin receptor (P = 0.61 and P < 0.001). A positive relationship was noted between free leptin and BMI (P < 0.001) and between free leptin and fat mass (P < 0.001), and a negative relationship was found between free leptin and IGF-I (P < 0.001) and, within patient, between free leptin and insulin (P < 0.001). There was no significant correlation between free leptin and WHR. Bound leptin had a positive association with IGF-I (P < 0.001) and insulin (P = 0.002) and a negative relationship with percent fat mass (P = 0.023). Soluble leptin receptor was also positively related to IGF-I (P < 0.001). In conclusion, our data suggest that the reduction in serum leptin with GH treatment, as noted by others, is mediated through a fall in free leptin. The fall in free leptin and in part the rise in bound leptin are most likely through a reduction in percent fat mass. However, the observed changes in free leptin and bound leptin and, more importantly, the rise in soluble leptin receptor, are not explained entirely by modifications in body composition and may be a direct result of GH/IGF-I.     

Normal growth despite GH, IGF-I and IGF-II deficiency.

A 6.5-year-old male with normal linear growth, despite septo-optic dysplasia, panhypopituitarism and a deficient GH/IGF axis, is presented. In addition to measuring IGF-I, IGF-II and IGFBP-3, serum IGFBP-1, -2, -4 and -5 were measured. A human osteosarcoma cell line was used to assess growth-promoting activity in the patient's serum. The role of leptin in linear growth in this case was investigated. There was no evidence for hyperinsulinism or hyperandrogenism. GH was undetectable upon multiple stimulation. GHBP was elevated. Serum IGF-I (25 microg/l), IGF-II (194 microg/l), IGFBP-3 (0.4 mg/l), and IGFBP-5 (87 microg/l) levels were low compared to age-matched prepubertal children. Serum IGFBP-4 level was normal. Molecular size of IGF-II in the patient's serum was normal, suggesting normal IGF-II bioavailability. Human osteosarcoma cell proliferation in response to the patient's serum was similar to sera from age-matched normal controls. Leptin levels were markedly elevated. Osteoblast cell proliferation was not stimulated by leptin. The data demonstrate that normal growth and osteoblast cell proliferation in this patient is not mediated by GH, total IGFs, insulin, or leptin, and suggest the presence of a yet unidentified growth factor or mechanism. The case offers a detailed picture of binding proteins in a case of growth without GH. It introduces osteoblast cell proliferation as a method of assessing serum growth-promoting activity in such cases. It adds IGF-II and leptin to the list of excluded growth-promoting candidates in GH-independent growth, and further demonstrates our incomplete understanding of the phenomenon of growth.     

Monitoring serum insulin-like growth factor-I (IGF-I), IGF binding protein-3 (IGFBP-3), IGF-I/IGFBP-3 molar ratio and leptin during growth hormone treatment for disordered growth

OBJECTIVE: Serum IGF-I levels are monitored during GH replacement treatment in adults with GH deficiency (GHD) to guide GH dose adjustment and to minimize occurrence of GH-related side-effects. This is not routine practice in children treated with GH. The aim of this study was to evaluate changes in (1) serum IGF-I, IGFBP-3 and IGF-I/IGFBP-3 molar ratio, and (2) serum leptin, an indirect marker of GH response, during the first year of GH treatment in children with disordered growth. DESIGN: An observational prospective longitudinal study with serial measurements at five time points during the first year of GH treatment was carried out. Each patient served as his/her own control. PATIENTS: The study included 31 patients, grouped as (1) GHD (n = 20) and (2) non-GHD (Turner syndrome n = 7; Noonan syndrome n = 4), who had not previously received GH treatment. MEASUREMENTS: Serum IGF-I, IGFBP-3 and leptin levels were measured before treatment and after 6 weeks, 3 months, 6 months and 12 months of GH treatment, with a mean dose of 0.5 IU/kg/wk in GHD and 0.7 IU/kg/wk in non-GHD groups. IGF-I, IGFBP-3 and the calculated IGF-I/IGFBP-3 molar ratio were expressed as SD scores using reference values from the local population. RESULTS: In the GHD group, IGF-I SDS before treatment was lower compared with the non-GHD (-5.4+/-2.5 vs. -1.8+/-1.0; P<0.001). IGF-I (-1.8 SDS +/- 2.2) and IGFBP-3 (-1.1 SDS +/- 0.6) levels and their molar ratios were highest at 6 weeks and remained relatively constant thereafter. In the non-GHD group, IGF-I levels increased throughout the year and were maximum at 12 months (0.3 SDS +/- 1.4) while IGFBP-3 (1.1 SDS +/- 0.9) and IGF-I/IGFBP-3 molar ratio peaked at 6 months. In both groups, IGF-I SDS and IGF-I/IGFBP-3 during treatment correlated with the dose of GH expressed as IU/m2/week (r-values 0. 77 to 0.89; P = 0.005) but not as IU/kg/week. Serum leptin levels decreased significantly during GH treatment in the GHD (median before treatment 4.0 microg/l; median after 12 months treatment 2.4 microg/l; P = 0.02) but not the non-GHD (median before treatment 3.0 microg/l; median after 12 months treatment 2.6 microg/l). In the GHD group, serum leptin before treatment correlated with 12 month change in height SDS (r = 0.70, P = 0.02). CONCLUSIONS: The pattern of IGF-I, IGFBP-3 and their molar ratio during the first year of GH treatment differed between the GHD and non-GHD groups. Calculation of GH dose by surface area may be preferable to calculating by body weight. As a GH dose-dependent increase in serum IGF-I and IGF-I/IGFBP-3 may be associated with adverse effects, serum IGF-I and IGFBP-3 should be monitored routinely during long-term GH treatment. Serum leptin was the only variable that correlated with first year growth response in GHD.     

Leptin concentrations in GH deficiency: the effect of GH insensitivity

Disorders of GH secretion are known to impair the physiological lipostat and to affect the secretion of leptin, a sensitive marker of regional fat accumulation and total body composition. In both children and adults with GH deficiency (GHD), leptin levels are increased proportionately with enhanced adiposity. In GHI, mutations of the GH receptor gene result in a phenotype similar to GHD, with increased adiposity and unfavorable lipid profiles. To examine the impact of different forms of growth disorders on leptin production, we measured leptin levels in 22 GHI patients homozygous for the E180 splice mutation (15 females and 7 males, aged 8-37 yr) and compared results with those obtained in 20 subjects heterozygous for the mutation (11 females and 9 males, aged 7-54), 17 idiopathic GHD patients (6 females and 11 males, aged 3-34), and 44 normal subjects (25 females and 19 males, aged 7-45). After the baseline evaluation, all subjects received two 7-d GH treatments at doses of 0.025 and 0.050 mg/kg x d in random order. Leptin, IGF-I, and IGF-binding protein-3 (IGFBP-3) were assayed by specific immunoassays. IGF-I and IGFBP-3 levels were significantly lower (P < 0.0001) in homozygous GHI and GHD patients compared with either controls or GHI heterozygotes. Circulating leptin levels were significantly higher in homozygous GHI patients than in normal controls (20.7 +/- 4.2 vs. 8.7 +/- 1.4 microg/liter) as well as when compared with heterozygous GHI subjects (14.4 +/- 3.4 microg/liter) and GHD patients (9.8 +/- 1.6 microg/liter; P < 0.01). Similar results were obtained when leptin was normalized for body mass index. When subjects were subgrouped by gender, leptin levels were significantly higher (P < 0.05) in GHI females than in females of all other groups and were significantly increased in GHD males (P < 0.01 vs. control males). Within the study groups, females had significantly higher leptin levels than males in controls (12.7 +/- 2 vs. 3.3 +/- 1 microg/liter; P < 0.001) and homozygous GHI patients (28.7 +/- 5.3 vs. 6.9 +/- 2.3 microg/liter; P < 0.05), but not in heterozygous GHI (20.1 +/- 5.4 vs. 7.3 +/- 2.4 microg/liter; P < 0.06) and GHD (10.9 +/- 2.6 vs. 9.2 +/- 2.1 microg/liter) patients. By multivariate analysis, log-normalized leptin levels were best predicted by gender and body mass index in homozygous GHI patients as well as in normal subjects. During the 1-wk courses of GH therapy, serum IGF-I and IGFBP-3 levels significantly increased (P < 0.0001) in GHD patients, heterozygous GHI patients, and control subjects at both GH doses. Inversely, leptin levels did not change significantly during either course of GH administration in the groups examined. These data demonstrate that leptin is increased in patients affected with long-standing homozygous GHI, probably reflecting abnormalities of body composition and metabolism typical of this condition.     

Metabolic parameters and adipokine profile during GH replacement therapy in children with GH deficiency

OBJECTIVE: GH replacement therapy in children with GH deficiency (GHD) mainly promotes linear growth. Not only have very few studies fully analyzed the metabolic consequences of GH therapy, but also the question as to whether GH may affect adipokine secretion has been insufficiently investigated. Our aim was to study the effects of GH replacement therapy on auxological data, lipid and glycemic profiles, insulin homeostasis (HOMA-IR) and serum adipokines in children. METHODS: This was a 1-year prospective study. Thirty-four GHD children (11.6 +/- 2.6 years) and thirty healthy matched controls were enrolled. Children affected by GHD were studied both before beginning continuous GH replacement therapy and again at 12 months. RESULTS: At the beginning of the study, total and LDL cholesterol were higher in GHD children than in controls (P<0.001), whereas HDL cholesterol, triglycerides, insulin, HOMA-IR, leptin, and adiponectin were similar. At 12 months of continuous GH replacement therapy in the GHD group, there was a significant increase in both auxological data and IGF-I (P<0.001); total cholesterol (P<0.001), LDL (P<0.001), triglycerides (P<0.005), and leptin (P<0.001) decreased significantly; HDL (P<0.003), insulin (P<0.001), HOMA-IR (P<0.001) increased while adiponectin was unmodified. Furthermore, IGF-IDelta showed an inverse correlation with leptin Delta (rho = -0.398, P = 0.02). CONCLUSIONS: In GHD children, the evaluation of metabolic parameters proves to be a useful tool for the evaluation of auxological parameters during GH replacement therapy. In our study, GH replacement therapy in GHD children improved final height, restored IGF-I levels, reduced leptin levels, and improved the lipid profile, without producing any unfavorable effects on glucose metabolism.     

Survivors of childhood acute lymphoblastic leukaemia, with radiation-induced GH deficiency, exhibit hyperleptinaemia and impaired insulin sensitivity, unaffected by 12 months of GH treatment

OBJECTIVE: Adult survivors of childhood acute lymphoblastic leukaemia (ALL) often exhibit GH deficiency (GHD), due to prophylactic cranial radiotherapy (CRT). It is not known whether the observed risk for adiposity in these patients is associated with impaired insulin sensitivity and whether the insulin sensitivity is affected by GH replacement therapy. SUBJECTS AND DESIGN: Eleven patients with GHD (median age 29 years), previously given prophylactic CRT for ALL, and 11 sex-, age- and body mass index (BMI)-matched controls were investigated with bioimpedance analysis (BIA) and analysis of serum leptin, serum free fatty acids (FFA) and serum insulin. Insulin sensitivity was measured by a euglycaemic-hyperinsulinaemic clamp technique (IS-clamp). Moreover, the effects of 12 months of individually titrated GH treatment (median dose 0.5 mg/day) on these parameters were investigated. RESULTS: At baseline, the patients had lower fat free mass (FFM) (P = 0.003), higher percentage fat mass (FM) (P = 0.05), serum insulin (P = 0.02) and serum leptin/kg FM (P = 0.01) than controls. The patients had a tendency towards impaired IS-clamp (P = 0.06), which disappeared after correction for body composition (IS-clamp/kg FFM; P > 0.5). In the patients, time since CRT was positively correlated with percentage FM (r = 0.70, P = 0.02), and there was an independent negative association between serum FFA and IS-clamp (P = 0.05). Twelve months of GH treatment increased serum IGF-I (P = 0.003) and FFM (P = 0.02) and decreased percentage FM (P = 0.03), but no significant changes were seen in serum leptin/kg FM, serum FFA, FFA-clamp, serum insulin or IS-clamp (all, P > or = 0.2). CONCLUSIONS: Young adult survivors of childhood ALL with GHD had increased fat mass, hyperleptinaemia and impaired insulin sensitivity, which could be a consequence of radiation-induced impairment of GH secretion or mediated by other hypothalamic dysfunctions, such as leptin resistance or other unknown factors, affected by CRT. Twelve months of individualized GH replacement therapy led to positive effects on body composition, but the hyperleptinaemia, hyperinsulinaemia and the impaired insulin sensitivity remained unchanged.     

A low individualized GH dose in young patients with childhood onset GH deficiency normalized serum IGF-I without significant deterioration in glucose tolerance

OBJECTIVE: Many GH deficient (GHD) patients have impaired glucose tolerance and GH substitution in these patients has caused deleterious effects on glucose tolerance with hyperinsulinaemia. This further impairment of glucose tolerance might be due to an unphysiologically high dose of GH. Whether such a deterioration can be avoided by an optimal GH replacement dose is not known. In most previous studies, the GH dose was calculated according to body weight or body surface area and not adjusted according to the serum IGF-I response. DESIGN: The study was of open design and investigations were performed before the start of GH substitution and after nine months of treatment. The GH dose was adjusted according to the response in serum IGF-I, and in patients with sub-normal serum IGF-I levels (all but two) we aimed for a serum IGF-I level in the middle of the normal range. The median GH dose at the end of the study was 0.14 IU/kg/week. PATIENTS: Ten patients, eight males and two females, with childhood onset GHD were examined. Their median age was 27 years (range 21-28). MEASUREMENTS: Overnight and 24-h fasting levels of glucose, insulin and IGFBP-1 were measured. Directly after the 24-h fast an oral glucose tolerance test (OGTT), with measurements of glucose, insulin and IGFBP-1 was performed. An intravenous glucose tolerance test (IVGTT) was performed after overnight fasting. Body composition was measured with bio-impedance analysis (BIA) and quality of life was assessed using a self-rating questionnaire, Qol-AGHDA. RESULTS: After GH treatment, there were no significant changes in glucose tolerance, measured by overnight and 24-h fasting levels of glucose, insulin and IGFBP-1, an oral glucose tolerance test (after 24-h fasting) and an intravenous glucose tolerance test (after overnight fasting). Percentage fat mass and BMI correlated negatively with both the 24 h fasting IGFBP-1 levels and the IGFBP-1 responses after the OGTT. All patients decreased their percentage of fat mass measured by BIA [median -2.9%; range -1.0-(-6.6); P = 0.005]. The administered GH dose correlated negatively with the relative change in whole body resistance (r = -0.66; P = 0.04). All, but one of the patients improved their quality of life score after GH therapy. CONCLUSIONS: In a group of young patients with childhood onset GH deficiency, 9 months of treatment with a low GH dose (median 0.14 IU/kg/week) caused no significant deterioration of glucose tolerance. The strong negative associations between BMI or percentage fat mass and IGFBP-1 suggest that serum IGFBP-1 is more closely related than insulin to body composition in GH deficient patients. It is important to consider which critical endpoints should determine the GH dose. We would suggest that, apart for normalizing the serum IGF-I level, another main endpoint should be normalization of, or at least avoidance of any deterioration in glucose tolerance.     

Growth hormone (GH) deficiency (GHD) of childhood onset: reassessment of GH status and evaluation of the predictive criteria for permanent GHD in young adults

GH secretion was reevaluated after completion of GH treatment at a mean age of 19.2 +/- 3.2 yr in 35 young adults with childhood-onset GH deficiency (GHD). The patients were subdivided into 4 groups according to their first pituitary magnetic resonance imaging (MRI) findings: group I, 11 patients with isolated GHD (IGHD) and normal pituitary volume (280 +/- 59.4 mm3); group II, 7 patients with IGHD and small pituitary gland (163.1 +/- 24.4 mm3; P = 0.0009 vs. group I); group III, 13 patients (5 with IGHD and 8 with multiple pituitary hormone deficiency) with congenital hypothalamic-pituitary abnormalities such as pituitary hypoplasia (95.8 +/- 39.3 mm3; P < 0.00001 vs. group I and P = 0.003 vs. group II), pituitary stalk agenesis, and posterior pituitary ectopia; and group IV, 4 patients with multiple pituitary hormone deficiency secondary to craniopharyngioma. Pituitary MRI and GH secretory status were reevaluated after GH withdrawal using arginine, insulin induced-hypoglycemia, and sequential arginine-insulin tests. Serum insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) were determined at the time of retesting and 6, 12, and 24 months after discontinuation of treatment in the patients with permanent GHD and after 6 months in those with normal GH responses to stimulation. The patients in groups I and II showed a normal response to stimulation after completion of GH treatment regardless of pituitary size, whereas all patients in groups III and IV still had a GH response of less than 3 microg/L to any of the tests. Pituitary volume normalized in 6 of 7 patients in group II, whereas in all patients in group III MRI studies confirmed the initial findings. Mean IGF-I and IGFBP-3 concentrations at the time of retesting were significantly higher in groups I and II than in groups III and IV. In patients of groups III and IV, mean IGF-I was significantly decreased after 6 and 12 months, whereas IGFBP-3 was significantly decreased 12 months after treatment withdrawal. Our results confirm that a high proportion of children with IGHD and normal or small pituitary show normalization of GH secretion at the completion of GH treatment, whereas GHD is permanent in all patients with pituitary hypoplasia, pituitary stalk agenesis, and posterior pituitary ectopia. IGF-I and IGFBP-3 determinations shortly after GH withdrawal had limited value in the diagnosis of GHD of childhood onset associated with congenital hypothalamic-pituitary abnormalities, but became accurate after 6-12 months. We suggest that patients with GHD and congenital hypothalamic-pituitary abnormalities do not require further investigation of GH secretion, whereas patients with IGHD and normal or small pituitary gland should be retested well before the attainment of adult height.     

High-affinity growth hormone (GH)-binding protein (GHBP), body fat mass, and insulin-like growth factor-binding protein-3 predict the GHBP response to GH therapy in adult GH deficiency syndrome

The study objective was to investigate which baseline factors can accurately predict plasma high-affinity growth hormone (GH)-binding protein (GHBP) levels after GH replacement therapy in patients with GH deficiency (GHD). The study group consisted of 36 GHD patients (22 men and 14 women; mean age, 43.1 years; (range, 21 to 60) known to have adult-onset GHD for many years (range, 4 to 22). They were randomly divided into a GH-treated group (n = 19) and a placebo group (n = 17). Body composition (assessed by bioelectrical impendance analysis [BIA]), plasma GHBP (fast protein liquid chromatography [FPLC] size-exclusion gel chromatography), insulin-like growth factor-I (IGF-I), and IGF-binding protein-3 ([IGFBP-3] radioimmunoassays) were measured before and after 6 months. A stepwise multiple linear regression analysis with the plasma GHBP level after 6 months as the dependent variable was used to unravel significant explanatory (or predictor) variables. In contrast to placebo therapy, GH replacement therapy increased the mean plasma levels of IGF-I and IGFBP-3 to the normal range, whereas a small but statistically significant increase in plasma GHBP was observed. The combination of baseline plasma GHBP, body fat mass, and IGFBP-3 predicts posttreatment GHBP levels accurately (adjusted R2 = .97), indicating that baseline variables such as age, gender, fat-free mass, and IGF-I have no contribution. Furthermore, reliability analysis showed that the observed and predicted values for GHBP fit a strict parallel model. These findings indicate that the variations in body fat mass and IGFBP-3 among adult GHD subjects explain the reported variable response of GHBP to GH replacement therapy.     

Effects of 36 hour fasting on GH/IGF-I axis and metabolic parameters in patients with simple obesity. Comparison with normal subjects and hypopituitary patients with severe GH deficiency

OBJECTIVE: Reduction of growth hormone (GH) secretion in obesity probably reflects neuroendocrine and metabolic abnormalities. Even short-term fasting stimulates GH secretion and distinguishes normal from hypopituitary subjects with growth hormone deficiency (GHD). Marked weight loss improves GH secretion in obesity but the effect of fasting is controversial. We studied the effects of a 36 h fasting on the GH/IGF-I axis and metabolic parameters in obesity. SUBJECTS: We studied nine obese patients (OB; three male and six female; age, 29.2+/-4.8; range, 18-59 y; body mass index (BMI), 43.4+/-2.7 kg/m(2); WHR, 0.9+/-0.1). Fifteen normal subjects (NS; eight male and seven female 28.9+/-0.6, 25-35 y; 21.6+/-0.4 kg/m(2)) and 10 adult hypopituitary patients with severe GH deficiency (GHD; seven male and three female; 37.6+/-2.3, 29-50 y; 24.5+/-1.0 kg/m(2); GH peak<3 microg/l after ITT and/or<9 microg/l after GHRH+arginine) served as control groups. STUDY DESIGN: We studied the effects of 36 h fasting on 8 h diurnal mean GH, insulin and glucose concentrations (mGHc, mINSc and mGLUc; assay every 30 min from 8.00 am to 4.00 pm) as well as on IGF-I, IGFBP-3, ALS, IGFBP-1, GHBP and free fatty acid (FFA) levels. RESULTS: Before fasting, basal IGF-I and ALS levels in OB were similar to those in NS and both were higher (P<0.001) than those in GHD. IGFBP-3 levels in OB were lower (P<0.01) than in NS but higher (P<0.02) than in GHD. GHBP levels in OB and GHD were similar and both were higher (P<0.01) than in NS. Glucose levels were similar in all groups. FFA levels in OB were higher (P<0.01) than in NS but similar to those in GHD. IGFBP-1 in OB were lower (P<0.05) than in NS and GHD which, in turn, were similar. On the other hand, mINSc in OB was higher (P<0.01) than that in NS and GHD which, in turn, were similar. The mGHc in OB was similar to that in NS but only the latter was higher (P<0.05) than in GHD. The individual mGHc in the three groups overlapped. After fasting, IGF-I levels in GHD were unchanged while they decreased in OB (P=NS) as well as in NS (P<0.01). IGFBP-3 and ALS levels did not change. GHBP levels in OB and GHD were unchanged while they increased in NS (P<0.01). Glucose and FFA levels were reduced and increased, respectively, in all groups (P<0.02 and P<0.01). IGFBP-1 increased while mINSc decreased in all groups (P<0.02 and P<0.01); in OB they persisted lower and higher (P<0.01) respectively, than in NS and GHD. Fasting significantly increased mGHc in NS (P<0.001) but not in OB as well as in GHD. Individual mGHc in OB showed persistent overlap with GHD. CONCLUSIONS: Short-term fasting does not increase GH secretion in obesity and does not distinguish somatotroph function in obese from that in severe GHD adults. Short-term fasting in obesity has attenuated effects on insulin and IGFBP-1 secretion while it normally increases free fatty acids in spite of any change in GH secretion.     

Individualized low-dose growth hormone (GH) treatment in GH-deficient adults with childhood-onset disease: metabolic effects during fasting and hypoglycemia.

Growth hormone (GH) has insulin-antagonistic effects, and GH secretion is augmented during fasting and hypoglycemia. In the present study, 10 patients aged 21 to 28 years with childhood-onset GH deficiency (GHD) were studied during a 24-hour fast and a hypoglycemic glucose clamp before and after 9 months of GH replacement. During the 24-hour fast, blood glucose, serum insulin, and serum free fatty acid (FFA) levels were measured. In the hypoglycemic clamp, the counterregulatory hormones (plasma catecholamines, serum glucagon, and serum cortisol), serum insulin-like growth factor (IGF) binding protein-1 (IGFBP-1), serum FFA, and glucose uptake were measured. The GH dose was adjusted to the response of serum IGF-I, and the median GH dose was 0.14 IU/kg/wk (range, 0.08 to 0.19). At the end of the study, serum IGF-I levels were normalized in all but one patient, in whom serum IGF-I was above the normal range. Nine months of GH treatment did not cause any significant changes in the blood glucose level, insulin to glucose ratio, or serum FFA level during the 24-hour fast, and none of the patients experienced hypoglycemia either before or after GH treatment. However, GH therapy resulted in increased insulin resistance during hypoglycemia, without changes in the counterregulatory hormonal responses, serum IGFBP-1, or serum FFA.     

Enhancement of the peripheral sensitivity to growth hormone in adults with GH deficiency

OBJECTIVE: Adults with severe GH deficiency (GHD) need recombinant human growth hormone (rhGH) replacement to restore body composition, structure functions and metabolic abnormalities. The optimal rhGH dose for replacement has been progressively reduced to avoid side effects. The aim of the present study was to define the minimal rhGH dose able to increase both IGF-I and IGF binding protein (BP)-3 levels in GHD and to verify the possible change in GH sensitivity. DESIGN AND PATIENTS: To this goal, we studied the effect of 4-day treatment with 3 rhGH doses (1.25, 2.5 and 5.0 microg/kg/day) on IGF-I and IGFBP-3 levels in 25 panhypopituitary adults with severe GHD (12 males and 13 females, age: 44.5+/-3.0 years, body mass index (BMI): 27.0+/-0.9 kg/m(2)) and 21 normal young adult volunteers (NV, 12 males and 9 females, age: 30.5+/-2.0 years, BMI: 20.8+/-0.5 kg/m(2)). RESULTS: Basal IGF-I and IGFBP-3 levels in GHD were lower (P<0.001) than in NV. In NV the 1.25 microg/kg dose of rhGH did not modify IGF-I levels. The dose of 2.5 microg/kg rhGH significantly increased IGF-I levels in men (P<0.001) but not in women, while the 5.0 microg/kg dose increased IGF-I levels in both sexes (P<0.001). IGFBP-3 levels were not modified by any of the administered rhGH doses. In GHD patients, all rhGH doses increased IGF-I levels 12 h after both the first (P<0.01) and the fourth rhGH dose (P<0.001). At the end of treatment percentage increases in IGF-I were higher (P<0.001) in GHD patients than in NV. In contrast with NV, in GHD patients the IGF-I response to short-term stimulation with rhGH was independent of gender. Moreover, GHD patients showed increases in IGFBP-3 after the fourth administration of both 2.5 and 5.0 microg/kg rhGH. CONCLUSION: The results of the present study demonstrate that the minimal rhGH dose able to increase IGF-I and IGFBP-3 levels in GHD patients is lower than in normal subjects, at least after a very short treatment. This evidence suggests an enhanced peripheral GH sensitivity in GH deprivation.     

Diagnostic efficiency of serum IGF-I, IGF-binding protein-3 (IGFBP-3), IGF-I/IGFBP-3 molar ratio and urinary GH measurements in the diagnosis of adult GH deficiency: importance of an appropriate reference population

OBJECTIVE: To analyse the diagnostic role of serum IGF-I, IGF-binding protein-3 (IGFBP-3), IGF-I/IGFBP-3 molar ratio and urinary GH (uGH) excretion in adult GH deficiency (GHD). DESIGN: Twenty-seven adults (age range: 18-71 years) with severe GHD, defined by a peak GH response to an insulin tolerance test below 3microg/l in patients with at least one additional pituitary hypofunction. Reference values were established from a selected age- and body mass index-matched population (154 healthy adults grouped in four age groups). METHODS: IGF-I and IGFBP-3 were measured by RIA (Nichols) and results expressed as standard deviation (s.d.) scores from our reference population and assay normative data (s.d. score Nichols). uGH was measured by IRMA. RESULTS: Within the control group, IGF-I, IGFBP-3, IGF-I/IGFBP-3 ratio standardisation regarding our control population and IGF-I with respect to the assay normative data resulted in disappearance of age-related differences. However, IGFBP-3 s.d. score Nichols resulted in mean values between +1.4 and +2.5 s.d. score. Greatest diagnostic efficiency was for IGF-I standardised with respect to our controls (97.2%), followed by s.d. score IGFBP-3 (92.9%). s.d. score IGF/IGFBP-3 ratio and uGH showed poor diagnostic efficiency. Any combination of at least two abnormal parameters raised specificity to 100%. IGF-I standardised with respect to assay reference (s.d. score Nichols) showed similar diagnostic value (95.0%) whereas IGFBP-3 showed low sensitivity (33. 3%). Within the GHD patients, those with three or more additional deficiencies had lower s.d. score IGF-I than those with only two or one. CONCLUSION: We underline the importance of an appropriate reference population for correct interpretation of GH secretion markers. Considering our results, specificity obtained with two simultaneous abnormal parameters when referred to an adequate reference population may add valuable information to alternative GH stimulation tests to confirm adult GHD.     

Ghrelin test for the assessment of GH status in successfully treated patients with acromegaly

OBJECTIVE: Posttreatment assessment of disease activity and definition of cure of acromegaly, using measurement of GH secretion, remains problematic. Furthermore, with our efforts to achieve tight biochemical control of the disease it is foreseeable that a proportion of patients may be rendered GH deficient, thus requiring testing for GH deficiency. The aim of our study was to evaluate residual GH secretion in cured patients with acromegaly. DESIGN AND METHODS: At baseline, circulating GH, IGF-I, IGFBP-3, leptin and lipid (cholesterol and tri-glycerides) levels were measured in 33 acromegalic patients nine years after treatment with surgery of whom 6 were additionally irradiated. Two tests were performed: the GH suppression test--oral glucose tolerance test (OGTT) and the GH provocation test--ghrelin test (1 microg/kg i.v. bolus) and the results were compared with 11 age- and sex-matched control subjects. RESULTS: According to the consensus criteria (normal IGF-I levels and post-OGTT GH nadir <1 microg/l), 21 treated acromegalic patients were cured, 6 had discordant IGF-I and GH nadir values during OGTT, while 6 had persistent acromegaly. After the GH provocative test with ghrelin (cut-off for severe GH deficiency is GH <3 microg/l), we detected 9 severely GH deficient patients (GHD) among 21 cured acromegalic patients. Mean GH peak (+/-s.e.m.) response to the ghrelin test in GHD acromegalics was significantly lower compared with acromegalics with sufficient GH secretory capacity and control subjects (1.2 +/- 0.2 microg/l vs 20.1 +/- 2.4 microg/l vs 31.1 +/- 2.5 microg/l respectively, P<0.0001). Mean IGF-I and IGFBP-3 levels were not different between GHD and GH-sufficient cured acromegalics. Leptin levels and body mass index (BMI) were significantly higher in GHD male acromegalics compared with GH-sufficient male acromegalics. GHD female acromegalics tended to have higher BMIs while leptin levels were not different. CONCLUSIONS: The assessment of residual GH secretory capacity by the GH provocation test is necessary in the long-term follow-up of successfully treated acromegalics since a large proportion of these patients are rendered GH deficient.     

Serum leptin and body composition in children with familial GH deficiency (GHD) due to a mutation in the growth hormone-releasing hormone (GHRH) receptor

OBJECTIVE: The relationship between GH, body composition and leptin in children remains ill-defined. We have therefore examined the impact of severe GH deficiency (GHD) due to a mutation in the GHRH receptor on serum leptin concentrations and body composition in childhood. PATIENTS: 12 affected children and young people (GHD) (4 M:8F, age 5.4-20.1 years, 8 Tanner stage (TS) 1-2, 4 TS 3-5) and 40 healthy controls (C) from the same region (13 M:27F, age 5.3-18.4 years, 20 TS 1-2, 20 TS 3-5). METHODS: Percent body fat was determined by infra-red interactance, from which the amounts of fat mass (FM, kg) and fat free mass (FFM, kg) were derived. Serum leptin concentrations were measured in a single fasted, morning serum sample and results expressed as a concentration and as leptin per unit fat mass (L/FM, ng/ml/kg). To control for differences in sex and pubertal maturation, leptin standard deviation scores (leptin SDS) were calculated using normative data from UK children. RESULTS: FFM was significantly lower in GHD children than in controls (TS 1-2 P < 0.05, TS 3-5 P < 0.001). FM did not differ significantly between the two groups. Serum leptin concentrations, leptin per unit fat mass and leptin SDS were significantly elevated in GHD children both peripubertal and pubertal compared with controls. Using all subjects, stepwise multiple linear regression with FM, FFM, age, puberty and sex as explanatory variables and leptin concentration as the dependent variable indicated that 59% of the variability in leptin could be accounted for by FM (+, 45%), FFM (-, 9%) and sex (+, 5%) (P < 0.001). However on inclusion of GH deficiency (coded GHD = 1, control = 2) as an explanatory variable 73% of the variability in leptin was explained by FM (+, 45%), GHD (-, 22%) and sex (+, 6%) (P < 0.001). CONCLUSIONS: These data indicate that severe GH deficiency in children is associated with elevated leptin concentrations, irrespective of sex or pubertal stage. This increase is not associated with differences in fat mass but is related to reduced fat free mass in GH deficiency. Furthermore in this population there may be an additional effect of GH deficiency on leptin, independent of the influences of sex and body composition.     

Insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP-4) concentration and stimulate IGFBP-3 independently of IGF receptors in human fibroblasts and epidermal cells.

Recent studies have provided a consensus that insulin-like growth factor-I (IGF-I) stimulates IGF-binding protein-3 (IGFBP-3) in vivo and in vitro. While it also appears well established that IGFBP-1 is inversely related to insulin concentrations, evidence regarding regulation of other IGFBP is inconclusive. Using immunoprecipitation and Western ligand blot, we have characterized the IGFBPs released into conditioned medium (CM) by cells from the adult human fibroblast cell line N3652 and the human epidermal squamous cell carcinoma line SCL-1. N3652 cells expressed IGFBP-3, IGFBP-2, a 24-kilodalton (kDa) IGFBP presumed to be IGFBP-4, and IGFBPs at 30 and 28 kDa. SCL-1 expressed IGFBP-3 and a putative IGFBP-4, with intermediate bands at 34 and 30 kDa. As determined by ligand blot of CM from confluent cells 72 h after the addition of peptides to serum-free medium, IGF-I and IGF-II potently stimulated IGFBP-3 in both cell lines, but otherwise IGFBP regulation in the two cells diverged. In N3652 cells, IGFBP-3 concentrations in CM increased to 700% and 800% of basal levels in the presence of IGF-I and IGF-II (at 100 ng/ml; n = 5 experiments), respectively. IGFBP-3 was not affected by insulin up to 10 micrograms/ml. In contrast, IGFBP-4 levels were diminished 54% and 73% by 100 ng/ml IGF-I and IGF-II, respectively, with no response to insulin. In SCL-1 cells, IGF-I and IGF-II were virtually identical in stimulating a mean 200% increase in IGFBP-3 (n = 5 experiments). Insulin was less potent, but caused a significant stimulation of IGFBP-3 levels. IGF-I, IGF-II, and insulin all stimulated an approximately 50% increase in IGFBP-4 concentrations. To test the hypothesis that IGF-induced alterations in IGFBP-3 and IGFBP-4 concentrations were regulated via the type 1 IGF receptor, we attempted to block IGFBP changes with type 1 IGF receptor antibody alpha IR-3 and to induce IGFBP changes with an IGF-II analog, [Leu27]IGF-II, with little affinity for the type 1 receptor. alpha IR-3 failed to block either the IGF-induced rise in IGFBP-3 in each cell line or the decline in IGFBP-4 in N3652 CM. [Leu27]IGF-II was as potent as IGF-II or IGF-I in inducing changes in IGFBP-3 and IGFBP-4 concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Common carotid intima-media thickness in growth hormone (GH)-deficient adolescents: a prospective study after GH withdrawal and restarting GH replacement

We prospectively investigated the risk of early atherosclerosis, by classical cardiovascular risk factors and intima-media thickness (IMT) at the common carotid arteries, in 23 adolescents diagnosed as GH deficient (GHD) during childhood and in 23 healthy sex-, age-, and BMI-matched controls. Measurements were performed in all subjects before stopping GH replacement. Because the diagnosis of GHD had been confirmed in 15 of the 23 adolescents, the protocol changed according to the diagnosis as follows: measurements were repeated after 6 months of GH withdrawal and 6 months of GH reinstitution in the 15 with GHD, and after 6 and 12 months of GH withdrawal, measurements were also taken in the eight non-GHD subjects. Serum IGF-I levels were in the normal range for age in all patients before GH withdrawal. When compared with controls, before GH withdrawal, GHD adolescents had reduced high-density lipoprotein cholesterol levels and increased total/high-density lipoprotein cholesterol ratio, fibrinogen, low-density lipoprotein cholesterol, and glucose levels; non-GHD adolescents had increased glucose, insulin, and homeostasis model assessment score. IMT at the common carotid arteries was similar in GHD and controls (0.52 +/- 0.03 vs. 0.55 +/- 0.06 mm; P = 0.23) and was higher in non-GHD than in controls (0.62 +/- 0.03 vs. 0.54 +/- 0.06 mm; P = 0.01). In GHD adolescents, 6 months of GH treatment withdrawal and 6 months of GH treatment reinstitution modified IGF-I levels, lipid profile, and insulin resistance but not IMT or systolic and diastolic peak velocities at the common carotid arteries. In non-GHD subjects, 12 months of GH treatment withdrawal significantly decreased IGF-I levels, IMT (to 0.54 +/- 0.06 mm; P < 0.001 vs. baseline), systolic and diastolic peak velocities, and improved insulin resistance. In conclusion, the discontinuation of GH in confirmed GHD adolescents is not followed by significant alterations of the common carotid arteries, despite the profound negative alterations of the lipid profile. In adolescents who were not confirmed to have GHD, IMT was increased while on GH therapy and normalized when they were taken off of GH.