Journal of Endocrinological Investigation (2021) 44:621–629 https://doi.org/10.1007/s40618-020-01367-6 ORIGINAL ARTICLE Glucagon stimulation test to assess growth hormone status in Prader–Willi syndrome L. Casamitjana1,2,3 · O. Giménez‑Palop1,2,3 · R. Corripio2,3,4 · R. Pareja1,3 · E. Berlanga3,5 · M. Rigla1,2,3 · JC. Oliva3,6 · A. Caixàs1,2,3 Received: 14 May 2020 / Accepted: 16 July 2020 / Published online: 27 July 2020 © The Author(s) 2020 Abstract Purpose Growth hormone deficiency (GHD) must be confirmed before starting treatment in adults with Prader-Willi syn- drome (PWS). Most studies use the growth-hormone-releasing hormone plus arginine (GHRH-arginine) test. No data are available on the glucagon stimulation test (GST) in PWS. We compared the utility of fixed-dose (1 mg) GST versus GHRH- arginine test in diagnosing GHD. Methods Adults and late adolescents with PWS underwent both tests on separate days. In the GHRH-arginine test, GHD was defined according to body mass index. In the GST, two cutoffs were analyzed: peak GH concentration < 3 ng/mL and < 1 ng/ mL. For analyses, patients were divided into two groups according to body weight (≤ 90 kg and > 90 kg). Results We analyzed 34 patients: 22 weighing ≤ 90 kg and 12 weighing > 90 kg. In patients weighing ≤ 90 kg, the two tests were concordant in 16 (72.72%) patients (k = 0.476, p = 0.009 with GST cutoff < 3 ng/mL, and k = 0.450, p = 0.035 with GST cutoff < 1 ng/mL). In patients weighing > 90 kg, the two tests were not concordant with GST cutoff < 3 ng/mL, but were concordant in 11 (91.6%) patients (k = 0.833, p = 0.003) with GST cutoff < 1 ng/mL. GH peaks on the two tests correlated (r = 0.725, p = 0.008). Conclusion Fixed-dose (1 mg) GST using a peak GH cutoff of < 3 ng/mL or < 1 ng/mL promises to be useful for screening for GHD in adults and late adolescents with PWS. However, in those weighing > 90 kg, the < 1 ng/mL cutoff seems better. Larger studies are necessary to establish definitive glucagon doses and cutoffs, especially in extremely obese patients. Keywords Prader–Willi · Growth hormone deficiency · Glucagon-stimulation test · GHRH-arginine test Introduction Prader-Willi syndrome (PWS) is the most common syn- dromic form of obesity, occurring in approximately one in * A. Caixàs acaixas@tauli.cat 10,000–30,000 live births, without sex differences in preva- lence. PWS results from the loss of expression of paternal 1 Endocrinology and Nutrition Department, Hospital alleles in the PWS region of chromosome 15 [1]. PWS is Universitari Parc Taulí, C/ Parc Taulí, 1, 08208 Sabadell, characterized by hypotonia, high adiposity, low lean mass, Spain hypogonadism, and growth hormone deficiency (GHD) [1, 2 Medicine Department, Universitat Autònoma de Barcelona, 2]. In children with PWS, it is widely accepted that treatment Bellaterra, Spain with growth hormone (GH) can be started without provoca- 3 Institut d’Investigació i Innovació Parc Taulí (I3PT), tive tests to demonstrate GHD. GH treatment usually ends Sabadell, Spain when the growth plates close and longitudinal bone growth 4 Pediatrics Department, Hospital Universitari Parc Taulí, finishes. However, after growth and body development, Sabadell, Spain GHD can cause a wide variety of physical and psychologi- 5 Clinical Laboratory Department, UDIAT, Corporació cal problems that can dramatically worsen quality of life. In Sanitària Universitària Parc Taulí, Sabadell, Spain early adolescence and adulthood, GH treatment can only be 6 Epidemiology Department, Fundació Parc Taulí, Sabadell, started if GHD is demonstrated by GH provocative tests [3]. Spain 13 Vol.:(0123456789) 622 Journal of Endocrinological Investigation (2021) 44:621–629 Depending on the provocative tests and body mass index Dyfed, UK). Body weight was measured to the nearest (BMI)-specific cutoffs used to evaluate GH status in differ- 0.1 kg with standard equipment. Body mass index (BMI) ent studies, the prevalence of GHD in PWS varies from 8 to was defined as weight in kilograms divided by the square of 55% [4, 5]. To date, most studies that evaluated GH status height in meters. According to the World Health Organiza- in PWS used a standard growth-hormone-releasing hormone tion classification [20], normal weight was defined as BMI (GHRH) plus arginine (GHRH-arginine) test [6, 7] or, less 18.5–24.9 kg/m2, overweight as BMI 25–29.9 kg/m2, and frequently, an insulin tolerance test (ITT) [8]. The ITT has obese as BMI ≥ 30 kg/m2. traditionally been accepted as the gold standard for assessing adult GHD, but it has potentially serious adverse effects and requires close medical supervision [9]. Furthermore, under- Endocrine protocol lying insulin resistance can prevent normoglycemic and/or hyperglycemic obese patients from achieving the hypoglyce- All subjects underwent a standard GHRH-arginine test and mia necessary to stimulate GH secretion [10]. The GHRH- a standard GST on separate days ranging from 8 to 15 days arginine test is considered the best alternative, but GHRH apart, being randomly assigned to start with one test or the is expensive and not widely available in some countries. other with a random–number generator [21]. Moreover, GHRH-arginine might not be the optimal test for Both tests started at 8 AM after overnight fasting and diagnosing GHD in PWS as GHRH stimulates both pitui- were done with patients in a recumbent position. tary GH synthesis and release [11, 12], whereas arginine potentiates the stimulatory effects of GHRH by inhibiting hypothalamic somatostatin release [13]. These effects can GHRH‑arginine test lead to false-normal GH responses in patients with GHD of hypothalamic origin such as in PWS. A catheter was placed in the antecubital vein; 15 min later The glucagon stimulation test (GST) is an easy-to-per- 1 µg/kg ­G HRH 1-29(GHRH, Ferring GmbH, Kiel, Ger- form, safe, inexpensive, and effective means of stimulating many) was injected as a bolus. In the 30 min after GHRH GH secretion with relatively few contraindications (e.g., administration, 0.5 g/kg (maximum dose 30 g) L-arginine pheochromocytoma or insulinoma) [14, 15]. Its mechanism hydrochloride (Torbay Pharmaceuticals, Paignton, Devon, of action is unclear, but it is apparently not influenced by UK) diluted in 300 cc of saline 0.9% sodium chloride was hypothalamic deficiency [16]. However, glucagon’s effect infused. Blood samples for GH determination were drawn on GH secretion seems to be weight-dependent [14], and at -15, 0, 30, 45, 60, 90, and 120 min after the intravenous most adults with PWS are obese. There is also no consen- bolus of GHRH. GH deficiency was defined as a peak con- sus on whether to use a fixed dose or a weight-based dose centration < 11 ng/mL if BMI < 25 kg/m2, < 8 ng/mL if BMI [17]. Some authors propose two different fixed glucagon 25–30 kg/m2, and < 4 ng/mL if BMI ≥ 30 kg/m2 [22]. doses depending on body weight (1 mg for patients weigh- ing ≤ 90 kg and 1.5 mg for those weighing > 90 kg) [14, 16, 17] or on BMI (1 mg for patients with BMI < 30 kg/m2 and GST 1.5 for those with BMI ≥ 30 kg/m2) [15, 18], whereas others propose weight-based dosing (0.017 mg/kg [19] or 0.03 mg/ All subjects received an intramuscular injection of 1 mg of kg body weight [17]). No prospective data are available glucagon (Novo-Nordisk, Bagsvaerd, Denmark) in the del- about the use of the GST to evaluate GH status in patients toid muscle. Blood samples for glucose and GH determina- with PWS. Thus, we aimed to compare the utility of a single tions were drawn every 30 min from baseline until 180 min fixed-dose (1 mg) GST versus the GHRH-arginine test for [14]. GHD was defined in two ways: according to the clas- diagnosing GHD in adults and late adolescents with PWS. sical definition (peak concentration < 3 ng/mL) [14, 15] and according to the definition recently proposed by Hamrahian et al. [17] (peak concentration < 1 ng/mL). Materials and methods GH concentrations were measured by chemiluminescent immunoassay (LIAISON hGH, Diasorin S.p.A, Saluggia, Patients Italy). The sensitivity was 0.05 ng/mL and intra-assay coef- ficients of variation (CV) were 2.26% at 3.48 ng/mL and We included all adult and late adolescent patients with PWS 1.93% at 17.3 ng/mL. treated at our center between January 1, 2016 and January 31, 2018 (N = 34). All subjects underwent cytogenetic analysis. Height was determined by a Harpenden Stadiometer (Holtain Ltd, 13 Journal of Endocrinological Investigation (2021) 44:621–629 623 Statistical analyses test. Median peak GH concentration was 8.19 ng/mL (IQR: 3.1–14.0 ng/mL). Continuous variables are reported as medians and inter- quartile ranges (IQR). Categorical variables are reported Glucagon‑stimulation test as frequencies and percentages. To assess the concordance between diagnostic tests, we used the Kappa index and its According to the classical cutoff (peak concentration < 3 ng/ standard error. To compare GH peak concentration between mL), 16 (72.7%) patients met the criterion for GHD. Of the two tests, we used the Mann -Whitney U test and for these 16 patients, 2 were normal weight, 5 overweight, and multiple comparisons we used the Kruskal–Wallis test. To 9 obese; of the 6 that did not meet the criterion for GHD, study the relationship between variables, we used regres- 3 were normal weight and 3 obese. The GST classified all sion analysis. Statistical significance was fixed at p < 0.05. patients weighing > 79.5 kg (n = 6) as GHD. Because using a single 1 mg dose of glucagon in patients According to the recently proposed cutoff (1 ng/mL), 10 weighing > 90 kg is not widely accepted, for the purpose of (45.5%) met the criterion for GHD. Of these 10 patients, 1 analysis we divided patients into two groups according to was normal weight, 1 overweight, and 8 obese; of the 12 that body weight (≤ 90 kg and > 90 kg). All analyses were done did not meet the criterion for GHD, 4 were normal weight, with IBM SPSS Statistics for Windows, version 25.0 (IBM 4 overweight, and 4 obese. Corp., Armonk, N.Y., USA). No adverse effects occurred during the test. In 18 (81.8%) patients, the concentration of GH peaked between 120 and 180 min after glucagon administration. Median peak GH concentration was 1.15 ng/mL (IQR: 0.24–3.97 ng/mL). Results Concordance between the GST and the GHRH‑arginine test Patients weighing ≤ 90 kg Using the < 3 ng/mL cutoff for GST, the two tests were con- We evaluated 22 patients with PWS who weighed ≤ 90 kg cordant in 16 (72.7%) patients and discordant in 6 (27.3%) [18 adults and 4 late adolescents; 5 male and 17 female; (k = 0.476, p = 0.009) (Table 2). Using the < 1 ng/mL cutoff, median age, 23.5 y (IQR: 19.7–36.2), range 15–47 y; median the two tests were concordant in 16 (72.7%) patients and BMI, 30.7 kg/m2 (IQR: 24.8–34.0)]. Of these, 5 were nor- discordant in 6 (27.3%) (k = 0.450, p = 0.035) (Table 3). mal weight, 5 overweight, and 12 obese, including 1 with BMI ≥ 40 kg/m2. Cytogenetic analysis revealed that 14 had Subgroup analyses microdeletions (6 type I, 8 type II), 5 had maternal uniparen- tal disomy, and 3 had imprinting defects. Prior to the study, In the 15 patients that underwent GH treatment during 15 patients had undergone GH therapy; all had stopped the childhood, peak GH concentration on the GHRH-argi- therapy at least 1 year before enrollment. At the time of nine test was higher than in those who did not receive GH the study, 10 patients were undergoing sex steroid replace- during childhood [12.5 ng/mL (IQR: 3.58–25.8 ng/mL) ment therapy. A total of 5 patients had type 2 diabetes (2 vs. 3.10 ng/mL (IQR: 1.52–6.13 ng/mL), respectively, treated only with oral agents, 2 with oral agents and GLP-1 p = 0.009]. On the GST, GH peaks were similar: [1.32 ng/ analogues, and 1 with oral agents and insulin); metabolic mL (IQR: 0.32–4.57 ng/mL) for patients with GH treatment control was acceptable (glycosylated hemoglobin < 7%). in childhood vs. 1.00 ng/mL (IQR: 0.18–1.26 ng/mL) for Table 1 reports the characteristics of each patient, peak GH those without, p = 0.332]. Those who received GH treat- concentrations in the two tests, and whether GHD was diag- ment during childhood were younger [23.0 years (IQR: nosed according to each test and cutoff. 19.6–26.7 years) vs. 37.0 years (IQR: 27.3–43.9 years), respectively, p = 0.007] and had lower BMI [28.9 kg/m2 (IQR: 25.9–32.0 kg/m2) vs. 34.3 kg/m2 (IQR: 30.2–36.2 kg/ GHRH‑arginine test m2), respectively, p = 0.039]. Regarding the genetic subtype, those with imprinting According to the established BMI-related cutoffs, 10 defects had the lowest peak concentrations of GH on both (45.5%) patients met the criteria for GHD. Of these 10 tests, being significant only for the GST [Deletion type I: patients, 4 were overweight and 6 obese; of the 12 that did 1.29 ng/mL (IQR: 0.51–5.83 ng/mL), Deletion type II: not meet the criteria for GHD, 5 were normal weight, 1 over- 1.82 ng/mL (IQR: 0.70–7.00 ng/mL), Imprinting defect: weight, and 6 obese. No adverse effects occurred during the 0.10 ng/mL (IQR:0.05-not calculable ng/mL), Maternal uniparental disomy: 1.24 ng/mL (IQR: 0.20–6.84 ng/mL), 13 624 13 Table 1  Characteristics of patients weighing ≤ 90 kg Sex (M/F) Age( years) Weight (kg) BMI (kg/m2) Genetic subtype GH treatment Peak GH GHRH- GHD Peak GH GHD (GH GHD (GH in childhood arginine (ng/mL) (GHRH- GST (ng/ peak < 3 ng/mL) peak < 1 ng/mL) arginine) mL) in GST in GST 1 M 17 73.1 23.3 D II Yes 13.7 No 14.4 No No 2 M 38 73 28.9 MUD Yes 3.58 Yes 1.38 Yes No 3 M 39 79.5 29.9 ID No 1.52 Yes 0.11 Yes Yes 4 M 28 53.6 32.4 MUD No 1.2 Yes 0.18 Yes Yes 5 M 47 84 34.3 D II No 0.42 Yes 0.99 Yes Yes 6 F 15 51 23.3 MUD Yes 14.7 No 12.3 No No 7 F 16 60 23.8 MUD Yes 25.8 No 1.24 Yes No 8 F 23 54.4 23.9 DI Yes 55 No 12 No No 9 F 24 65 24.2 ID Yes 12.5 No 0.1 Yes Yes 10 F 23 55 25.1 DI Yes 50.3 No 1.32 Yes No 11 F 22 74.6 27.1 D II Yes 6.38 Yes 2.57 Yes No 12 F 37 72 28.3 D II No 5.08 Yes 1.06 Yes No 13 F 33 65 30.5 DI Yes 50.5 No 3.77 No No 14 F 23 70.7 31 DI Yes 8.05 No 0.57 Yes Yes 15 F 17 72 31.2 D II Yes 11.4 No 4.57 No No 16 F 23 80 32.8 MUD Yes 3.02 Yes 0.22 Yes Yes 17 F 30 82.5 32.9 ID Yes 3.13 Yes 0.05 Yes Yes 18 F 19 89 33.9 D II Yes 12.5 No 0.6 Yes Yes 19 F 36 68.5 34.5 D II No 2.67 Yes 0.25 Yes Yes 20 F 42 77.6 35.4 D II No 6.13 No 7.81 No NO 21 F 20 84.4 37.5 DI No 10.8 No 1.26 Yes No 22 F 24 87 43.1 DI Yes 2.67 Yes 0.32 Yes Yes BMI body mass index, GH growth hormone, GHD growth hormone deficiency, GHRH-arginine growth-hormone-releasing hormone plus arginine, GST glucagon stimulation test, D I type I deletion, D II type II deletion, MUD maternal uniparental disomy, ID imprinting defect Journal of Endocrinological Investigation (2021) 44:621–629 Journal of Endocrinological Investigation (2021) 44:621–629 625 Table 2  Contingency table in patients weighing ≤ 90 kg (n = 22). Regression analysis growth hormone deficiency (GHD) according to the glucagon stimu- lation test (GST) using peak growth hormone concentration < 3 ng/ mL as the cutoff versus according to the growth-hormone-releasing Peak concentrations of GH observed on the two tests did hormone (GHRH)-arginine test not correlate (r = 0.330, p = 0.134). GH peak on the GHRH- arginine test correlated negatively with BMI (r = − 0.465, GHD with GST (GH GHD with the GHRH-arginine peak < 3 ng/mL), n (%) test n (%) p = 0.029) and weight (r = − 0.562, p = 0.06), but not with age. Peak GH on the GST correlated negatively with BMI No Yes (r = − 0.469, p = 0.028) (Fig. 1), but not with age (r = 0.293, No 6 (100.0%) 0 (0.0%) p = 0.186) or with weight (r = 0.368, p = 0.092). Yes 6 (37.5%) 10 (45.5%) Patients weighing > 90 kg We evaluated 12 patients with PWS who weighed > 90 kg Table 3  Contingency table in patients weighing ≤ 90 kg (n = 22). [12 adults; 7 male and 5 female; median age, 26 y (IQR: growth hormone deficiency (GHD) according to the glucagon stimu- 19.0–32.3), range 18–51 y; median BMI, 41.0 kg/m2 (IQR: lation test (GST) using peak growth hormone concentration < 1 ng/ mL as the cutoff versus according to the growth-hormone-releasing 34.9–49.2)]. All were obese, 7 with BMI ≥ 40 kg/m 2. hormone (GHRH)-arginine test Cytogenetic analysis revealed that 10 had microdeletions (5 type I, 4 type II, and 1 atypical BP2-BP4 microdeletion) GHD with GST (GH peak < 1 ng/ GHD with the GHRH-arginine mL), n (%) test n (%) and 2 had maternal uniparental disomy. Prior to the study, 4 patients had undergone GH therapy; all had stopped the No Yes therapy at least 1 year before enrollment. At the time of the No 9 (75.0%) 3 (25.0%) study, 8 patients were undergoing sex steroid replacement Yes 3 (30.0%) 7 (70.0%) therapy. Two patients had type 2 diabetes (1 treated with oral agents, GLP-1 analogues, and insulin, and the other with oral agents and insulin); metabolic control was accept- able (glycosylated hemoglobin < 7%). Table 4 reports the p = 0.048]. This significance did not disappear after adjust- characteristics of each patient, peak GH concentrations in ment for age, weight, or BMI. the two tests, and whether GHD was diagnosed according Peak GH was higher in females than in males on the to each test and cutoff. GHRH-arginine test [10.8 ng/mL (IQR: 4.11–20.3 ng/ mL) vs. 2.10 ng/mL (IQR: 1.36–8.64 ng/mL), respec- GHRH‑arginine test tively, p = 0.039)], but not on the GST [1.24 ng/mL (IQR: 0.29–4.17 ng/mL) in females vs. 1.0 ng/mL (IQR: According to the established BMI-related cutoffs, 7 (58.3%) 0.15–7.89 ng/mL) in males, p = 0.820)]. patients met the criteria for GHD. Median peak GH concen- There were no differences in GH response on either test tration was 2.90 ng/mL (IQR: 1.38–6.47 ng/mL). between patients treated with sex steroids versus those not treated with sex steroids in the whole group or separated by sex (data not shown). Fig. 1  Correlation between peak growth hormone (GH) concen- tration and body mass index (BMI) on the glucagon-stim- ulation test (GST) in patients weighing ≤ 90 kg. Spearman correlation test, r =− 0.469, p = 0.028 13 626 Journal of Endocrinological Investigation (2021) 44:621–629 Table 5  Contingency table in patients weighing > 90 kg (n = 12): BMI body mass index, GH growth hormone, GHRH-arginine growth-hormone-releasing hormone plus arginine, GHD GH deficiency, GST glucagon stimulation test, D I type I deletion, D II GHD with GST ( peak < 1 ng/mL) growth hormone deficiency (GHD) according to the glucagon stimu- lation test (GST) using peak growth hormone concentration < 1 ng/ mL as the cutoff versus according to the growth-hormone-releasing hormone (GHRH)-arginine test Yes Yes Yes Yes Yes Yes NO No No No No No GHD with GST ( GH GHD with the GHRH-arginine test peak < 1 ng/mL) n (%) GHD with GST ( peak < 3 ng/mL) No Yes No 5 (83.3%) 1(16.7%) Yes 0 (0.0%) 6 (100.0%) Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Glucagon‑stimulation test GST (ng/ Peak GH 0.14 0.14 1.53 1.57 0.05 1.48 0.09 1.18 1.19 1.24 0.27 0.05 mL) According to the classical cutoff (peak concentration < 3 ng/ mL), all patients, met the criterion for GHD. GHRH -argi- According to the recently proposed cutoff (peak concen- GHD with tration < 1 ng/mL), 6 (50%) met the criterion for GHD. Of these 6 patients, 4 had BMI > 40 kg/m2. nine Yes Yes Yes Yes Yes Yes Yes No No No No No No adverse effects occurred during the test. In 10 (83.3%) patients, the concentration of GH peaked between 120 and Peak GH GHRH- arginine (ng/mL) 180 min after glucagon administration. Median peak GH concentration was 0.73 ng/mL (IQR: 0.10–1.42 ng/mL). 1.78 1.25 6.22 6.55 1.48 4.17 3.31 8.84 2.48 0.78 1.34 10.6 Concordance between the GST and the GHRH‑arginine test GH treatment Using the < 3 ng/mL cutoff, because all patients met the cri- in childhood terion for GHD on the GST, the two tests were not concord- ant (k = 0). Yes Yes Yes Yes No No No No No No No No Using the < 1 ng/mL cutoff, the two tests were concord- ant in 11 (91.7%) patients (k = 0.833, p = 0.003) (Table 5). Genetic subtype type II deletion, AD atypical microdeletion, MUD maternal uniparental disomy Regression analysis MUD MUD D II D II D II D II D II AD DI DI DI DI There was a good correlation between peak GH concentra- tions on the two tests (r = 0.725, p = 0.008) (Fig. 2). No cor- BMI (kg/m2) relations were observed between peak GH and BMI, weight, Table 4  Characteristics of patients weighing more than 90 kg or age on either test. 30.6 34.8 34.8 35.3 40.1 44.2 41.8 47.5 49.7 62.9 54 35 Discussion Weight(kg) 127.2 103.5 129.5 We aimed to evaluate the utility of a GST using a fixed- 93.8 98.4 90.5 91.5 110 104 109 115 125 dose (1 mg) of glucagon administered intramuscularly in diagnosing GHD in adults and late adolescents with PWS by Age (years) comparing the results with those achieved with the GHRH- arginine test. In patients weighing ≤ 90 kg, the GST diagno- 51 19 18 19 30 30 33 22 39 19 28 24 sis was concordant with the GHRH-arginine test diagnosis, regardless of whether the peak GH cutoff was < 3 ng/mL Sex (F/M) or < 1 ng/mL. However, in patients weighing > 90 kg, only the < 1 ng/mL peak GH cutoff had good concordance with M M M M M M M F F F F F the GHRH-arginine test. These findings suggest that GST could be used in cases where other, more sophisticated tests 10 11 12 1 2 3 4 5 6 7 8 9 13 Journal of Endocrinological Investigation (2021) 44:621–629 627 Fig. 2  Correlation between peak growth hormone (GH) concentration on the growth- hormone-releasing hormone (GHRH)-arginine test and on the glucagon-stimulation test (GST) in patients weigh- ing > 90 kg. Spearman correla- tion test, r = 0.725, p = 0.008 such as ITT or GHRH-arginine test might be complicated to those with BMI > 40 kg/m2 [25, 26]. In our cohort, all sub- perform because they require intravenous access (sometimes jects weighing > 79.5 kg (n = 18) failed to achieve peak GH difficult in patients with PWS) and have other drawbacks. concentrations > 3 ng/mL in the GST. However, in those Moreover, the GHRH-arginine test might not be the best weighing > 90 kg, defining GHD as a peak GH concentra- test for diagnosing GHD in PWS as it stimulates mainly the tion < 1 ng/mL yielded results more concordant with those pituitary secretion and can lead to false-negative diagnoses of the GHRH-arginine test and peak GH in the two tests for GHD in hypothalamic diseases such as PWS. showed a good correlation. The GST does not require intravenous access, is repro- In the analysis of subgroups of the patients weigh- ducible, safe, and inexpensive; moreover, it is not influenced ing ≤ 90 kg, patients who received GH treatment during by sex or hypothalamic GHD [16]. GST has mild adverse childhood had a higher GH peak on the GHRH-arginine effects including nausea, vomiting, and headaches; more test, probably because this group was younger and had lower rarely and mostly in elderly patients, GST can cause hypo- BMI than the group not treated with GH during childhood. tension, hypoglycemia, or seizures [23]. The lowest GH peak found in the imprinting defect Most [9, 14, 17] but not all [15] authors propose a fixed genetic subtype, even after adjusting for age and BMI, must intramuscular dose of 1 mg glucagon for patients weigh- be interpreted with caution given the lack of power due to ing < 90 kg and 1.5 mg for those weighing ≥ 90 kg. More- the sample size. However, other authors have also reported over, some authors have suggested that using these fixed that the pattern of GH secretion varied by genetic subtype, doses and a peak GH concentration cutoff < 3 ng/ml as rec- with higher GH responses in typical deletion subjects than ommended in the 2009 update of the American Association in patients with disomy [27]. Further studies are needed to of Clinical Endocrinologists guidelines [24] may poten- elucidate the significance of this finding. tially overdiagnose adult GHD in many overweight/obese The higher GH peak on the GHRH-arginine test in subjects and in those with glucose intolerance [25]. For this females than in males could be related to an estrogenic reason, Hamrahian et al. [17] suggested changing the peak effect. Several earlier studies have shown that estrogens can GH cutoff to < 1 ng/mL for the same glucagon fixed doses enhance the GH response to most stimuli, such as hypogly- mentioned above and < 2 ng/mL when glucagon was used cemia and arginine, in men and in premenopausal and post- in a weight-based dose (0.03 mg/kg). These authors also menopausal women [28]. By contrast, no sex-based differ- concluded that the 3 ng/mL cutoff GH peak misdiagnosed ence in GH response to glucagon was observed in this study some GH-sufficient adults. or in others [29, 30], and exogenous estrogen administration In the present study, we used a fixed dose of 1 mg of is ineffective in enhancing the GH response to glucagon in glucagon for all patients, and we analyzed those weigh- men [29]. ing ≤ 90 kg and those weighing > 90 kg separately. The mechanisms by which glucagon stimulates GH secre- In the group weighing ≤ 90 kg, peak GH concentrations tion remain unclear [9]. It seems that peak GH concentra- on GST correlated negatively with BMI. These results are tions do not directly depend on glycemic levels [31]. One in accordance with those of a previous study where peak possible mechanism could be that breakdown of glucagon GH concentrations correlated negatively with weight and in the muscles produces a peptidyl fragment that promotes BMI in healthy controls [14]. Other studies also found that the release of GH [19]. Additionally, glucagon induces nor- the negative correlation between BMI and peak GH con- epinephrine secretion, which might stimulate the release of centrations in the GST was stronger in patients with BMI GH via α-receptors [16]; this mechanism could be of great between 30 kg/m2 and 40 kg/m2 and seemed to plateau for 13 628 Journal of Endocrinological Investigation (2021) 44:621–629 interest in PWS because patients with PWS have underlying Regional (Grant PI-18/00734). This work was also supported by a grant autonomic dysfunction [32]. from Pfizer (2015/594). The route of glucagon administration may also be impor- tant in the release of GH. Intravenous glucagon causes less Compliance with ethical standards GH release than both intramuscular and subcutaneous Conflicts of interest The authors have no conflicts of interest. glucagon [33]. We chose the intramuscular route because it has been suggested to be more reliable and effective than Ethics approval The study complied with all provisions in the Declara- the subcutaneous route [30]. However, new routes, such as tion of Helsinki and was approved by the local ethics committee (Com- intranasal glucagon, recently approved by FDA [34] to treat itè d’Ètica d’Investigació amb. medicaments (CEIm) del Parc Taulí). hypoglycemia, might also prove effective, although to our Consent to participate All patients agreed to participate after being knowledge there is insufficient information about its effec- informed together with their parents or caregivers. Their legal guard- tiveness in provoking the release of GH. ians (usually theirparents) signed the informed consent before enroll- ment. • Our study has several limitations. First, we compared Consent for publication All authors agree to publish these results. the results of the GST against GHRH-arginine test rather than against the gold standard (ITT). However, the ITT Open Access This article is licensed under a Creative Commons Attri- has serious drawbacks in PWS patients, and the GHRH- bution 4.0 International License, which permits use, sharing, adapta- arginine test is more widely used. Second, our analyses tion, distribution and reproduction in any medium or format, as long may have been underpowered to detect small effects, as you give appropriate credit to the original author(s) and the source, given the relatively small sample size including patients provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are with a wide range of BMI. The number of patients included in the article’s Creative Commons licence, unless indicated weighing > 90 kg was low (n = 12), and they received otherwise in a credit line to the material. If material is not included in 1 mg glucagon rather than the 1.5 mg recommended by the article’s Creative Commons licence and your intended use is not other authors. Nevertheless, using the cutoff < 1 ng/mL, permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a we obtained good concordance between the two tests in copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. this group of patients. Finally, we did not take glycemic status into account. Whether glucose levels can interfere with the response in the GST is controversial. Higher blood glucose levels (whether fasting or peak and nadir) References during the GST have been associated with lower peak GH responses in some studies [25, 35]; however, other 1. Cassidy SB, Schwartz S, Miller JL, Driscoll DJ (2012) Prader- authors consider that blood glucose levels do not impair Willi syndrome. Genet Med 14:10–26 glucagon-induced GH release [29, 36]. 2. Angulo MA, Butler MG, Cataletto ME (2015) Prader-Willi syn- drome: a review of clinical, genetic, and endocrine findings. J Endocrinol Invest 38:1249–1263 In conclusion, a fixed-dose (1 mg) GST using a peak GH 3. Deal CL, Tony M, Hoÿbye C et al (2013) Growth hormone cutoff of < 3 ng/mL or < 1 ng/mL seems useful for screening research society workshop summary: consensus guidelines for for GHD in adults and late adolescents with PWS. How- recombinant human growth hormone therapy in Prader-Willi syndrome. J Clin Endocrinol Metab 98(6):E1072–1087 ever, in those weighing > 90 kg, the < 1 ng/mL cutoff is 4. Moix Gil E, Giménez-Palop O, Caixàs A (2018) Treatment with more suitable. Larger studies are necessary to corroborate growth hormone in the Prader-Willi syndrome. Endocrinol Dia- our findings and establish definitive glucagon doses and cut- betes Nutr 65:229–236 offs, especially in patients with extreme obesity or glucose 5. Grugni G, Sartorio A, Crinò A (2016) Growth hormone therapy for Prader-Willi syndrome: challenges and solutions. Ther Clin intolerance. Risk Manag 2(12):873–881 6. Marostica E, Grugni G, De Nicolao G et al (2013) The Acknowledgements We thank the Associació Catalana de Síndrome GHRH+arginine stimulated pituitary GH secretion in children de Prader-Willi for their crowdfunding and all the patients and their and adults with Prader-Willi syndrome shows age- and BMI- families for kindly agreeing to participate. dependent and genotype-related differences. Growth Horm IGF Res 23:261–266 Author contributions LC, OG-P, and AC designed the study, recruited 7. Grugni G, Marzullo P, Ragusa L et al (2006) Impairment of GH the patients, and wrote the first draft of the manuscript. MR, RC, and responsiveness to combined GH-releasing hormone and arginine AC contributed to additional versions of the manuscript. RP, LC, AC, administration in adult patients with Prader-Willi syndrome. Clin and OG-P did all the tests. EB did the laboratory work and JCO ana- Endocrinol (Oxf) 65:492–499 lyzed the data. 8. Beauloye V, Dhondt K, Buysse W et al (2015) Evaluation of the hypothalamic-pituitary-adrenal axis and its relationship with Funding This study was supported in part by the Fondo de Investi- central respiratory dysfunction in children with Prader-Willi gación Sanitaria del Instituto Carlos III-Fondo Europeo de Desarrollo 13 Journal of Endocrinological Investigation (2021) 44:621–629 629 syndrome rare endocrinological diseases. Orphanet J Rare Dis 23. Tavares ABW, Seixas-da-Silva IA, Silvestre DHS et al (2015) 10:4–11 Potential risks of glucagon stimulation test in elderly people. 9. Yuen KCJ, Tritos NA, Samson SL et al (2016) American associa- Growth Horm IGF Res 25:53–56 tion of clinical endocrinologists and American college of endo- 24. Cook DM, Yuen KCJ, Biller BMK et al (2009) American Associa- crinology disease state clinical review: update on growth hor- tion of Clinical Endocrinologists medical guidelines for clinical mone stimulation testing and proposed revised cut-point for the practice for growth hormone use in growth hormone-deficient glucagon stimulation test in the diagnosis of adult. Endocr Pract adults and transition patients - 2009 update. Endocr Pract 15:1–29 22:1235–1244 25. Yuen KCJ, Biller BMK, Katznelson L et al (2013) Clinical char- 10. Lee P, Greenfield JR, Ho KKY (2009) Factors determining inad- acteristics, timing of peak responses and safety aspects of two dos- equate hypoglycaemia during insulin tolerance testing (ITT) after ing regimens of the glucagon stimulation test in evaluating growth pituitary surgery. Clin Endocrinol (Oxf) 71(1):82–85 hormone and cortisol secretion in adults. Pituitary 16:220–230. 11. Barinaga M, Bilezikjian LM, Vale WW et al (1985) Independent https​://doi.org/10.1007/s1110​2-012-0407-7 effects of growth hormone releasing factor on growth hormone 26. Toogood A, Brabant G, Maiter D et al (2012) Similar clinical release and gene transcription. Nature 314:279–281 features among patients with severe adult growth hormone defi- 12. Donze SH, Damen L, van der AlfenVelden JAEM et al (2019) ciency diagnosed with insulin tolerance test or arginine or gluca- Prevalence of growth hormone (GH) deficiency in previously GH- gon stimulation tests. Endocr Pract 18:325–334 treated young adults with Prader-Willi syndrome. Clin Endocrinol 27. Grugni G, Giardino D, Crinò A et al (2011) Growth hormone (Oxf) 91(1):118–123 secretion among adult patients with Prader-Willi syndrome due 13. Alba-Roth J, Müller OA, Schopohl J, Von Werder K (1988) to different genetic subtypes. J Endocrinol Invest 34:493–497 Arginine stimulates growth hormone secretion by suppressing 28. Wiedemann E (1981) Adrenal and gonadal steroids. In: endogenous somatostatin secretion. J Clin Endocrinol Metab Daughaday WH (ed) Endocrine control of growth. Elsevier Ltd, 67(6):1186–1189 New York, pp 67–119 14. Gómez JM, Espadero RM, Escobar-Jiménez F et al (2002) Growth 29. Mitchel ML, Byrne MJ, Sanchez Y, Sawin CT (1970) Detection hormone release after glucagon as a reliable test of growth hor- of growth-hormone deficiency. N Eng J Med 5:539–541 mone assessment in adults. Clin Endocrinol (Oxf) 56:329–334 30. Rao RH, Spathis GS (1987) Intramuscular glucagon as a pro- 15. Conceição FL, da Costa e Silva A, Leal Costa AJ, Vaisman M vocative stimulus for the assessment of pituitary function: growth (2003) Glucagon stimulation test for the diagnosis of GH defi- hormone and cortisol responses. Metabolism 36(7):658–663 ciency in adults. J Endocrinol Invest 26:1065–1070 31. Giuffrida FMA, Berger K, Monte L et al (2009) Relationship 16. Leong KS, Walker AB, Martin I et al (2001) An audit of 500 sub- between GH response and glycemic fluctuations in the glucagon cutaneous glucagon stimulation tests to assess growth hormone stimulation test. Growth Horm IGF Res 19:77–81 and ACTH secretion in patients with hypothalamic-pituitary dis- 32. DiMario FJ, Dunham B, Burleson JA et al (1994) An evaluation of ease. Clin Endocrinol (Oxf) 54:463–468 autonomic nervous system function in patients with Prader-Willi 17. Hamrahian AH, Yuen KCJ, Gordon MB et al (2016) Revised GH syndrome. Pediatrics 93:76–81 and cortisol cut-points for the glucagon stimulation test in the 33. Cain JP, Williams GH, Dluhy RG (1970) Glucagon stimulation of evaluation of GH and hypothalamic–pituitary–adrenal axes in human growth hormone. J Clin Endocrinol Metab 31:222–224 adults: results from a prospective randomized multicenter study. 34. https:​ //www.fda.gov/news-events​ /press-​ announ​ cemen​ ts/fda-appro​ Pituitary 19:332–341 ves-first​-treat​ment-sever​e-hypog​lycem​ia-can-be-admin​ister​ed- 18. Lin T, Tucci JR (1974) Provocative tests of growth hormone witho​ut-injec​tion. Last accessed 23 June 2020 release. A comparison of results with seven stimuli. Ann Intern 35. Wilson JR, Utz AL, Devin JK (2016) Effects of gender, body Med 80(4):464–469 weight, and blood glucose dynamics on the growth hormone 19. Arvat E, Maccagno B, Ramunni J et al (2000) Interaction between response to the glucagon stimulation test in patients with pitui- glucagon and Hexarelin, a peptidyl GH secretagogue, on soma- tary disease. Growth Horm IGF Res 26:24–31. https​: //doi. totroph and corticotroph secretion in humans. Eur J Endocrinol org/10.1016/j.ghir.2015.12.005 143:601–606 36. Spathis GS, Bloom SR, Jeffcooate J (1974) Subcutaneus glucagon 20. https​://www.euro.who.int/en/healt​h-topic​s/disea​se-preve​ntion​/ as a test of the ability of the pituitary to secrete GH and ACTH. nutri​tion/a-healt​hy-lifes​tyle/body-mass-index​-bmi. Last accessed Clin Endocrinol (Oxf) 3:175–186 23 June 2020 21. Domenech J (1981) Tablas de Estadística, 4th edn. Herder, Publisher’s Note Springer Nature remains neutral with regard to Barcelona jurisdictional claims in published maps and institutional affiliations. 22. Corneli G, Di Somma C, Baldelli R et al (2005) The cut-off limits of the GH response to GH-releasing hormone-arginine test related to body mass index. Eur J Endocrinol 153:257–264 13