Vol. 8/2009 Nr 4(29) Endokrynologia Pediatryczna Pediatric Endocrinology Evaluation of Correlations among Secretion Rates of Ghrelin, the Growth Hormone and the Insulin-like Growth Factor I in Children with Short Stature Ocena zależności pomiędzy wydzielaniem greliny, hormonu wzrostu i insulinopodobnego czynnika wzrostowego I u dzieci z niedoborem wzrostu 1,2 Renata Stawerska, 1,2 Joanna Smyczyńska, 2,3 Andrzej Lewiński, 4 Hanna Pisarek, 1,2 Maciej Hilczer 1 Klinika Endokrynologii Wieku Rozwojowego Uniwersytetu Medycznego w Łodzi 2 Klinika Endokrynologii i Chorób Metabolicznych Instytutu Centrum Zdrowia Matki Polki w Łodzi 3 Klinika Endokrynologii i Chorób Metabolicznych Uniwersytetu Medycznego w Łodzi 4 Pracownia Diagnostyki Hormonalnej Zakładu Neuroendokrynologii Katedry Endokrynologii Uniwersytetu Medycznego w Łodzi Adres do korespondencji: Renata Stawerska, Klinika Endokrynologii i Chorób Metabolicznych, Instytut Centrum Zdrowia Matki Polki, 93-338 Łódź, ul. Rzgowska 281/289, tel./fax: 42 271 13 43, e-mail: endo-iczmp@lodz.home.pl Key words: ghrelin, growth hormone, insulin-like growth factor I, short stature, children Słowa kluczowe: grelina, hormon wzrostu, insulinopodobny czynnik wzrostu I, niedobór wzrostu, dzieci The study was supported with the funds from the Ministry of Science and IT (Project No. 2 P05E 01030) Praca finansowana ze środków Ministerstwa Nauki i Informatyzacji (nr 2 P05E 01030) ABSTRACT/STRESZCZENIE Introduction. Ghrelin is a hormone which stimulates growth hormone (GH) secretion, acting independently on somatoliberin. The aim of the study was to assess the secretion of ghrelin in children with short stature, taking into account the GH secretion and the insulin-like growth factor type I (). Material and methods. The analysis concerned 34 children (17 girls and 17 boys) with short stature, aged: 3.9-16.6 years, with normal thyroid function and without any ailments from gastrointestinal tract. Fasting plasma ghrelin and serum concentrations were measured in each child. was expressed as SDS for age and sex. The maximal GH concentration was assessed every half an hour the period of three hours, falling asleep, and in two stimulating tests (clonidine and glucagon). The patients were qualified into the following groups: GHD (growth hormone deficiency maximal GH concentration in both stimulating tests below 10 ng/ml), NSD (neurosecretory dysfunction decreased GH secretion falling asleep and normal secretion in stimulating test), ISS (idiopathic short stature) normal GH secretion. Results. No correlations were observed between ghrelin and maximal GH concentrations, both in stimulating tests or and falling asleep. No differences were found in ghrelin concentrations among the analyzed groups. Significantly higher (p < 0.01) ghrelin concentrations were noticed in children with decreased concentrations than in children with normal secretion rates. A negative correlation between ghrelin concentrations and SDS (r = -0.51; 9
Praca oryginalna Endokrynol. Ped., 8/2009;4(29):9-16 p < 0.05) was observed in the unde group of studied subjects analysed group of children. Conclusions. A negative correlation between ghrelin and concentrations, with no relationship between ghrelin and GH secretion in children with short stature, indicates the necessity of further studies on the mechanisms of the regulation of GH and secretion. Pediatr. Endocrinol. 8/2009;4(29):9-16. Wstęp. Grelina stymuluje wydzielanie hormonu wzrostu (GH), działając niezależnie od somatoliberyny. Celem pracy było porównanie stężenia greliny u dzieci z niedoborem wzrostu w zależności od wydzielania GH i insulinopodobnego czynnika wzrostowego I (). Materiał i metody. Do badań zakwalifikowano 34 dzieci z niedoborem wzrostu (17 dziewcząt i 17 chłopców) w wieku 3,9-16,6 lat (średnia±sd: 10,1±3,6 lat) z prawidłową czynnością tarczycy oraz bez dolegliwości ze strony przewodu pokarmowego. U każdego dziecka rano na czczo oceniano stężenie całkowitej ghreliny i w surowicy krwi. Stężenia wyrażono wartością SDS dla wieku i płci. Maksymalne stężenie GH (maxgh) w surowicy oceniano na podstawie pomiarów przeprowadzanych co pół godziny w ciągu 3 godzin po zaśnięciu oraz w 2 rutynowych testach stymulacyjnych (po podaniu klonidyny i glukagonu). Na podstawie wartości maxgh pacjentów zakwalifikowano do następujących grup: GHD (growth hormone deficiency) maxgh w obu testach stymulacyjnych oraz po zaśnięciu obniżone (poniżej 10 ng/ml); NSD (neurosecretory dysfunction) maxgh po zaśnięciu obniżone, a prawidłowe w testach stymulacyjnych (powyżej 10 ng/ml); ISS (idiopathic short stature) prawidłowe wydzielanie GH (maxgh powyżej 10 ng/ml w przynajmniej jednym z testów stymulacyjnych oraz po zaśnięciu). W zależności od wyniku dzieci zostały dodatkowo podzielone na grupy z prawidłowym oraz z obniżonym stężeniem. Wyniki. Nie stwierdzono korelacji pomiędzy stężeniem greliny a maksymalnym wydzielaniem GH w testach stymulacyjnych oraz po zaśnięciu. Nie wykazano znamiennych różnic w stężeniu greliny pomiędzy analizowanymi grupami. Stwierdzono natomiast znamiennie wyższe (p < 0,01) stężenie greliny u dzieci z obniżonym wydzielaniem niż u dzieci z prawidłowym wydzielaniem oraz znamienną ujemną korelację pomiędzy stężeniem greliny a SDS (r = -0,51; p < 0,05) w całej analizowanej grupie dzieci z niedoborem wzrostu. Wniosek. Istnienie ujemnej korelacji pomiędzy stężeniem greliny i przy braku zależności pomiędzy stężeniem greliny a wydzielaniem GH u dzieci z niedoborem wzrostu wskazuje na celowość dalszych badań nad mechanizmami regulacji wydzielania GH i. Endokrynol. Ped. 8/2009;4(29):9-16. Introduction Although ghrelin is regarded to be a growth hormone (GH) secretion stimulating factor, its effects on the hypothalamus, or the pituitary gland have not been explained yet, as for as suggested effects on the somatoliberin (GHRH) GH insulin-like growth factor type I () axis is concerned. It is known that ghrelin is influenced by negative feedback mechanisms, other than GHRH. Nevertheless, its activity is synergistic and complementary with the activity of the above neurohormone [1, 2]. Following somatostatin (GHIH) administration, ghrelin concentration in blood decreases. Even thongh it has been proven that ghrelin, when administered in pharmacological doses, stimulates GH secretion, there is no evidence whether ghrelin is able to stimulate GH secretion also in physiological conditions [3]. Ghrelin effects on GH and secretion rates have been studied for the last few years. Nontheless, the explanation of their mechanism is still incomplete. It has been found that, in GHD-affected adults, fasting ghrelin concentration is not significantly different from that of normal subjects. So far, very few studies have attempted to evaluate ghrelin concentrations in children with short stature due to different etiology. Therefore, the goal of the study was to assess ghrelin concentrations in children with short stature, taking into account GH and concentrations. Material & methods A consent for the study was obtained from the local Bioethical Commission. Subjects: of 34 children (17 girls and 17 boys), aged: 3.9-16.6 years (the mean ± SD: 10.1 ± 3.6 years) with short stature, defined as height below - 2.0 SDS from the median value for child s age and sex, determined on the basis of the actual population standards. Children without chronic diseases or sufferings in history were qualified into the study. Body height and weight were measured in all the qualified patients, followed by the calculation of height standard deviation score (HSDS) and body mass index standard deviation score (BMI SDS). The stage of puberty was assessed in each child by the Tanner scale. The lack of any pubertal features was identified in 30 out of 34 children, qualifying the patients to stage I of the Tanner scale; the other 10
Stawerska R. i inni: Evalution of Correlations among Secretion Raters of Ghrelin, the Growth Hormone and the Insulin-like Growth Factor I in Children... four cases presented puberty at stage II or III of the Tanner scale. The children were in-patients of the Department of Endocrinology and Metabolic Diseases, Polish Mother s Memorial Hospital Research Institute. Thyroid dysfunction was excluded in each of children by TSH and FT 4 tests. Girls were also submitted to karyotype test to exclude Turner s syndrome. A 3-hour nocturnal profile of GH secretion was recorded every half an hour in each child during sleep, starting from the first hour falling asleep. Then two stimulation tests were performed on subsequent days of hospitalisation. Both tests are routinely used at the Department and include: the oral clonidine test in dose of 0.15 mg/m 2, during which GH was measured at 0, 30 th, 60 th, 90 th, 120 th minute of the test, and the test with intramuscular administration of glucagon in dose of 30 µg/kg, during which GH was measured at 0, 90 th, 120 th 150 th and 180 th minute. Peak GH concentration ( ) was determined in both tests and falling asleep. Following individual values, the patients were qualified to the following groups: 1. GHD (growth hormone deficiency) if < 10 ng/ml in both stimulation tests and in sleep; 2. NSD (neurosecretory dysfunction) if < 10 ng/ml (decreased) in sleep but > 10 ng/ml (normal) in, at least, one stimulation test; 3. ISS (idiopathic short stature) if > 10 ng/ml (normal) in sleep and in, at least, one stimulation test. On the first hospitalisation day, fasting serum concentrations of total ghrelin and were measured. concentrations were expressed by SDS for age and sex. The children were divided into the following two groups, according to levels: normal (if SDS > -1.0), decreased (if SDS -1.0). The growth hormone was measured by the immunometric method at the Immunohistochemical Laboratory of the Department of Laboratory Diagnostics of Polish Mother Memorial Hospital Research Institute in Lódź. The measurements were performed by IMMULITE, DPC assay sets, calibrated vs the WHO IRP 80/505 standard set, with sensitivity: 0.01 ng/ml, range: up to 40 ng/ml, the conversion index: ng/ml x 2.6 = miu/l, the intra-assay CV: 5.3-6.5% and inter-assay CV: 5.5-6.2%. The total ghrelin was measured by radioimmunoassay at the Hormonal Diagnostic Laboratory of the Department of Neuroendocrinology, Medical University of Lódź. The measurements were performed by LINCO Research, with sensitivity 93 pg/ml, the intra-assay CV: 4.4-10.0% and inter-assay CV: 14.7-17.8%. When GHD was identified, other pituitary hormone tests and MR examination of pituitary region were performed. The isolated GHD was identified in all the cases, without any supporting justification in MR images. One-way ANOVA was applied for statistical analysis with post-hoc tests to account for median differences (because of the differences in the number of patients, Tukey s test was used for assays with a different number of patients. The analysis of correlations among parametric data was based on a typical regression and correlation analysis. The level of significance at p < 0.05 was accepted for all the tests and comparisons. Results Following the obtained results, five (5) patients were qualified into the GHD group, 10 patients into the NSD group and 19 into the ISS group (see Table 1 for age, clonidine and glucagons stimulation tests, and falling asleep, as well as blood serum concentrations of and ghrelin in particular groups of children). Significant differences were found, regarding the maximal GH secretion in particular groups, which was at the base of their isolation, while no significant differences were found in SDS among GHD, NSD and ISS groups. No significant differences were observed among ghrelin concentrations in the analysed groups of patients (see Table I). No significant correlations between ghrelin concentrations and maximal GH secretion rate in the clonidine stimulation test, in the glucagon stimulation test and falling asleep were found in the entire group of children. A significant negative correlation was found between ghrelin concentration and SDS (r = -0.51; p < 0.05), (see Figure 1). See Table II for data regarding age, in stimulation tests, and falling asleep, as well as serum and ghrelin concentrations in the groups, isolated by SDS results. concentrations did not reveal any significant differences 11
Praca oryginalna Endokrynol. Ped., 8/2009;4(29):9-16 Table I. Age, maximal GH concentration stimulation tests and falling asleep, as well as and ghrelin serum concentrations in GHD, NSD and ISS groups Tabela I. Wiek, maksymalne stężenie hormonu wzrostu (maxgh) w testach stymulacyjnych i po zaśnięciu oraz stężenie i ghreliny w surowicy krwi w grupach GHD, NSD i ISS Group The number and sex of patients (f/m) GHD n = 5 (3/2) NS ISS n = 10 (4/6) n = 19 (10/9) Age (mean ±SD) 10.91 ±2.52 10.64 ±4.04 9.56 ±3.07 clonidine 5.58 ±1.16 13.86 ±10.30 16.37 ±7.04 glucagon 6.13 ±2.74 10.73 ±6.33 9.37 ±4.91 in sleep (ng/ml) 5.25 ±3.91 7.27 ±1.15 15.21 ±5.89 (ng/ml) 203.30 ±153.46 217.52 ±175.43 171.84 ±84.41 SDS -1.07 ±1.02-0.94 ±1.31-0.86 ±0.91 Total ghrelin (pg/ml) 1604.92 ±656.08 1698.95 ±1754.62 1485.10 ±858.83 p > 0.05 p > 0.05 p > 0.05 p > 0.05 Table II. Age, maximal GH concentration stimulation tests and falling asleep, as well as and ghrelin serum concentrations in the group of children with normal and decreased concentrations Tabela II. Wiek, maksymalne stężenie hormonu wzrostu (maxgh) w testach stymulacyjnych i po zaśnięciu oraz stężenie i ghreliny w surowicy krwi w grupie dzieci z prawidłowym i z obniżonym stężeniem Group The number and sex of patients (f/m) Age (mean ±SD) clonidine glucagon in sleep (ng/ml) (ng/ml) SDS Total ghrelin (pg/ml) < -1.0 SD n = 16 (5/11) 9.91 ±3.64 14.44 ±10.34 8.42 ±4.43 11.63 ±7.48 116.21 ±65.45-1.77 ±0.78 2080.91 ±1435.60 > -1.0 SD n = 18 (11/7) 10.25 ±3.03 13.58 ±6.67 10.00 ±5.85 11.02 ±5.41 252.32 ±131.76-0.21 ±0.56 1140.68 ±603.17 p > 0.05 p > 0.05 p > 0.05 p > 0.05 p < 0.05 p < 0.05 p < 0.05 among the groups, taking into accaunt both stimulation tests and sleep. Significantly higher (p < 0.01) ghrelin concentrations were found in children with decreased than secretion values (in comparison with children displaying normal secretion values see Table II). Discussion Ghrelin effects, exerted on the stimulation of GH secretion by somatotropic cells of the pituitary gland, one still a controversial subject. Ghrelin secretion presents a pulsatile pattern, with maintained circadian rhythms and higher nocturnal concentrations, similarly to GH [3]. Although the equal secretion patterns of ghrelin and GH during 24 h are suggestive of GH secretion rates to be biased by endogenous ghrelin, no such a correlation has been confirmed so for. It is also possible that the mutual relations between GH and ghrelin concentrations result from GHIH effects. GHIH inhibits GH secretion by its direct effects onto the pituitary gland and by GHRH suppression. It also inhibits ghrelin secretion. Conversely, low GHIH concentrations (resulting from the negative feedback mechanism under high concentrations of GH) stimulate ghrelin secretion. It may also be possible that high ghrelin concentrations suppress SMS secretion, which increases GH concentration. 12
Stawerska R. i inni: Evalution of Correlations among Secretion Raters of Ghrelin, the Growth Hormone and the Insulin-like Growth Factor I in Children... Fig. 1. Correlation between SDS and serum ghrelin concentrations in the analysed group of children with short stature Ryc. 1. Korelacja pomiędzy wartością SDS i stężeniem ghreliny w analizowanej grupie dzieci z niedoborem wzrostu No differences were observed in our study of ghrelin concentration levels among the groups of children with GH, NSD or ISS. Similarly, Tauber et al. [4] did not find any significant differences between morning ghrelin concentrations in children with idiopathic GHD vs the group of healthy children with normal height. In turn, Ghizzoni et al. made a very interesting observation in their study [3]. The authors evaluated circadian ghrelin secretion rhythms in children with NSD and ISS, demonstrating that, unless there are no differences between diurnal ghrelin concentrations in either of the studied groups, then nocturnal ghrelin concentrations were significantly higher in children with NSD than in those with ISS [3]. Therefore, an extension of our study by nocturnal profile may bring similar results. The criterion of normal anatomical structure of the hypothalamus and of the pituitary, applied in the group of our patients, was an important aspect of the study, as it is certain that children with pituitary stalk interruption syndrome (PSIS) present significantly higher ghrelin concentrations, similar to those of the Prader Willi syndrome [4]. Therefore, hyperghrelinemia, as observed in the syndrome, does not increase GH secretion. Consequently, Tauber et al. presented a thesis that ghrelin acts mainly at the hypothalamic level and a primary injury of this region and of its links with the pituitary is at the base of the ghrelin-resistance syndrome [4]. This thesis is supported by the fact that ghrelin administration in subjects with GHD causes a significantly higher stimulation of GH secretion in cases of a normal pituitary stalk than in cases of PSIS. Therefore, it seems that ghrelin activity predominantly targets the hypothalamus and a normal link between the hypothalamus and the pituitary is necessary to demonstrate its effect onto GH [5, 6]. Nevertheless, taking into consideration studies of other authors, concerning adult patients with GHD, no correlations were found in their reports between ghrelin concentrations and in the stimulation tests, while the base ghrelin concentration in subjects of the control group did not differ from those with GHD [7]. The authors suggest that ghrelin concentration in patients with GHD may seem unchanged vs the persons of the control group, as obesity develops in GHD, reducing ghrelin secretion rates [7]. Also in healthy children, a clear correlation between ghrelin concentration and child s BMI SDS is observed the higher BMI SDS, the lower serum ghrelin concentration. Moreover, it is suggested that decreased GH secretion in stimulation tests in children with obesity may be associated with decelerated stimulation of GH by ghrelin in this group [9, 10]. However, no correlation was found between BMI SDS and ghrelin in our group of patients which did not include any case of obesity. 13
Praca oryginalna Endokrynol. Ped., 8/2009;4(29):9-16 Analysing the results of our studies, we also took into account the child s age, puberty stage and sex. It is known that ghrelin concentration negatively correlates with the child s age. It also depends on the stage of puberty there is some evidence that the concentration of this hormone is higher in children before puberty than in those during this period. Since no sexual dysmorphism is observed regarding ghrelin secretion in non-pubertal children, the more distinctive is the trend of ghrelin concentration decrease in subsequent stages of puberty, much more in boys than in girls [2]. Therefore, it is extremely important that our group of patients should include children in the prepubertal age. The mean age of patients was almost the same, without any significant differences among groups. For this reason, we may assume that the results of our studies were not affected both by the age or puberty stage of the participating patients and taking into account the described lack of sexual dysmorphism in the children before pubertal age the sex of patients. Therefore, all the evaluated groups were uniform with regards to that observation. We demonstrated, however, a close negative correlation between and ghrelin concentrations in children with short stature. It should be noted that also in healthy children a negative correlation is observed between concentration (also IGF BP3) and ghrelin concentration. It is not surprising, as secretion increases with child s age and puberty progression, while, as it is known, ghrelin concentration demonstrates a parallel drop. Thus, these two facts do not need to be mutually dependent but they may be associated with other factors affecting the child s growth and pubertal development. By contrast, the results of our studies demonstrate that in the group of children, uniform with regards to age and pubertal stage, ghrelin concentrations are closely related with concentrations, which, in our opinion, is a reliable evidence for mutual relationship between these two hormones. Also, because there is no simultaneous dependence of ghrelin and on GH secretion, it seems that ghrelin cannot be the factor directly stimulating GH secretion. The fact that ghrelin secretion was significantly higher in the group of children with normal concentrations vs the group with decreased secretion (regardless of the diagnosis obtained from results) is another confirmation for the hypothesis. The oligosymptomatic disorders of the gastric functionality should also be considered. They may be a potential case of short stature and low secretion, while simultaneously suppressing ghrelin secretion by reducing the number of ghrelin-positive gastric cells [11 13]. It should be then remembered that ghrelin is produced not only by cells of the hypothalamus but also of the stomach and the pancreas and its secretion depends on the energetic status of the body, being stimulated by fasting and suppressed meals and under hyperglycaemia [14]. On the other hand, one should keep in mind the imperfection of stimulation tests results with regards to GH secretion. Conclusion The existence of a negative correlation between ghrelin and concentrations, with a parallel lack of correlation between ghrelin concentration and GH secretion in children with short stature justifies further studies to be undertaken with regards to the mechanism of GH and secretion control. 14