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Department of Pediatrics (Y.H.C., K.-H.P., E.K.K., D.-K.J.), Department of Pathology (S.Y.S.), Department of Dietetics (E.M.K., M.Y.R.), Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea; and Clinical Research Center (Y.J.O., S.-H.C., S.H.Y.), Samsung Biomedical Research Institute, Seoul 135-710, Korea
Address all correspondence and requests for reprints to: Dong-Kyu Jin, M.D., Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Il-Won Dong, Gang-Nam Gu, Seoul, 135-710, Korea. E-mail: jindk{at}smc.samsung.co.kr.
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Abstract |
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Design: We investigated the densities of ghrelin-expressing cells (GECs), the amounts of ghrelin in gastric tissues, and ghrelin levels in plasma in 16 PWS patients and compared these results with those of 13 GHD patients and comparison groups (19 normal lean and 10 normal obese subjects).
Results: In the gastric body and fundus, 2- to 3-fold increases in the numbers of GECs (P < 0.001) and in the amounts of ghrelin (P < 0.018) were noted in PWS patients vs. comparison groups, whereas GEC numbers in GHD patients were similar to those of the comparison group despite elevated fasting plasma ghrelin levels. In addition, IGF-I SD scores in PWS were not found to be correlated with GEC densities, the amounts of ghrelin expressed in gastric tissues, or plasma ghrelin levels.
Conclusions: Our results suggest that IGF-I or GH/IGF axis deficiency appears to be unrelated to observed GEC increases in the stomach of patients with PWS.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Prader-Willi syndrome (PWS) is the most common form of human syndromic obesity. Ghrelin levels have been reported to be elevated in PWS (5, 6, 7). Moreover, unlike normal obese individuals, who have low fasting-plasma ghrelin concentrations, PWS patients can have 3- to 4-fold higher ghrelin fasting concentrations (5, 6, 7). Elevated plasma ghrelin levels in PWS may be related to low plasma insulin levels, because insulin plays an important role in the suppression of plasma ghrelin (8, 9, 10).
Recently, plasma ghrelin levels were reported to be elevated in patients with GH deficiency (GHD) (11, 12). Because some PWS patients show attenuated plasma GH levels, and because total and free IGF-I have been reported to be low in PWS compared with obese individuals, partial GHD may also contribute to the hyperghrelinemia in PWS.
In the present study, we hypothesized that the hyperghrelinemia observed in PWS is related to IGF-I or GH/IGF axis deficiency. We investigated plasma ghrelin levels, ghrelin-expressing cell (GEC) densities, and ghrelin quantities in the gastric tissues of PWS and GHD patients and in those of lean and obese normal subjects and correlated these results with total IGF-I levels. We found that GEC densities and ghrelin quantities were increased in the fundus and body of the stomachs of PWS patients and that these increases were not related to IGF-I levels, whereas gastric GEC densities and ghrelin quantities in GHD were similar to those observed in lean or obese normal subjects.
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Subjects and Methods |
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Sixteen PWS patients [age, 8.1 ± 2.6 yr; 12 males, 4 females; body mass index (BMI), 25 ± 1.5 kg/m2], 19 healthy normal lean subjects (age, 8.9 ± 1.4 yr; 14 males, 5 females; BMI, 16.5 ± 0.42 kg/m2), 13 GHD patients (age, 9.1 ± 1.8 yr; 9 males, 4 females; BMI, 27 ± 1.8 kg/m2), and 10 healthy normal obese subjects (age, 8.7 ± 2.3 yr; 7 males, 3 females; BMI, 26 ± 1.7 kg/m2) were enrolled in the study. The subjects had no history of GH treatment and were not being treated with GH at study commencement.
PWS patients were genetically confirmed using the standard methylation test. Of the 16 PWS patients, 11 showed deletion of the paternally transmitted chromosome 15q11–13, four had uniparental disomy, and one had an imprinting defect. In terms of the diets of PWS patients, all were routinely monitored monthly by our dietetics department. In addition, nutritional guidelines were provided, and all patients submitted a food diary. Only PWS patients who adhered to the study guidelines were included. These were as follows: total daily calories 10–11 kcal/height (in centimeters) per day were recommended for patients of less than 130% of ideal body weight vs. age- and sex-matched Korean standards, and total daily calories were reduced by 20% for PWS patients 30% over ideal body weight (13).
GHD was diagnosed using a GH stimulation test. Of the 13 GHD patients, seven had developed GHD due to hypopituitarism caused by a brain tumor (six craniopharyngioma, one germinoma). The mean ± SD of peak GH levels in these seven patients was 1.36 ± 1.69 ng/ml. The six craniopharyngioma patients underwent transcranial tumor removal with resection of the pituitary stalk, and the single germinoma patient had completed radiation treatment more than 6 months before study commencement. The 13 GHD patients had no history of GH treatment and were not being treated with GH at study commencement; however, thyroxine, 1-disamino-ß-D-arginine vasopressin, and cortisol were being administered to four of the six GHD patients who had been treated surgically for craniopharyngioma. The other two patients were receiving 1-disamino-ß-D-arginine vasopressin only at the time of the study.
The six patients with idiopathic GHD were diagnosed using a GH stimulation test and had typical manifestations of the disease, i.e. stunted growth, relatively small gonads, and a retarded bone age. The mean ± SD of peak GH levels of idiopathic GHD was 1.86 ± 1.72 ng/ml, and the height SD score (SDS) ranged from –3.64 to –2.37.
Brain magnetic resonance imaging revealed an ectopic posterior pituitary gland and a small pituitary gland volume in four of these six cases, whereas the remaining two cases had normal brain magnetic resonance imaging findings. Blood samples were taken, and stomach biopsies were performed 3 months after GH stimulation tests in the 13 GHD patients.
The lean and obese normal subjects (comparison group) enrolled were the children or siblings of hospital staff who understood the purpose of and the procedures used. These comparison group subjects were recruited on the basis of age and BMI status and were then allocated to obese or lean groups according to BMI status. The criteria used to recruit the comparison group were an absence of a history of significant major illness (especially gastrointestinal disease) and of unusual dietary habits. These were verified by interview with a dietician using a 1-month food diary, and these and subjects were enrolled in the study only if the dietician considered that food habits and food uptake were normal.
Informed consent was obtained from all participants or parents, as appropriate. The study design was reviewed and approved by the Samsung Medical Center Institutional Review Board.
Gastroduodenal endoscopy and biopsy samples
All subjects underwent gastroduodenal endoscopy after overnight fasting and were intravenously injected with midazolam (0.1 mg/kg) 5 min before endoscopy. In addition, blood samples were taken for fasting plasma ghrelin determinations before the midazolam injection. Six biopsy specimens were taken at three sites along the greater curvature of the stomach, i.e. two from the antrum, two from the body, and two from the fundus. One specimen from each of these sites was forwarded to a pathologist for immunohistochemical staining. The other three specimens were stored at –70 C for ELISA.
Peptide extraction from stomach tissue
Peptides levels in stomach biopsy specimens were assayed using enzyme immunoassay kits, according to the instructions of the manufacturer (Phoenix Pharmaceuticals, Belmont, CA; http://www.phoenixpeptide.com) (14). Briefly, 200 µl of 0.1 M acetic acid was added to each tissue sample (wet weight, 14.4 ± 1.16 mg) and placed in a boiling water bath for 10 min. After cooling on ice, samples were homogenized in polypropylene tubes and centrifuged at 13,000 x g for 15 min. Pellets obtained were resuspended in 3 N NaOH (200 µl) and analyzed for total protein using a bicinchoninic acid assay kit (Sigma, St. Louis, MO). For this protein assay, 50 µl of supernatant was removed from centrifuged suspension; the remainder was used for ghrelin ELISA. The interassay and intraassay coefficients of variance of the protein assay were 9.4 and 7.2%, respectively, and the test sensitivity was 2 ng/ml.
ELISA for ghrelin and RIA for total IGF-I
Ghrelin levels in stomach biopsy specimens and in plasma were measured using a commercially available human ghrelin ELISA kit (Phoenix Pharmaceuticals), as described previously (15). The interassay and intraassay coefficients of variance were less than 10%, and the lower and upper assay detection limits were 0.75 and 100 ng/ml, respectively. The quantities of ghrelin in stomach-biopsy specimens are described as ratios of ghrelin to total protein (nanograms per milligram). Values represent the averages of four experiments. Total IGF-I was determined by RIA using a commercially available kit (Immunotech, Marseille, France), which had interassay and intraassay coefficients of variance of 6.3 and 6.8%, respectively, and a test sensitivity of 2 ng/ml. Samples and standards were incubated in tubes coated with the first monoclonal antibody in the presence of the second antibody labeled with iodine 125. After incubation for 60 min at 18–25 C with shaking (350 rpm), bound radioactivity was measured. Radioactivity recovery percentages were between 78 and 111%. Data are expressed as concentrations and as SDS, the latter of which were deduced from age, sex, and Tanner stage-related data (16).
Immunohistochemistry for stomach ghrelin
A total of 174 tissues samples (three biopsy specimens from each of the 58 participants) were examined. Tissues were immediately fixed with 10% neutrally buffered formalin for 18 h and embedded in paraffin, and sections were taken serially at 5 µm. Conventional hematoxylin and eosin staining was performed on the first cut-section to confirm the histopathological architecture of the biopsy site. Twenty serial cut-sections from formalin-fixed paraffin-embedded tissues were collected on poly-L-lysine-coated slides for immunohistochemical study. Immunohistochemical staining was performed using a standard streptavidin-peroxidase procedure (LSAB2 kit; Dako, Glostrup, Denmark) using a polyclonal antihuman ghrelin antibody (amino acids 13–28; dilution 1:500; Phoenix Pharmaceuticals). Primary antibody was incubated for 1 h at room temperature with citrate buffer (pH 6.0) microwave (1200 W for 5 min) to retrieve antigen. Finally, slides were stained with 3,3-diaminobenzidine tetrachloride for 3 min and counterstained with hematoxylin. The negative immunohistochemical controls used either lacked primary antibody or had primary antibody replaced with normal rabbit IgG. The positive control used for ghrelin immunohistochemistry was healthy gastric mucosa, as described previously (17).
Morphometric analysis of ghrelin expression in the stomach
We measured the densities of gastric neuroendocrine cells showing ghrelin expression in the antrum, body, and fundus. Immunohistochemical ghrelin expressions in the stomach were evaluated in alternate serial sections to avoid recounting cells under a BX50 Olympus Optical (Tokyo, Japan) light microscope. Ghrelin densities were expressed as the number of GECs per 0.024 mm2. Biopsy slides were read in a blind manner by two pathologists.
Statistical analysis
Data normality was tested using the Shapiro-Walk’s test, and, if data were not normally distributed, the Wilcoxon’s signed rank test was used; otherwise the paired t test was used to determine differences. Data are expressed as means and SDs throughout, and P values < 0.05 were regarded as statistically significant. All statistical analyses, including correlation analysis, were performed using SPSS (Chicago, IL) software.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Fasting plasma ghrelin levels were as follows: 5050 ± 1600 pg/ml in PWS patients; 6440 ± 1190 pg/ml in GHD patients; 1780 ± 480 pg/ml in normal lean subjects; and 1610 ± 580 pg/ml in normal obese subjects.
GEC density
Immunohistochemically, ghrelin was found to be mainly located in the cytoplasm of gastric glandular cells located in the body base or fundus. Morphometric analysis was used to quantitate the number of positive cells per 0.024 mm2. Although GEC staining in the antrum was not entirely negative, it was slight compared with that in the fundus, and no differences in GEC density were observed between the four subject groups. However, GEC staining was noted in the body and fundus of the stomach in all four groups (Fig. 1A
). Remarkably, 2- to 3-fold increases in GEC density were noted in PWS patients vs. lean or obese normal subjects (P < 0.001) (Table 1 and Fig. 1B). Moreover, GEC increases were remarkable, without exception, in the stomachs of PWS patients (Fig. 1B), regardless of genotype, and these correlated with plasma ghrelin levels (body, r = 0.84, P = 0.03; fundus, r = 0.81, P = 0.03). However, GEC densities in GHD were similar to those in comparison groups.
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To support the immunohistochemical findings, ghrelin in the gastric body and fundus were quantitated by ELISA (Table 1
) and were found to be significantly higher in PWS patients than in normal obese subjects (P = 0.018). Moreover, plasma ghrelin levels as determined by ELISA were found to be correlated with ghrelin quantities in the gastric body (P = 0.001; r = 0.761) and fundus (P = 0.04; r = 0.536) of PWS patients.
Mean IGF-I levels in the study groups
The mean ± SD of serum IGF-I in the 16 PWS patients was 80.4 ± 66.7 ng/ml (median, 53.0; interquartile range, 12–204), and the mean ± SD of serum IGF-I SDS in PWS patients was –2.70 ± 2.69 (median, –2.58; interquartile range, –4.9 to –0.40).
The mean ± SD of serum IGF-I of the 13 GHD patients was 49.4 ± 27.4 ng/ml (median, 45.0; interquartile range, 26.2–75.8), and the mean ± SD of IGF-I SDS corresponds to –3.83 ± 3.57 (median, –3.20; interquartile range, –4.59 to –0.49).
Correlations between IGF-I (or IGF-I SDS) with plasma ghrelin, immunohistochemistry data (in fundus, body, and antrum), ghrelin levels (in fundus, body, and antrum), or age
Because pituitary GHD is not a universal finding in PWS, whereas IGF-I levels are almost always low (as demonstrated by this study), a decision was made to study the correlation between study variables and IGF-I rather than GH. Thus, we searched for correlations between IGF-I and the following variables: plasma ghrelin, immunohistochemistry data (in fundus, body, and antrum), ghrelin levels (in fundus, body, and antrum), and age. However, of these variables, only age was found to be significantly correlated with IGF-I (r = 0.79; P = 0.003). We then reexamined the relationships between the above variables and IGF-I SDS after adjusting for age. However, no significant correlations were found: plasma ghrelin (r = 0.23, P = 0.40); immunohistochemistry data (fundus, r = 0.08, P = 0.74; body, r = 0.34, P = 0.18; antrum r = 0.17, P = 0.51); and ghrelin quantity data (fundus, r = 0.27, P = 0.30; body, r = 0.13, P = 0.62; antrum, r = –0.007, P = 0.98).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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One potential explanation for the difference between PWS and GHD may be in their different sites of fat accumulation and insulin levels and sensitivities. PWS patients have a significantly higher percentage of body fat than obese subjects in the arms and legs but not in the trunk (18, 19). Therefore, the ratio of lean mass in the trunk to that in the limbs is significantly higher in PWS patients than in obese and normal-weight subjects (20). Moreover, fasting insulin levels were reported to be significantly lower in obese PWS subjects than in subjects with simple obesity (21, 22), and levels of insulin sensitivity were significantly higher in obese PWS subjects (23). In contrast, in GHD patients, the excess fat mass has a predominantly truncal distribution (24).
Adults with hypopituitarism including both severe GHD and GH insensitivity were reported to be insulin resistant under conditions of insulin stimulation (25). Plasma ghrelin levels have been reported to increase after carbohydrate-rich meals, and decreases in plasma ghrelin levels are accompanied by increases in plasma insulin (26, 27, 28). Evidence supporting an inhibitory role of insulin with respect to ghrelin secretion has been reported (8, 9, 10). Therefore, relative hypoinsulinemia may act as an additive signal to enhance hyperghrelinemia in PWS.
It should be noted that pituitary GHD is not a universal finding in PWS, although IGF-I levels are almost always low. A reduced IGF-I level may be a common characteristic of both diseases. However, IGF-I, which mediates most of the effects of GH, apparently does not mediate changes in GEC numbers, because the present study found that both the PWS and GHD groups had a low IGF-I.
The relationship between hyperghrelinemia and hyperphagia needs additional study. The GHD subjects in the present study were presumably not hyperphagic. In addition, the direct orexigenic effect of physiological ghrelin concentrations has not been demonstrated in rodents or humans.
In conclusion, our findings demonstrate that both PWS and GHD patients have increased serum ghrelin levels, but that only PWS patients show an increase in GEC numbers. These observations suggest that high serum ghrelin levels in these two conditions occur via different mechanisms.
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Footnotes |
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First Published Online June 14, 2005
Abbreviations: BMI, Body mass index; GEC, ghrelin-expressing cell; GHD, GH deficiency; PWS, Prader-Willi syndrome; SDS, SD score.
Received October 18, 2004.
Accepted May 31, 2005.
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