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Departments of Pediatrics (K.H.P., Y.H.C., E.K.K., W.Y.S., D.-K.J.) and Orthopedic Sports Medicine (W.H.P.), Samsung Medical Center, Sungkyunkwan University School of Medicine, and Clinical Research Center (Y.J.O., A.H.K., S.H.C., S.W.K., S.J.H.), 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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Objective: The objective of the study was to analyze changes in acylated ghrelin (AG) and des-acylated ghrelin (DAG) levels after glucose loading and characterize correlations between insulin sensitivity and ghrelin suppression.
Design: Plasma glucose, insulin, AG, and DAG levels were measured in PWS children (n = 11) and normal obese controls (n = 10) during oral glucose tolerance testing.
Setting: All subjects were admitted to the Samsung Medical Center.
Interventions: Oral glucose tolerance testing was performed in all subjects after an overnight fast.
Main Outcome Measures: Plasma levels of the hormones AG, DAG, and insulin, and those of glucose at 0, 30, 60, 90, and 120 min after glucose challenge were measured, and whole-body insulin sensitivity index (WBISI) values were calculated.
Results: AG levels fell markedly more from fasting levels in PWS children than normal healthy obese controls at 30, 60, and 90 min after glucose challenge, but no significant differences in DAG levels were observed at any time between PWS patients and controls. Fasting AG and DAG levels were found to correlate with WBISI in PWS, and absolute suppressions (
from baseline) in AG at 30 min after glucose challenge (nadir) were also correlated with WBISI in PWS (r = 0.64, P = 0.035).
Conclusions: Our results suggest that AG is sensitively suppressed by insulin and that this suppression correlated with insulin sensitivity in PWS children.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In addition to its central effects, ghrelin also acts peripherally. Its metabolic effects include the inhibition of insulin secretion, increase of glucose levels, and modulation of several nonendocrine actions (12, 13, 14, 15). It is generally believed that the biological activity of ghrelin is dependent on acylation at its serine 3 residue (14, 16). In fact, in humans and animals the acute administration of non-acylated ghrelin (AG) does not modify GH, prolactin, ACTH, insulin, or glucose levels (17), which concurs with the observation that des-acylated ghrelin (DAG) is unable to bind classical GH secretagogue receptor 1a (14, 16). However, DAG is not biologically inactive because it shares with AG some cardiovascular actions and the ability to modulate cell proliferation (14, 18, 19). More recently it was reported that acylated or non-AG exert the direct adipogenic effect to bone marrow adipocytes (20). Thus, DAG is not an inactive peptide and probably acts via GH secretagogue receptors that recognize ghrelin independently of its acylation. In this context, it is not surprising that DAG is present in the circulation at 2.5-fold higher concentrations than its acylated form (14, 16).
Insulin is a physiological and dynamic modulator of plasma ghrelin levels (7). Moreover, it has been reported that insulin has an inhibitory effect on ghrelin secretion and that this is independent of plasma glucose levels (21, 22).
Inverse relationships have been reported between fasting ghrelin and insulin levels and insulin resistance indices (23, 24, 25). Also, postmeal ghrelin suppression was found to be correlated with a rise in insulin (24).
To our knowledge, no report has been issued on changes in plasma AG or DAG levels during oral glucose tolerance test (OGTT) in PWS children. Furthermore, the dynamics of AG (or DAG) secretion (suppression after meals) and insulin sensitivity have not been evaluated in PWS children.
Therefore, we investigated plasma AG and DAG response to glucose load in children with PWS and obese non-PWS children and compared insulin sensitivity indices.
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Subjects and Methods |
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The study population consisted of 11 children with PWS [mean body mass index (BMI) 23.06 kg/m2, interquartile range 18.96–27.95 kg/m2] and 10 obese normal controls (mean BMI 26.15 kg/m2, interquartile range 24.5–27.36 kg/m2). Subjects with diabetes mellitus (fasting plasma glucose > 126 mg/dl with a 2-h OGTT value of > 200 mg/dl) were excluded.
Only two children in the PWS group had a BMI between the normal 90th and 95th percentiles; the others had BMIs greater than the 95th percentile. All subjects were in the prepubertal state. Patients’ clinical characteristics are detailed in Table 1
. To recruit controls, several middle and high schools located in southern Seoul were visited. We explained the purpose of the study to teachers, and a written study protocol was sent to all parents. Informed consent was obtained from parents or guardians as appropriate. The study design was reviewed and approved by the Samsung Medical Center Institutional Review Board.
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All subjects were admitted to the Pediatric Ward at the Samsung Medical Center. After an overnight fast of 10–12 h, children received an OGTT (1.75 g/kg, maximum 75 g). Blood samples were drawn at 0, 30, 60, 90, and 120 min to determine serum glucose, insulin, plasma AG, and DAG levels. Samples were collected on ice, centrifuged immediately at 4 C, and stored at –70 C until required for assay.
To prepare plasma samples for AG and DAG analysis, whole blood was drawn directly into a centrifuge tube containing 500 U aprotinin and 1.25 mg EDTA-2Na per 1 ml of whole blood collected and centrifuged immediately at 4 C. One hundred microliters of 1 mol/liter HCl per milliliter of collected plasma were then immediately added and stored at –70 C until required for assay.
Body fat percent was assessed by dual-energy x-ray absorptiometry using an Expert-XL (Lunar Corp., Madison, WI).
Hormonal assay
Plasma glucose was measured using an YSI 2300 dual analyzer (Yellow Springs Instrument Co., Yellow Springs, OH). Serum insulin was measured using a commercially available immunoradiometric assay kit (BioSource Europe S.A., Nivelles, Belgium) with a detection limit of 1 µU/ml and intra- and interassay coefficients of variation of less than 10%.
Plasma AG was measured in duplicate using a commercial ELISA kit (Linco Research, Inc., St. Charles, MO); inter- and intraassay coefficients of variance were less than 10%, and the lower and upper detection limits for this assay were 8.4 and 540 pg/ml.
Plasma DAG was measured in duplicate using a commercial ELISA kit (Linco Research); inter- and intraassay coefficients of variance were less than 10%, and the lower and upper detection limits for this assay were 40.6 and 2595 pg/ml.
Statistical analysis
Whole-body insulin sensitivity indexes (WBISIs) were calculated using OGTT, as proposed by Matsuda and DeFronzo (26), as 10,000/
(fasting glucose x fasting insulin) x (mean glucose x mean insulin during OGTT). This index was used to assess the relationship between insulin sensitivity and ghrelin suppression during OGTT. WBISI was validated in obese children and found to be correlated with insulin sensitivity as determined using the hyperinsulinemic-euglycemic clamp test (r = 0.78, P < 0.0005) (27).
All values are expressed as means ± SE or median (interquartile range). The t test with Bonferroni’s correction was used to compare the PWS and normal obese control group AG levels at different times after glucose challenge, and the Mann-Whitney test with Bonferroni’s correction was used to compare the other hormone levels in the PWS and control groups.
Two-way ANOVA with repeated measures was used to compare these two groups with respect to temporal hormone levels changes. Correlations between WBISI and fasting AG, DAG, or absolute ghrelin suppression were determined using Spearman’s correlation analysis. P < 0.05 was regarded as statistically significant. All statistical analyses were performed using SAS (version 8.2; SAS Corp., Cary, NC).
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Results |
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. The PWS and control groups did not differ significantly with respect to age, BMI, or sex ratio, but percent body fat levels in the PWS group [median 50.5% (46.7–52.9%)] were higher than in the control group [median 42.5% (38.78–47.73%)](P = 0.024), when measured by dual-energy x-ray absorptiometry. No significant intergroup difference of insulin sensitivity index was observed (Table 1). Plasma glucose and insulin
Fasting glucose levels were not significantly different between PWS patients [median 86 (76–91) mg/dl] and controls [median 89 (83–96) mg/dl], and plasma glucose levels at all times during OGTT also showed no significant differences between the two groups (Fig. 1).
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). Moreover, repeated-measures ANOVA revealed no significant group x time interaction between the two groups, meaning that temporal plasma insulin patterns were similar in the two groups. Acylated ghrelin
AG levels reached a nadir at 30 min after glucose loading in PWS children and at 90 min in controls (Fig. 2). Baseline (fasting) AG was significantly higher in PWS subjects [0.12 (0.091–0.188) vs. 0.044 (0.029–0.102) ng/ml, P = 0.007] and higher at 30 min [0.068 (0.051–0.090) vs. 0.032 (0.010–0.067) ng/ml, P = 0.047] and 60 min during OGTT [0.080 (0.052–0.117) vs. 0.037 (0.012–0.066) ng/ml, P = 0.024] in PWS patients (Fig. 2). Repeated-measures ANOVA showed a significant AG group x time interaction, meaning that changes in AG with time were significantly different for the two groups (P = 0.041). Mean absolute AG suppressions (abbreviated mean absolute AG) between 0 and 60 min after glucose loading were higher in PWS patients than controls (Fig. 2). However, percentage of acylated ghrelin suppressions was not different between the two groups at 30 min [42.6% (34.5–48.5%) in PWS vs. 30.0% (19.9–44.8%) in controls; P = 1.0] and 60 min after glucose loading [(32.6% (15.5–41.7%) in PWS vs. 24.3% (20.7–27.1%) in controls; P = 1.0 by t test with Bonferroni’s correction].
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Baseline DAG was not different significantly between PWS and obese normal children [0.29 ng/ml (0.19–0.38) vs. 0.18 ng/ml (0.12–0.25), P = 0.679]. DAG levels reached a nadir at 90 min after glucose loading in PWS children and at 60 min in controls (Fig. 3). However, no significant differences in DAG levels were observed at any time during OGTT in the two groups (P = 0.45 at 30 min after glucose loading, P = 0.179 at 60 min after glucose loading). Repeated-measures ANOVA showed no significant group x time interaction between the two groups with respect to DAG levels, and absolute mean DAG values were no different at any time between the two groups. In addition, DAG percent values were no different between the two groups at 30 and 60 min after glucose loading.
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In normal obese controls, no correlation was found between WBISI and fasting basal plasma AG or DAG levels. However, in PWS patients, WBISI was found to correlate positively with fasting AG (r = 0.76, P = 0.006) and DAG (r = 0.87, P < 0.001) (Fig. 4). Absolute AG values at 30 min after glucose loading (nadir) were correlated with WBISI in PWS children (r = 0.64, P = 0.035) (Fig. 4), but no significant correlation between AG and WBISI was found at any time among controls.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In addition, our results show that changes in AG with time were significantly different for the two groups, whereas no significant differences in DAG levels were observed at any time during OGTT in the two groups. Therefore, it is plausible that AG may exert dominant ghrelin action in PWS. One of the big issues in the ghrelin research is whether voracious appetite, observed in PWS, is really related to increased plasma ghrelin levels in PWS. Several reports demonstrated that appetite appears not to be mediated by ghrelin homeostasis (29, 30). However, these studies measured total ghrelin and did not differentiate AG from total ghrelin. It was reported that DAG has antagonistic effect to ghrelin in relation to appetite stimulation (31). In addition, recently a novel hormone, obestatin, which suppresses appetite, was discovered (32). Interestingly, obestatin is derived from the same ghrelin gene. It is observed that both of ghrelin and obestatin derived from the same proprotein act through distinct receptors and exerts opposing physiological actions. Moreover, obestatin needs to be amidated, which is reminiscent of acylation of ghrelin to be biologically active. Therefore, physiological significance of increased ghrelin in PWS needs to be evaluated in the light of interaction of AG, DAG, and obestatin effect on appetite in PWS, especially active forms.
The second issue in the present study is the relationship between WBISI and ghrelin suppression (AG and DAG). This relationship between WBISI and total ghrelin levels was reported using OGTT data in the children previously by Bacha and Arslanian (33). Specifically, fasting ghrelin levels were observed to be significantly lower in overweight children than lean children, and obese children showed a blunted insulin sensitivity to total ghrelin (33). A direct comparison is not possible because we measured AG and their study measured total ghrelin. However, in the present study, mean AG at any time during OGTT was more marked in PWS children, i.e. AG was more sensitively suppressed by insulin in PWS. It appears that baseline AG levels and AG in PWS children, as found in the present study, resemble those of lean controls in their study rather than those of obese controls (33).
In general, it has been reported that PWS patients are relatively insulin sensitive in relation to total ghrelin (34, 35). In the present study, PWS group and obese normal children group had similar BMIs. But baseline (fasting) plasma insulin levels were not significantly different, and neither were plasma insulin levels at any time during OGTT. We speculate that the higher percent body fat of PWS children might have contributed to our finding of similar insulin levels and insulin sensitivities in our two study groups.
In relation to WBISI and ghrelin, we observed distinct difference between AG and DAG. We noted that both baseline AG and baseline DAG have significant correlations with WBISI, but this correlation is lost after glucose challenge in DAG, but correlation between absolute AG and WBISI prevails. Differential suppression of AG and DAG might be important for two reasons. First, because our study subjects were obese, a certain degree of insulin resistance is to be expected in both study groups. However, our results indicate that AG is more sensitively suppressed by insulin in PWS, which may represent another manifestation of higher insulin sensitivity in PWS. This finding is also supported by the finding that absolute AG at 30 min (nadir) after glucose loading is correlated with WBISI in PWS children, although no significant correlation was found between WBISI and absolute AG at any time in the control group. Second, our results show that AG and DAG differ in the timing of maximal suppression. AG promptly decreased with insulin surge, whereas DAG response was delayed for up to 90 min. Thus, our results suggest that the mechanism of AG suppression by insulin differs from that of DAG suppression. So far, the insulin action to ghrelin has been studied mainly using total ghrelin, which as stated earlier is composed of AG and DAG. Because the response patterns of AG and DAG to insulin were dissimilar in the present study, we speculate that ghrelin response to insulin should be reevaluated vs. the two major ghrelin forms, i.e. AG and DAG.
According to our results, insulin can be considered an important modulator of fasting AG because insulin sensitivity to glucose as represented by WBISI appears to parallel insulin sensitivity to AG in PWS children. In this respect, we hypothesize that insulin suppresses AG better in PWS patients. However, it can be assumed inversely that ghrelin has a suppressing role on insulin secretion; thus, larger decreases in AG should induce greater insulin responses in PWS.
Our study has several limitations. First, contact of gastric mucosa with dextrose has been shown to reduce ghrelin levels in mice (10), and our recent study revealed that gastric emptying in PWS is not increased and tends to be slow despite higher ghrelin levels (36). Thus, a slower gastric motility in PWS patients might alter the rate of absorption of glucose or prolong contact between dextrose and the stomach and thus be expected to contribute to the different acylated ghrelin response observed after glucose challenge in PWS patients. Second, we used WBISI as parameter of insulin sensitivity. This method has been validated in normal controls (26, 27) but not in PWS patients, and this mitigates against accurate interpretation. Third, in interpreting the present data, we had to decide the relative significance of absolute AG and percentage AG values because absolute AG values and percentage AG values differ in the statistical significance. Therefore, our conclusion is based on the assumption that absolute AG might be a better parameter for insulin sensitivity to AG in PWS patients. But this assumption needs to be proved in another study.
In summary, fasting AG levels were higher and AG suppression during OGTT was greater in PWS children than obese normal children. In addition, fasting plasma AG and AG levels were found to correlate with WBISI in PWS children. Our results reveal that ghrelin acylation may be of physiological significance in PWS. Moreover, it appears that the regulatory mechanisms of AG and DAG differ and that the regulation of AG is closely related to insulin sensitivity.
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Footnotes |
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All the authors have nothing to declare.
First Published Online February 28, 2006
Abbreviations: AG, Acylated ghrelin; BMI, body mass index; DAG, des-acylated ghrelin; OGTT, oral glucose tolerance test; PWS, Prader-Willi syndrome; WBISI, whole-body insulin sensitivity index.
Received September 30, 2005.
Accepted February 16, 2006.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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