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Division of Pediatric Endocrinology, Metabolism and Diabetes Mellitus, Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15213
Address all correspondence and requests for reprints to: Dr. Silva A. Arslanian, Division of Endocrinology, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue at DeSoto Street, Pittsburgh, Pennsylvania 15213. E-mail: silva.arslanian{at}chp.edu.
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Abstract |
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Introduction |
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Several lines of evidence suggest that glucose and insulin metabolism may be implicated in the regulation of ghrelin levels. Plasma ghrelin levels decrease after oral and iv glucose administration (1). An inverse pattern of ghrelin and insulin levels has been described during 24-h observation in normal subjects (2). Also, a reciprocal relationship between insulin and ghrelin has been observed during hyperinsulinemic-euglycemic clamp studies (4). Moreover, a study that evaluated ghrelin concentrations in normal vs. type 1 diabetics revealed that insulin is required for prandial ghrelin suppression in humans (5). More recently, an inverse relationship between fasting ghrelin and insulin levels and insulin resistance indices has been reported by several investigators (6, 7, 8). Also, in a previous study of adults, postmeal suppression of ghrelin correlated with the rise in insulin (6).
In the pediatric literature, the data for the influence of nutrient consumption and insulin resistance on plasma ghrelin is controversial. One study reported no suppression of ghrelin after feeding in children (9). On the other hand, in obese Japanese children, ghrelin levels inversely correlated with fasting insulin and insulin resistance indexes (10). Also, in Pima Indian children, ghrelin levels inversely correlated with fasting insulin (11).
The relationship between the dynamics of ghrelin secretion (suppression after meals) to insulin sensitivity and insulin secretion has not been evaluated in children. Based on various studies, insulin may be involved not only in mediating the acute effects of feeding on ghrelin levels, but also in the chronic effects of obesity and positive energy balance leading to chronically suppressed ghrelin levels in obesity states. Therefore, we hypothesized that 1) in overweight (OW) children, ghrelin levels are not adequately suppressed after food intake, contributing to lack of satiety; and 2) ghrelin suppression in OW children is resistant to the effect of insulin. To test this hypothesis, we evaluated 1) ghrelin suppression in response to an oral glucose tolerance test (OGTT) in normal weight (NW) vs. OW children, and 2) the relationship of ghrelin and its dynamics to insulin sensitivity (IS).
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Subjects and Methods |
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The study population consisted of 60 healthy prepubertal children, 7–12 yr of age. They included 37 NW subjects [body mass index (BMI), 5th to <95th percentile for age and sex] and 23 OW (BMI, 
95th percentile for age and sex) children. There were only three children (one male and two females) in the NW group whose BMI was between the 85th and 95th percentile. All studies were approved by the human rights committee of Children’s Hospital of Pittsburgh. Children were recruited through newspaper advertisement and flyers posted in the health center. All research participants and their parents or guardians gave informed consent/assent after receiving detailed explanation of the research study. All subjects were documented to be in good health by a thorough medical interview and physical examination. OW children were free of any associated comorbidities or syndromes leading to obesity. Subjects were not receiving any medications. All subjects were assessed to be in Tanner stage 1 of puberty by careful physical examination and confirmatory hormonal levels. The characteristics of study subjects are detailed in Table 1
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After an overnight fast of 10–12 h, subjects were studied at the General Clinical Research Center of Children’s Hospital of Pittsburgh. Anthropometric measurements, including height and weight, were obtained without shoes and in light clothing. Children received an oral glucose tolerance test (OGTT; 1.75 g/kg; maximum, 75 g). Blood samples were drawn at 0, 15, 30, 60, 90, 120, and 180 min for determination of glucose, insulin, and ghrelin levels. Fasting adiponectin and IGF-binding protein-1 (IGFBP-1) were determined. Fasting dehydroepiandrosterone sulfate, estradiol (in females), and testosterone (in males) were obtained to confirm pubertal staging. Body composition was assessed by dual energy x-ray absorptiometry using an absorptiometer (Lunar Corp., Madison, WI).
Measurements
Blood specimens were collected on ice, centrifuged immediately at 4 C, and stored at –80 C. They were thawed only at the time of the ghrelin assay. Plasma glucose was measured by the glucose oxidase method with the use of a glucose analyzer (YSI, Inc., Yellow Springs, OH). Plasma insulin was measured by RIA (Linco Research, Inc., St. Charles, MO), which is 100% specific for human insulin with less than 0.2% cross-reactivity with human proinsulin and no cross-reactivity with C peptide or IGF. Adiponectin was measured by RIA as reported by us previously (12). IGFBP-1 levels were measured in Esoterix, Inc. (Calabasas Hills, CA), by immunochemiluminescent assay. Ghrelin levels were determined by RIA specific for total ghrelin (Linco Research, Inc). It uses 125I-labeled ghrelin tracer and rabbit antighrelin serum with a specificity of 100%. The intra- and interassay coefficients of variation are 1.7–5.5% and 3.2–6.6%, respectively. Estradiol was measured at Children’s Hospital of Pittsburgh laboratory by chemiluminescent assay. A level less than 21 pg/ml (77.1 pmol/liter) is consistent with prepubertal status. A testosterone panel, including total and free testosterone, was performed by HPLC tandem mass spectrometry at Esoterix, Inc. (Calabasas Hills, CA). A level less than 18 ng/dl (624.1 pmol/liter) was considered consistent with prepubertal status.
Calculations
Fasting insulin sensitivity index, calculated as the fasting glucose to insulin ratio (GF/IF) was used as a surrogate estimate of fasting insulin sensitivity. This estimate was used to assess the relationship of insulin sensitivity to fasting ghrelin. Our group has demonstrated that this index correlates well with the insulin sensitivity measured with the hyperinsulinemic-euglycemic clamp (r = 0.92) in normoglycemic children (13). The whole body insulin sensitivity index (WBISI) was also calculated during the OGTT, as proposed by Matsuda et al. (14), as 10,000/
(fasting glucose x fasting insulin) x (mean glucose x mean insulin during OGTT). This index was used to assess the relationship of insulin sensitivity to ghrelin suppression during the OGTT. WBISI was validated in obese children and was found to correlate with insulin sensitivity derived from the hyperinsulinemic-euglycemic clamp (r = 0.78; P < 0.0005) (15).
Statistical analysis
The distribution of the different variables was examined, and the appropriate statistical test was applied. Student’s t test or Mann-Whitney test was used for two-group comparisons. Pearson or Spearman’s correlations were used to examine bivariate relationships. Multiple regression analysis was used to examine multivariate relationships. Ghrelin and insulin areas under the curve (AUCs) during OGTT were calculated by the trapezoidal rule. Repeated measures ANOVA was used for comparison of ghrelin levels at different time points after OGTT. Ghrelin levels reached a statistical nadir at 60 min, and insulin peaked at 30 min after the glucose load. Log transformation of nonparametric variables was performed before the analysis. P < 0.05 was considered statistically significant. Results are reported as the mean ± SEM.
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Results |
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The two groups had similar age and pubertal stage. The OW group had significantly higher BMI and percent body fat compared with the NW children, by design (Table 1
). There were no significant differences between the two groups in serum testosterone [7.7 ± 2.2 ng/dl (267.0 ± 76.3 pmol/liter) in NW vs. 7.6 ± 2.6 ng/dl (263.5 ± 93.6 pmol/liter) in OW], free testosterone [0.7 ± 0.2 pg/ml (2.4 ± 0.7 pmol/liter) in NW vs. 1.0 ± 0.3 pg/ml (3.5 ± 1.0 pmol/liter) in OW] among the males or estradiol among the females [14.8 ± 2.3 pg/ml (54.3 ± 8.4 pmol/liter) in NW vs. 14.6 ± 2.6 pg/ml (58.7 ± 9.5 pmol/liter) in OW].
Baseline fasting data
There was no statistically significant difference in fasting glucose between the two groups [83.4 ± 1.4 mg/dl (4.6 ± 0.1 mmol/liter) in NW vs. 84.4 ± 1.4 mg/dl (4.7 ± 0.1 mmol/liter) in OW]. GF/IF was significantly higher in the NW subjects (Table 1). Fasting insulin was significantly higher, and fasting ghrelin, adiponectin, and IGFBP-1 levels were significantly lower in the OW group (Table 1).
There was no significant difference in fasting ghrelin levels between males and females in each group [605.7 ± 132.6 vs. 598.9 ± 55.6 pmol/liter (P = 0.21) in NW and 445.9 ± 54.8 vs. 312.8 ± 36.5 pmol/liter (P = 0.11) in OW; males vs. females, respectively].
OGTT data
Ghrelin levels reached a nadir at 60 min after oral glucose load in NW and OW children. The mean absolute suppression in ghrelin in NW children was 140.2 ± 14.0 pmol/liter vs. 72.6 ± 10.6 pmol/liter in OW children (P < 0.001; Fig. 1). Percent suppression in ghrelin was not significantly different between the NW and OW children (24.5 ± 1.7% in NW vs. 19.8 ± 2.6% in OW; P = 0.099). However, this similar percent suppression occurred at significantly higher insulin increment in OW children. Insulin reached a peak level at 30 min after the glucose load, with the change (
) in insulin at 30 min being 50.4 ± 5.9 µU/ml (302.4 ± 35.4 pmol/liter) in NW and 138.9 ± 18.6 µU/ml (833.4 ± 111.6 pmol/liter) in OW (P < 0.001). The whole body insulin sensitivity index was lower in the OW group (2.7 ± 0.3 vs. 6.8 ± 0.8 in NW; P < 0.001).
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Fasting ghrelin levels correlated inversely with BMI (r = –0.49; P < 0.001), percent body fat (r = –0.35; P = 0.006) (Fig. 2), and fat mass (r = –0.40; P = 0.001). Fasting ghrelin levels correlated positively with GF/IF (r = 0.59; P < 0.001; Fig. 3) and inversely with fasting insulin levels (r = –0.54; P < 0.001). The correlation with fasting insulin levels and GF/IF persisted after controlling for BMI (r = –0.38, P = 0.003 and r = 0.41, P = 0.002 respectively). Ghrelin AUC during the OGTT correlated inversely with insulin AUC (r = –0.45; P < 0.001) in the total group of subjects, but not in NW and OW groups separately. Ghrelin suppression at 60 min of the OGTT correlated inversely with insulin increment at 30 min. (r = –0.31; P = 0.02) and positively with WBISI (r = 0.43; P = 0.001; Fig. 3). Eliminating the outlier in Fig. 3 from the analysis did not change the statistical significance of the correlations between WBISI and ghrelin suppression (r = 0.40; P = 0.002) or between GF/IF and fasting ghrelin (r = 0.57; P < 0.001), respectively. When the same correlations were performed using percent suppression in ghrelin at 60 min of the OGTT, percent suppression correlated positively with glucose 60 min (r = 0.33; P = 0.009), but not with insulin or WBISI. IGFBP-1, as a marker of insulin sensitivity, correlated with fasting ghrelin (r = 0.45; P < 0.001), absolute ghrelin suppression at 60 min (r = 0.58; P < 0.001), and percent ghrelin suppression at 60 min (r = 0.28; P = 0.03). Adiponectin, another marker of insulin sensitivity, also correlated with fasting ghrelin (r = 0.48; P < 0.001) and absolute ghrelin suppression at 60 min OGTT (r = 0.36; P = 0.005), but not with percent ghrelin suppression. The correlation of fasting ghrelin to fasting adiponectin levels persisted after controlling for BMI (r = 0.31; P = 0.02).
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The fasting insulin level remained associated with fasting ghrelin independent of BMI (r2 = 0.37; P < 0.001) or percent body fat (r2 = 0.36; P < 0.001). Similarly, approximately 38% of the variance in fasting ghrelin was explained by GF/IF independent of BMI (P < 0.001) and percent body fat (P < 0.001). When IGFBP-1, adiponectin, fasting insulin, and BMI (or percent body fat) were included as independent variables, fasting insulin (r = –0.35; P = 0.04) and adiponectin (r = 0.30; P = 0.03) together and independently explained 42% of the variance in fasting ghrelin. Similarly, GF/IF (r = 0.41; P = 0.02) and adiponectin (r = 0.28; P = 0.03) independent of BMI (or percent body fat) and of IGFBP-1 explained 44% of the variance in fasting ghrelin (r2 = 0.44; P < 0.001).
In a multiple regression analysis with absolute ghrelin suppression as the dependent variable and fasting ghrelin, glucose, and insulin as the independent variables, the significant determinants of ghrelin suppression were fasting ghrelin (ß = 0.36; P = 0.008), insulin (ß = –0.28; P = 0.033), and glucose (ß = 0.28; P = 0.011); with total r2 = 0.42; P < 0.001. With percent ghrelin suppression as the dependent variable and insulin and glucose as independent variables, only glucose (ß = 0.32, P = 0.02) contributed significantly to the variance in percent suppression in ghrelin (total r2 = 0.14; P = 0.019). Performing the same analysis using percent change in insulin was not revealing. However, the accepted standard of analyzing insulin levels in response to OGTT is by absolute numbers and not percent change.
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Discussion |
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Our results are consistent with a role for ghrelin in maintaining energy homeostasis (1, 11, 16), with levels being significantly lower in obese children and correlating inversely with measures of adiposity, including percent body fat and fat mass. This is in contradistinction to a study in Japanese children (10) that did not find a relationship between ghrelin and percent body fat. However, that study examined only obese children (BMI, 28 ± 4.5 kg/m2) at different stages of puberty (5–19 yr), which might have introduced a confounding variable, especially because they report a trend toward a negative relationship of ghrelin levels to age (r = –0.268; P = 0.054) in their study.
Our findings of ghrelin suppression after OGTT are in contradistinction to a previous report that ghrelin secretion is refractory to the inhibitory effect of feeding in childhood (9). In the latter study, the ghrelin response to a standardized meal was evaluated at baseline and 60 min after feeding. The difference in composition of the stimulus OGTT vs. a meal could have contributed to the difference in results. Because insulin inhibits ghrelin secretion (4, 5), the insulin responses to the different meals might modulate ghrelin response differently. Alternatively, by sampling only at 60 min after the meal, ghrelin suppression in between the two time points might have been missed. Moreover, the subject population was different from ours, because it compared prepubertal children with adults.
The interplay between ghrelin and insulin in relation to energy balance is unraveling, although it has not yet been completely clarified. One study in adults using hyperinsulinemic hyperglycemic clamp concluded that ghrelin is not directly regulated by changes in circulating glucose and insulin, but only by supraphysiological levels of insulin (17). Similar conclusions were made by another group using iv glucose load and sc insulin (18). However, the latter study did demonstrate an effect of feeding to suppress ghrelin, and the suppression was correlated with the AUC of glucose (18). Also, in contradistinction to the former study (17), another investigation demonstrated a decrease in ghrelin after iv glucose administration (1). In other studies, insulin at physiological doses has been shown to suppress ghrelin levels during hyperinsulinemic clamp studies (4, 19, 20, 21). Moreover, a study that evaluated ghrelin concentrations in normal vs. type 1 diabetics revealed that insulin is required for prandial ghrelin suppression (5). Therefore, it is believed that insulin mediates the effect of nutritional intake on ghrelin levels acutely after meal ingestion. In agreement with these findings, in our study the increment in insulin during the OGTT and insulin sensitivity modulate ghrelin suppression after OGTT.
In obesity, fasting ghrelin levels are lower than those in NW adults (1, 16, 22) and children (11, 23). Chronic hyperinsulinemia appears to mediate this suppression. Many studies have demonstrated an inverse relationship between fasting ghrelin levels and fasting insulin and insulin resistance indices in adults (8, 16) and children (10, 23). Also, ghrelin levels are lower in conditions associated with insulin resistance; they are lower in lean Pima Indians compared with Caucasians (16), they are lower in obese women with polycystic ovarian syndrome (7, 24), and they are negatively associated with insulin resistance and the prevalence of type 2 diabetes (25). In Japanese children, ghrelin levels correlated inversely with insulin resistance indices and plasminogen activator inhibitor-1 (10).
In agreement with the literature, we demonstrate a positive relationship between fasting ghrelin and insulin sensitivity independent of body adiposity. Moreover, fasting ghrelin is a good reflection of its dynamics (suppression) after OGTT. The obese subjects have lower levels of fasting ghrelin, with lower absolute suppression in response to the glucose load. The percent suppression of ghrelin is similar in the two groups. However, the fact that a comparable percent suppression in OW youth occurs despite much higher insulin responses would suggest that there is an element of insulin resistance to ghrelin suppression. This raises the question of whether this may be yet another manifestation of insulin resistance in obesity. In support of this hypothesis, ghrelin suppression during hyperinsulinemic clamp studies has been shown to be positively associated with insulin sensitivity (20, 26) and to be reduced in noninsulin-treated type 2 diabetics compared with nondiabetics of similar BMI (26).
In addition, in the present study, fasting ghrelin and ghrelin suppression after the glucose load correlate positively with IGFBP-1 and with adiponectin levels. Both IGFBP-1 and adiponectin levels have been shown to be reduced in obesity (27, 28) and to correlate positively with insulin sensitivity and inversely with components of the insulin resistance syndrome (12, 28, 29). These findings are consistent with a role for insulin sensitivity in regulating ghrelin levels.
In summary, the results of this study reveal that OW children have lower fasting ghrelin and less absolute, although similar, percent suppression of ghrelin levels in response to feeding compared with NW youth. Ghrelin suppression in OW children appears to be resistant to the effect of insulin. This may be yet another manifestation of insulin resistance in obesity. Whether alterations in meal-induced ghrelin suppression in OW individuals could be responsible for differences in satiety needs to be investigated.
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Acknowledgments |
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Footnotes |
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First Published Online February 22, 2005
Abbreviations: AUC, Area under the curve; BMI, body mass index; , change; GF/IF, fasting glucose to insulin ratio; IGFBP-1, IGF-binding protein-1; NW, normal weight; OGTT, oral glucose tolerance test; OW, overweight; WBISI, whole body insulin sensitivity index.
Received August 9, 2004.
Accepted February 8, 2005.
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) vs. OW (•) subjects during the 180-min OGTT. Right panel, Absolute ghrelin suppression at 60 min of the OGTT (
) vs. OW (
) subjects. To convert ghrelin concentrations to picograms per milliliter, divide by 0.296.
