Systematic Review of Ghrelin Response to Food Intake in Pediatric Age, From Neonates to Adolescents

Objective:

Food intake and energy balance are regulated during the lifespan with critical changes in each specific period (infancy, adulthood, aging). Some of ghrelin's changes may contribute to the regulation of food intake and weight in children. We aimed to analyze the ghrelin response to feeding in lean or obese subjects from birth to adolescence.

Methods:

We searched PubMed, Scopus, Google Scholar, Cochrane, and EMBASE (December 1999 to February 2013) and identified 62 relevant articles, of which 29 were suitable to be included.

Results and Conclusions:

Total ghrelin response to meals is particular, with refractoriness in neonates and lean children and an inhibition that starts from puberty. Total ghrelin levels are decreased after meals, irrespective of pubertal stages in obese children and adolescents. Conversely, total ghrelin is decreased after an oral glucose tolerance test in all ages, with the exception of neonates. Data on unacylated ghrelin response are scant but resemble those of total ghrelin. The acylated ghrelin response to meals or oral glucose tolerance test is discordant, although a precocious inhibition followed by a rise back is present in both lean and obese children. The post-feeding profile in children with Prader-Willi syndrome is also peculiar, with a conserved and deeper inhibition of all ghrelin forms.

Ghrelin has an emerging role on appetite, glucose and lipid metabolism, and body composition. Combined with ghrelin's GH-releasing activity, these have provided an important strength to this research field, introducing new perspectives to neuroendocrinology and metabolism. In particular, ghrelin is the only known appetite-stimulating hormone in humans, and it seems to be one of the principal factors involved in appetite, craving, and weight regain after weight loss (1, 2).

Ghrelin is a 28-amino acid peptide predominantly produced by the stomach (3). It was discovered as the first natural ligand of the orphan GH secretagogue receptor (GHS-R) type 1a (3–5). Ghrelin presents a characteristic acylation with a fatty n-octanoic acid on the Ser3 residue (3). The n-octanoyl group appears to be essential for its binding to and activation of the GHS-R1a (3, 4). Unacylated ghrelin (UAG), which is devoid of the n-octanoil group at Ser3, is the most abundant circulating form (3–6). Numerous studies have demonstrated that UAG is also a biologically active molecule, suggesting the existence of GHS-R subtypes that are activated independently of its acylation (7). The mechanism of acylation of the preproghrelin or UAG is largely unknown. Yang et al (8) have identified the acyltransferase that octanoylates ghrelin, which was named GOAT (ghrelin O-acyltransferase).

Ghrelin has emerged as one of the most powerful orexigenic and adipogenic agents (4–6, 9–11). As a result of central and peripheral actions, acylated ghrelin (AG) administration in rodents causes weight gain that occurs even in the absence of overfeeding (12). AG influences energy balance involving neuropeptide Y and agouti-related peptide in the arcuate nucleus, as well as decreasing the melanocortin tone (4, 12–14) and acting on fibers of the vagal nerve (4, 12). The active vaccination with ghrelin immunoconjugates decreases feed efficiency, adiposity, and body weight gain in rats (15). More recently, by acting on the modification of fatty acid chain length, it enhances or reduces the chronic actions of AG on adiposity in rodents (16). On the other hand, the role of UAG on food intake is not fully clarified, but it seems able to induce a negative energy balance by decreasing food intake and delaying gastric emptying via the hypothalamus (2).

In humans, ghrelin secretion is pulsatile, with a higher secretion during the night. It decreases after food ingestion, thus suggesting a metabolic control in vivo. Ghrelin could contribute to meal initiation or to nutrient-type ingestion (4, 10, 17). The circulating levels of ghrelin are modulated by chronic and acute energy imbalance. In fact, ghrelin secretion is increased in anorexia and cachexia, reduced in obesity, and normalized by the recovery of ideal body weight (4, 9, 12, 18). More recently, GOAT was detected in human circulation in healthy, obese, and anorexic adults, with a positive correlation with body mass index (BMI) and a negative correlation with ghrelin levels, suggesting that GOAT counteracts the functional changes of ghrelin observed under these conditions (19). The increase of ghrelin levels, which has been reported after weight loss induced by diet and lifestyle modifications, may help in promoting the regain of weight. GOAT inhibition attenuates food foraging, food intake, food hoarding, and hedonic feeding in mice (20, 21). Furthermore, the overall ghrelin profile is partially abnormal in adult obesity; there is absent or changed ghrelin elevation during fasting (22), an abolished or blunted increase during the night or sleep deprivation (23, 24), and a blunted suppression after a meal (25). The only clinical exception seems to be Prader-Willi syndrome (PWS), characterized, among many other features, by severe obesity and hyperphagia. Ghrelin hypersecretion has been hypothesized to participate in the development of at least some symptoms of PWS, such as hyperphagia and weight excess (26).

As anticipated, circadian ghrelin secretion is modulated by acute variations in the energy balance and nutritional status. Although some stimulatory effects of short-term fasting on ghrelin secretion have been suggested by some authors (4, 17, 27, 28), this observation has not definitively been confirmed (29–31), probably because the assay methods allow only for the evaluation of total ghrelin levels. Notably, during fasting, AG decreases to nadir levels seen postprandially and UAG remains near peak levels as seen preprandially, suggesting that long-term fasting inhibits acylation and that this may be regulated independently by nutrient availability in the gut or esterases that cleave the acyl group (32, 33). In fact, the lipid group that is attached by GOAT is likely derived from free fatty acids in the lumen of the gut rather than circulation (34). Indeed, with prolonged fasting, AG levels are suppressed, whereas UAG is tonically secreted (32). However, the mechanisms mediating the metabolic control of ghrelin secretion at present are still a matter of debate. Gastric secretion per se has been reported not to play a role, with results derived from studies evaluating the effects of nutrients (4, 12, 26, 35). The depth and duration of ghrelin decrease after meals is related to the total amount of calories ingested and the type of the macronutrients, in particular carbohydrates and proteins (4, 9, 35). Insulin and glucose are among the major determinants of ghrelin secretion that, in turn, modulate insulin secretion and glucose metabolism (4, 9, 12, 18).

Overall, published data suggest that ghrelin acts to optimize energy metabolism during food restriction as well as to prepare the metabolic pathway to use fuel. Food intake, appetite, and energy balance are strictly regulated during the lifespan, with critical changes in each specific period (infancy, adulthood, aging). There is increasing evidence, although not conclusive, that some ghrelin changes may also contribute to the regulation of food intake and weight in children, initiating with neonates. As such, the aim of this review has been to obtain an overview of the influence of feeding on ghrelin secretion in the pediatric age group by evaluating healthy and obese subjects from birth to adolescence.

Subjects and Methods

PICO (Patient, Intervention, Comparison, Outcomes) strategy was reported (Supplemental Table 1, published on The Endocrine Society's Journals Online web site at http://jcem.endojournals.org). Guidelines of the Preferred Reporting for Systematic Reviews and Meta-Analyses (PRISMA) statement were followed, and a PRISMA checklist is provided (Supplemental Table 2).

Literature search

We systematically searched PubMed, Cochrane, Scopus, Google Scholar, and EMBASE (December 1999 to February 2013) to identify studies evaluating ghrelin regulation by feeding in neonates, children, and adolescents.

The electronic database search was conducted using the following keyword combinations: “ghrelin,” “acylated ghrelin,” “unacylated ghrelin,” “desacylated ghrelin,” “neonates,” “newborns,” “child,” “adolescents,” “food intake,” “feeding,” “meal,” “oral glucose tolerance test (or OGTT),” “intravenous glucose tolerance test (or IVGTT),” and “food questionnaire.” The Boolean operators NOT, AND, and OR were used with the aforementioned terms, also in various combinations. The terms were combined in various ways to generate a wide search. In addition, we checked the references of eligible articles for further papers that were not captured by our search strategy. After the initial screening of titles and abstracts, studies included by three reviewers were compared; disagreement was resolved by consensus on full-length articles. The inter-review agreement was calculated with K statistics.

Inclusion and exclusion criteria

We included only full-length articles that met the following predetermined criteria: 1) population—neonates, infants, children, and adolescents (from 0 to 18 y); healthy, overweight, and obese subjects were included; obesity was considered as essential obesity or monogenic obesity; for neonates, small, appropriate, and large for gestational age (SGA, AGA, and LGA, respectively) were all included; 2) sample size—studies with > 10 subjects in the test group of healthy subjects, and studies with > five subjects in the test groups of monogenic obesity; 3) publication date—articles from December 1999 (discovery of ghrelin) to February 2013; 4) language—full-length articles in the English language; 5) study design—test meal (solid or liquid) or oral/iv glucose load (OGTT or IVGTT) with pre- and postprandial analysis of ghrelin (total ghrelin, AG, and/or UAG) concentrations; postprandial analyses in sequential days were also included; ghrelin and placebo administration and feeding response by food questionnaire or scheduled meal consumption; and weight-loss program with pre- and postanalysis of ghrelin (total ghrelin, AG, and/or UAG) concentrations; 6) study exclusion—studies on pharmacological intervention that might interfere with ghrelin; studies including patients with chronic systemic diseases (with the exception of essential and monogenic obesity), or taking concomitant therapy with antiepileptic agents, glucocorticoids, or metformin; and 7) measures—total ghrelin, and/or AG, and/or UAG concentrations; food questionnaire items or scheduled meal consumption.

Outcome measures

In the analysis of the studies concerning ghrelin, we focused on the following measures: 1) changes with respect to baseline of total ghrelin, and/or AG, and/or UAG concentrations (after a meal consumption or OGTT or IVGTT); and 2) food consumption (scheduled or food questionnaire) after ghrelin administration or its association with baseline total ghrelin, and/or AG, and/or UAG concentrations.

Data extraction, synthesis, and quality assessment

A form was generated to register whether individual studies met eligibility criteria and to collect data regarding the study design and methodological quality. Three investigators independently reviewed and extracted data from the papers according to the predetermined criteria. Any difference of opinion about the studies was resolved by discussion between the investigators. The following data were extracted from each retrieved article: name of the first author, year of publication, design of the study, country where the study was performed, ethnic group, total number of individuals, sex and puberty distribution, weight category, type of intervention, fasting time and feeding time before and after the intervention, type of measurement of ghrelin peptides, type of stabilization of blood samples during the collection, specific outcomes, and other measurements (correlations).

Study quality was independently assessed by three reviewers according to the Newcastle-Ottawa Scale for quality assessment of cohort studies and case-control studies (36). The scales allocate stars, with a maximum of nine; the criteria were quality of selection (maximum, four stars), comparability (maximum, two stars), and exposure (maximum, three stars). High quality was assessed for ≥ eight stars.

Results

The literature search, we performed, identified 62 potentially relevant articles. After reviewing titles, abstracts, and full-length texts, we selected 29 articles for closer assessment and inclusion in this review (37–47, 49–67). Agreement between reviewers on which studies to include was good: the K for agreement was 80% after screening titles and abstracts and 100% after screening full-text articles. Overall, data including 830 neonates, children, or adolescents were reported. They are summarized in Supplemental Tables 1–4. Concluding remarks are reported in Table 1.

Study quality score averaged 5.27, with no high-quality studies (quality score ≥ 8) and with a proportion of medium-quality studies (quality score ≥ 6) of 41.4% (Supplemental Table 3). Risk of a bias was high because: 1) there was an absence of blinded studies; 2) most of the studies did not describe exclusion criteria for controls; 3) most of the studies did not describe how the allocation was performed in cases and controls; 4) some of the studies had a selective reporting bias; 5) the methods to perform the intervention (OGTT) were unconventional; and 6) methods to stabilize and measure ghrelin were different.

Neonates

Three of the selected studies were with regard to the role of ghrelin in food intake in newborns and infants (37–39) (Supplemental Table 1). Two of these (37, 39) were observational studies, and one (38) was interventional. In the observational studies, the stimulus was represented by ordinary nutrition of the newborns, breast- or formula-feeding, in the study by Bellone et al (37) and enteral associated or not with parenteral nutrition in the study by Hübler et al (39). In the interventional study by Iñiguez et al (38), the stimulus was represented by IVGTT, 25% dextrose 0.5 g/kg, administered by constant infusion over 3 minutes. Overall, data from a total of 189 subjects were reported. In the studies by Bellone et al (37) and Hübler et al (39), the patient populations were represented by preterm newborns, 2–4 days old. Iñiguez et al (38) reported data about 1-year-old infants, born full term. The three included studies analyzed total ghrelin levels.

The study by Hübler et al (39) analyzed ghrelin levels in preterm neonates. They found that total ghrelin was positively correlated with the enteral nutritional state, but not with the overall intake of energy or fluids in this age group. The strong positive association of ghrelin and enteral caloric intake did not change after adjustment for the covariates body weight, gestational age, serum glucose, IGF-1, and infant's age.

Bellone et al (37) reported that the meal did not modify ghrelin levels as compared to fasting condition in preterm AGA newborns. Ghrelin levels were negatively correlated to birth weight and body weight and did not vary between breast- and formula-fed newborns.

Iñiguez et al (38) reported a decrease of about 20% in ghrelin levels 10 minutes after glucose infusion in both SGA and AGA 1-year-old infants. No differences in ghrelin levels between SGA and AGA infants either at fasting or after IVGTT were observed, but a higher interindividual variability in changes was detected after glucose infusion. In SGA infants, post-IVGTT ghrelin levels were positively correlated to body weight and length at 1 year and to Δ weight SD score (SDS) between birth to 1 year.

Lean children and adolescents

Eight of the selected studies described ghrelin's role with respect to food intake in normal-weight children (40–47) (Supplemental Table 2). They were all interventional studies. In five of these eight studies, the intervention was a standardized meal: liquid mixed meal in two of them (40, 41), and solid meal in three (42–44), whose composition is summarized in Supplemental Table 2. In the remaining three studies (45–47), the intervention was an OGTT, with a nonstandard World Health Organization dose in a study that used exclusively the maximum dose (75 g) (46). Data from a total of 138 normal-weight subjects were analyzed. Of the eight selected studies, six evaluated total ghrelin levels (40, 41, 43, 44, 46, 47), one measured only AG (42), and one measured both AG and UAG levels (45).

Meal intervention: total ghrelin

In the study by Bizzarri et al (40), total ghrelin response after a standard liquid high-fat meal was evaluated. No significant change in plasma ghrelin levels was found.

Stock et al (41) evaluated the total ghrelin response to a liquid mixed meal in normal-weight female adolescents. They found a fall in ghrelin concentrations after the meal, with a maximum drop of 25% compared with baseline after 60 minutes, followed by a rise back to baseline values at the end of the study period. They evaluated hunger and satiety perception by a four-item questionnaire and found no correlations between ghrelin and self-reported satiety.

Bellone et al (43) reported a total ghrelin response to a standardized light breakfast in prepubertal lean children and found no change in ghrelin levels after the meal.

In the study by Lomenick et al (44), normal-weight children were given a standardized breakfast and lunch. The authors showed that total ghrelin was not suppressed after eating, did not rise before the next meal, and did not correspond to hunger ratings. However, a decrease in total ghrelin levels was reported 4 hours after the lunch.

One further study (48) measuring total ghrelin levels in children born to healthy mothers or to mothers with a history of gestational diabetes could not be included because it reported exclusively ghrelin trends in figures (actual values not available) and evaluated the ghrelin response to the meal challenge only as a comparison between the two groups. However, a trend toward an initial suppression after the meal (with a nadir at 60 min) followed by a rise back to initial values could be seen.

Meal intervention: AG, UAG

Misra et al (42) assessed AG levels in normal-weight adolescent girls in fasting condition and after the administration of a high-carbohydrate, high-protein, and high-fat breakfast on three different days. Ghrelin levels did not significantly change with respect to baseline values. The study was restricted to girls because normal-weight girls were compared to obese ones because girls are at a higher risk for obesity than boys, with a greater fat accumulation during adolescence.

No studies included UAG evaluations.

OGTT intervention: total ghrelin

Baldelli et al (46) measured total ghrelin after an OGTT in normal-weight prepubertal children. They found a ghrelin suppression by OGTT, with a 31% nadir at 120 minutes. No correlation was found between ghrelin and insulin or glucose levels.

In the study by Bacha et al (47), normal-weight prepubertal children were recruited. The authors considered as normal weight those subjects whose BMI values were between the fifth and 95th percentiles for age and sex, and they included three children (one male and two females) whose BMI values were between the 85th and 95th percentiles. Total ghrelin levels were suppressed, with a nadir at 60 minutes. The suppression of ghrelin correlated positively with the whole-body insulin sensitivity index (WBISI) and negatively with the change in insulin at 30 minutes. Only the change in glucose (but not insulin) contributes to the variance in the percentage of ghrelin suppression.

OGTT intervention: AG, UAG

Prodam et al (45) found that AG levels decreased at 60 minutes after OGTT and subsequently increased at 120 minutes, returning to basal concentrations, with similar dynamics in prepubertal and pubertal subjects. AG variation, particularly at 60 minutes, and the AG nadir were associated with the waist-to-height ratio and insulinogenic index at 120 minutes, which remained after correction for puberty and BMISDS. Regression analyses adjusted for sex, puberty, and BMISDS revealed that waist-to-height ratio explained 34.4% of the AG variation at OGTT.

Prodam et al (45) also found that UAG decreased for the entire OGTT session with a maximal inhibition at 120 minutes. Pubertal subjects presented a lower total UAG and a lower inhibition of UAG concentrations with respect to prepubertal individuals. Homeostasis model assessment for insulin resistance (HOMA-IR) was the only predictor that explained the 30.1% UAG variation. Area under the curve (AUC) of UAG was negatively associated with insulin AUC and positively associated with Matsuda index.

Obese children and adolescents

Twenty-three studies analyzed ghrelin's role in the food intake of overweight or obese children (40–42, 44–47, 49–64) (Supplemental Table 3). All trials were single-center studies with the exception of a multicenter study (40). Most the studies (n = 14) had an interventional, case-control study design. The cases were all overweight or obese patients without other known endocrine or genetic disorders, and controls were lean healthy matched subjects (40–42, 44–47, 53, 56, 57, 59–62). In three studies, overweight or obese subjects were compared to anorexic subjects (41) and PWS (63) and craniopharyngioma patients (58). In one study, the cases were obese children undergoing a Mandometer intervention to relearn how to slow down their eating; they were compared as controls with obese subjects in a standard lifestyle care (64). In seven studies (49–52, 54, 55, 64), an overweight or obese population was evaluated without a control group. Interventions were variable: a mixed solid meal as breakfast or lunch (42, 44, 49–53, 56), a liquid meal (40, 41, 54), or an OGTT (45–47, 55, 57–64). In three of 10 studies with OGTT (55, 59, 62), OGTT was performed not according to the World Health Organization standard dose (1.75 g/kg with a maximum of 75 g), but instead with a lower dose (0.75 g/kg). In two studies (52, 57), the response of ghrelin to feeding was evaluated before and after a weight-reduction program, whereas in another study (64), it was evaluated before and after a Mandometer intervention or a standard care to improve lifestyle behavior. A trial compared ghrelin response to feeding with a glass of water ingestion (51). Overall, data from a total of 467 overweight or obese children and adolescents with ages ranging from 8 to 18 years were reported, including one subject under paroxetine and methylphenidate treatment (41). Eighteen studies analyzed changes in total ghrelin levels (40, 41, 44, 46, 47, 49–61), eight studies analyzed changes in AG (42, 45, 53, 57, 60, 62–64), and two studies analyzed changes in UAG (45, 63), respectively.

Meal interventions: total ghrelin

Stock et al (41) studied overweight and obese adolescents who consumed a liquid mixed meal, where total ghrelin concentrations fell after feeding and reached a nadir after 60–90 minutes, with a following rise back to baseline. Ghrelin changes did not correlate with peptide YY (PYY), glucose-dependent insulinotropic polypeptide, and markers of satiety. Fasting total ghrelin levels correlated with total ghrelin nadir (adjusted r² = 0.85) and total ghrelin AUC (adjusted r² = 0.89) after the meal.

Maffeis et al (49) observed that total ghrelin levels rapidly decreased with a nadir at 60 minutes, after a solid mixed meal in prepubertal overweight boys. Total ghrelin nadir was correlated with insulin sensitivity (r = 0.803; P < .01) and total absolute ghrelin AUC with that of insulin (r = −0.709; P < .03).

Lomenick et al (44) studied obese subjects who received a breakfast and lunch. Total ghrelin levels did not change after eating with respect to baseline levels. Hunger ratings assessed by using a visual analog scale decreased after both breakfast (P < .003) and lunch (P < .0001), compared to the premeal values. No correlations were found between fasting total ghrelin and fasting insulin or HOMA-IR.

Gil-Campos et al (56) described a decrease in total ghrelin levels at 1 hour (P < .007) and 2 hours (P < .001) after feeding a standard breakfast, with a recovery to baseline levels 3 hours after the meal. Total ghrelin levels were associated with glucose (r = −0.507; P < .001), insulin (r = −0.507; P < .001), and the quantitative insulin sensitivity check index (r = 0.714; P < .001). Total ghrelin levels were independently associated with HOMA-IR (r = 0.266; P = .049). In the group as a whole, the nadir of total ghrelin was associated with glucose (r = −0.251; P = .017) and C-peptide (r = −0.314; P < .004).

Maffeis et al (50) offered to obese boys, in a random order, two different solid meals comparable in terms of energy and protein content but different in terms of carbohydrate-to-fat ratio (a moderate-fat meal and a high-fat meal). Total ghrelin levels decreased at 60 minutes after ingestion of both meals, with a following increase for 150 minutes. After 150 minutes, total ghrelin levels continued to increase with the high-fat meal, whereas it decreased with the moderate-fat meal, with no changes in the AUC of total ghrelin levels after both menus. Food intake induced a reduction in appetite (P < .05) that was higher after the moderate-fat meal than the high-fat meal. No correlation between the AUC of total ghrelin and the AUC of hunger was reported.

Mittelman et al (53) studied obese adolescents who consumed to satiation in random order two mixed meals, one small and one large. Only the large meal suppressed total ghrelin levels (P < .005) compared to baseline values. The response of total ghrelin levels to caloric intake had a caloric-dependent suppression of about −18.0 pg/mL/100 kcal.

Rigamonti et al (52) studied obese adolescents who consumed standard meals before and after a 3-week weight-reduction intervention. Total ghrelin levels decreased (P < .05) after both meals with respect to the fasting state, without differences between pre- and post-weight-reduction programs. No differences were found between fasting morning total ghrelin levels and appetite before and after body weight loss.

Maffeis et al (51) observed that total ghrelin levels decreased (P < .02) after breakfast consumption.

Bizzarri et al (40) observed that total ghrelin levels remained unchanged after a standard liquid mixed meal in prepubertal or obese children with early puberty. No correlations were found between changes in total ghrelin and PYY.

Ellis et al (54) enrolled overweight girls in peripubertal and pubertal stages who underwent a standard liquid meal. No total ghrelin changes after feeding were reported. However, total ghrelin levels tended to be higher than baseline values among peripubertal girls 4 hours after the liquid meal test.

Meal interventions: AG, UAG

Misra et al (42) recruited obese adolescent girls and assigned them to three kinds of breakfast in random order. Subjects also received a standard lunch of macaroni and cheese 4 hours later. No suppression in AG levels was recorded after meals, but an increase in AG levels from baseline after the high-carbohydrate breakfast was reported. Δ Percentage AUC of AG correlated with basal AG levels after the high-protein (r = −0.44; P = .03), high-carbohydrate (r = −0.66; P = .005), and high-fat breakfasts (r = −0.41; P = .04). In the obese group, Δ percentage AUC of AG was associated with insulin AUC after the high-carbohydrate breakfast (r = −0.60; P = .04).

Mittelman et al (53) also observed a modest suppression of AG by the small meal (P = .034) but not by the large meal. Within 15 minutes of starting both meals, an increase in AG levels with respect to baseline was reported. No significant correlations were found among analyzed parameters. The response of AG levels to feeding has a caloric-dependent suppression of about −1.3 pg/mL/100 kcal.

No studies included UAG evaluations.

OGTT interventions: total ghrelin

Soriano-Guillén et al (55) observed that total ghrelin levels decreased at each time point, reaching a nadir at 60 minutes after a nonstandard OGTT in obese children and adolescents. No correlation was found at any time between ghrelin and insulin, glucose, or HOMA-IR; however, total ghrelin was associated with IGF binding protein-1 (IGFBP-1) at fasting (r = 0.58; P < .01).

Bacha et al (47) described, in prepubertal obese children, decreased total ghrelin levels after an OGTT with a nadir at 60 minutes. Ghrelin suppression at 60 minutes correlated with the insulin increment at 30 minutes (r = −0.31; P = .02), the WBISI (r = 0.43; P < .001), and changes in glucose at 60 minutes (r = 0.33; P = .009). Ghrelin nadir was predicted by fasting total ghrelin, and by the change in insulin and glucose levels during the OGTT (r² = 0.42; P < .001). The AUC of total ghrelin correlated with AUC of insulin (r = −0.45; P < .001). Absolute total ghrelin suppression at 60 minutes was associated with IGFBP-1 and adiponectin at fasting (r = 0.58, P < .001; and r = 0.36, P = .005, respectively).

Baldelli et al (46) studied prepubertal overweight and obese children with the maximum dose of OGTT. Total ghrelin levels were inhibited at each time point (P < .005) compared to baseline with a nadir at 90 minutes. No correlation between total ghrelin and insulin or glucose levels was described.

Galli-Tsinopoulou et al (61) described total ghrelin levels that decreased with a nadir at 60 minutes and a surge at 90 minutes (P = .026) in prepubertal obese children. Fasting total ghrelin levels and fasting glucose-to-insulin ratio were correlated (r = 0.441; P = .006). The AUC of total ghrelin was correlated with the AUC of insulin (r = 0.586; P = .007), the insulin sensitivity index composite (r = 0.504; P = .024), and the fasting glucose-to-insulin ratio (r = −0.527; P = .017). Total ghrelin levels after OGTT were predicted by the AUC of insulin. The percentage of the decrease in total ghrelin levels at 60 minutes was associated with the insulin sensitivity index composite (r = −0.451; P = .046) and the percentage rise in insulin levels at 60 minutes (r = −0.454; P = .044).

Wang et al (59) studied prepubertal and pubertal obese subjects with nonstandard OGTT. Total ghrelin levels decreased, with the maximum total ghrelin decrease occurring at 60 minutes with respect to basal values in boys of Tanner I, but not in the other pubertal categories and in females. No significant correlation between total ghrelin changes and insulin or glucose were found.

Roth et al (60) described a decrease in total ghrelin levels after a standard dose of glucose administrated to obese children.

O'Gorman et al (58) observed that total ghrelin levels progressively decreased from 0 to 60 minutes after a maximum dose of OGTT in prepubertal and pubertal obese subjects. The total ghrelin decrease from 0 to 30 minutes correlated with fasting total ghrelin (r = −0.55; P < .001), but not with insulin.

Martos-Moreno et al (57) studied prepubertal obese children at the first visit and after a reduction of over 2 SD of BMI (n = 21). Total ghrelin levels decreased after OGTT in both conditions, with no differences between them.

OGTT interventions: AG

Paik et al (63) observed that AG levels fell from fasting levels and reached a nadir at 90 minutes in prepubertal obese children, without a significant correlation between absolute suppression and WBISI at any time.

Lányi et al (62) studied pubertal obese subjects with a nonstandard OGTT. AG levels decreased from 0 to 60 minutes, with a following rebound at 120 minutes that was higher than baseline values.

Roth et al (60) observed an increase in AG levels 180 minutes after a standard OGTT in obese children, describing a positive correlation between changes in AG levels and HOMA-IR (r = 0.5; P = .034), quantitative insulin sensitivity check index (r = −0.47; P = .047), fasting glucose-to-insulin ratio (r = −0.40; P = .004), and fasting ghrelin (r = −0.63; P = .005).

In the study by Prodam et al (45), prepubertal and pubertal obese subjects with and without metabolic syndrome had similar basal and post-OGTT AG circulating profiles. AG levels decreased at 60 minutes in both groups, with a rebound at 120 minutes to basal levels. The AG nadir (P < .01) and rebound (P < .002) correlated with the insulinogenic index at 120 minutes (r = −0.245; P < .01). The AUC of AG was explained for 49.5% positively by insulinogenic index at 120 minutes and negatively by insulin AUC (P < .001).

Galhardo et al (64) studied prepubertal and pubertal obese children at baseline and at 12 months after a randomized intervention trial to lose weight. At baseline, AG levels decreased with a nadir at 60 minutes. Only subjects in the Mandometer intervention arm had a lower mean AG AUC (P < .001) after OGTT, with a more pronounced suppression of AG levels at 60 minutes (P < .001). Changes in fasting AG levels correlated with premeal satiety (r = −0.77; P = .002; 95% confidence interval [CI], −0.93 to −0.37). Changes in AG AUC after OGTT were correlated with variations in the portion size (r = 0.69; P = .009; 95% CI, 0.26 to 0.89) in the Mandometer intervention arm.

Martos-Moreno et al (57) also showed an increase in AG levels after OGTT, both at the diagnosis of obesity and after weight reduction, thus increasing the AG-to-total ghrelin ratio throughout the entire test. At diagnosis, the AG-to-total ghrelin ratio correlated with HOMA-IR index (r = 0.26; P < .05).

OGTT interventions: UAG

Paik et al (63) recorded a decrease in UAG levels with respect to baseline levels reaching a nadir at 60 minutes.

Prodam et al (45) showed decreased UAG levels for the entire OGTT (P < .0001) with a maximum inhibition at 60 minutes (P < .0001) and a lower inhibition of UAG levels at 60 minutes in pubertal subjects. Subjects with metabolic syndrome had a lower fasting UAG (P < .01) due to insulin resistance but similar UAG dynamics after OGTT. HOMA-IR was the only predictor of the UAG variation (P < .01). The AUC of UAG was negatively associated with insulin AUC and positively associated with the Matsuda index (P < .04).

Obese children and adolescents with PWS

Five studies examined ghrelin and food intake in young subjects with PWS (40, 63, 65–67) (Supplemental Table 4). Four were single-center studies (63, 65–67), and one was a multicenter study (40). All were interventional, case-control studies. The cases were PWS patients matched with controls represented by healthy overweight or obese subjects, without other known endocrine or genetic disorders (40, 63, 66, 67). One other study (65) also included a normal-weight subject in the control sample. In two studies, PWS patients were also compared with a normal-weight population (40), with short-stature children (67), or with PWS patients on treatment with recombinant human GH (rhGH) for a period ranging from 2 to 18 months (40). Interventions were: a mixed meal as a breakfast (67), a liquid meal (40), OGTT (63), or euglycemic-hyperinsulinemic clamp (66). One study (65) was designed as a 24-hour ghrelin profile, with meals served at defined times according to a standard hospital diet. Overall, data were reported from 36 PWS children and adolescents, with an age range of 6 to 16 years. Total ghrelin levels were analyzed in four studies (40, 65–67) and AG and UAG in one (63).

Meal interventions

Bizzarri et al (67) observed in prepubertal and pubertal subjects with PWS that total ghrelin levels were suppressed at 120 minutes after a standard mixed breakfast (P < .01). A negative correlation between fasting total ghrelin and insulin levels was found (P < .01), however only when all subjects were considered a group.

Paik et al (65) monitored total ghrelin levels for 24 hours in children and adolescents under a standard hospital diet. A postmeal decrease in total ghrelin levels was recorded; however, the data were inconsistent and showed wide individual variations.

Bizzarri et al (40) described, in prepubertal or in early pubertal PWS subjects, decreased total ghrelin levels (P < .05) at 45, 60, and 90 minutes after a standard liquid mixed meal. Fasting total ghrelin correlated with fasting PYY levels (r = −0.68; P < .05).

OGTT/clamp interventions

In the study by Paik et al (63), AG and UAG levels fell from fasting levels and reached nadir at 30 and 90 minutes, respectively, after OGTT in prepubertal PWS children. Fasting AG and UAG levels (r = 0.76, P = .006; r = 0.87, P < .001, respectively), and Δ nadir of AG correlated with WBISI (r = 0.64; P = .035).

Paik et al (66) investigated PWS children by euglycemic-hyperinsulinemic clamp and observed that total ghrelin levels were maximally suppressed at 90 minutes after the infusion of insulin. Total ghrelin levels were also relatively constant at this reduced level, 15 minutes after the discontinuation of the insulin infusion.

Discussion

The first aim of our review was to critically assess studies evaluating total ghrelin, AG, and UAG responses to food intake in the pediatric age group, including neonates to adolescents. The main limitations of this analysis were the low number of patients included in most studies and the intermediate quality and the high heterogeneity of the study populations regarding weight, age, puberty, food interventions, and ghrelin measurements. Despite this, we can, however, summarize that total ghrelin response to meals is peculiar in children, with refractoriness in neonates and lean prepubertal children and an inhibition starting from puberty. Total ghrelin levels decreased after meals irrespective of pubertal stages in obese children and adolescents. Conversely, total ghrelin inhibition after OGTT was present in all ages, with the exception of neonates. Doubts remain regarding the UAG response because data are scant; however, these limited data resemble those on total ghrelin. Data regarding the AG response to meals or OGTT, while numerous, are discordant although an inhibition with a following rise back appears to be present in both lean and obese children. Total ghrelin, AG, and UAG postfeeding profiles in PWS children are also peculiar, with a more conserved and a deeper inhibition from what is observed in PWS adults.

The results regarding the neonatal cohort differ in some aspects from those reported in normal-weight and obese children. A reasonable number of studies have been performed in this specific population, but just three studies have been included due to the inclusion criteria. It has to be underlined that all three studies are discordant with respect to ghrelin's response to food intake, despite being conducted in a sufficiently large cohort. Two of the studies evaluated preterm AGA and SGA neonates and observed an increase in total ghrelin levels after food intake (39) or, conversely, a refractoriness where no significant increases were reported (37). These contradictory findings could be linked to the smaller group studied by Bellone et al (37) or, more likely, to the time of observation, the second (39) and fourth days (37) after delivery. If true, one could hypothesize that the ghrelin response to feeding changes over time. This idea could explain the third study where total ghrelin levels were inhibited by glucose at 1 year of age (38). Consistent with this hypothesis, in the fetus, the pancreas appears to be a major source of ghrelin expression during perinatal life that is secreted primarily by epsilon cells, whereas low levels are detected in the fetal stomach (68, 69). The expression of ghrelin by the stomach increases gradually after birth to reach adult-like levels by 3–5 weeks of life in mice, whereas pancreatic expression declines from birth to weaning and is almost undetectable in the adult pancreas (69). Furthermore, the degradation of AG into UAG and non-UAG metabolites is faster in the fetus and in the rat neonate compared to the adult (70). However, we must not forget that the meal composition could have a role, as demonstrated in the third study where there was a stronger inhibition due to glucose and a blunted action due to milk enteral feeding or breast formula.

No studies on feeding regulation of AG and UAG levels in newborns were found. Findings on LGA neonates are lacking, and those regarding SGA neonates are scant. More information is needed in this specific phase of life, with attention to all ghrelin forms and gestational ages and with the specific goal to unravel whether ghrelin responses are stimulated or blunted by meals in a time after delivery dependency.

The results regarding the cohort of normal-weight children are derived from a larger number of studies, although the cumulative number of subjects is lower than that of studies in neonates. Two different interventions were performed—meals and OGTT—with more observations made in the prepubertal category. The strength of these studies is the design, with similar meals in terms of carbohydrate composition ranging from 45–60%. Studies conducted in prepubertal children are concordant with the finding that total ghrelin levels are refractory to an inhibition by both solid and liquid meals (40, 43, 44). Conversely, from puberty total ghrelin levels are inhibited by meals (41). The two studies performed with an OGTT demonstrated an inhibition of total ghrelin levels after glucose ingestion in children, although there was a discrepancy with respect to the maximum inhibition with a nadir at 60 minutes (47) or 120 minutes (44). No powered studies with OGTT have been performed in normal-weight adolescents.

An explanation of this peculiar profile of the ghrelin system in prepubertal children is not evident. One could argue that the insensitivity of ghrelin to the inhibitory effect of food intake in childhood fits well with the hypothesis that the functional profile of the ghrelin system in children is oriented to anabolic processes of growth. This hypothesis is in agreement with the fact that children with poor weight gain and with a failure to thrive have higher fasting total ghrelin and AG levels (71, 72), again suggesting that the system is driven to an anabolic balance. It is conceived that, as in anorexia, in conditions of caloric restriction, ghrelin increases blood glucose concentrations in order to maintain glucose homeostasis (73). On the other hand, the findings of an inhibition in prepubertal children after an OGTT suggest that a meal would be able to inhibit ghrelin only when sufficient amounts of adsorbed carbohydrates elevate glucose and insulin levels. However, we have demonstrated in adults that differently from carbohydrates and independently from their modulatory effects on insulin secretion and glucose levels, both lipids and amino acids play a negligible role in the acute control of ghrelin secretion (35). Indeed, we can hypothesize that prepubertal lean children are partially refractory to carbohydrates and that this is overcome when a large amount is administered, or more probably when glucose is administered. An OGTT stimulus could expose the ghrelin-secreting cells in the stomach to more rapid and dramatic increases in simple carbohydrates, which could suppress ghrelin levels more rapidly and strongly than do mixed solid meals at this age. This would seem feasible if we consider that all studies with OGTT conducted in obese children and adolescents are concordant in demonstrating an inhibition of total ghrelin levels at 60 minutes after glucose administration (46, 47, 55, 58–61, 64). Because the inhibition of total ghrelin after a mixed meal starts from puberty to adulthood in lean subjects, we can deduce that a progressive maturation of the stomach in concert with the physiology of growth is present.

Although lean children are refractory to ghrelin inhibition by meals, obese children presented a decrease in total ghrelin levels after mixed or liquid meals, irrespective of pubertal stage (41, 49–53, 56, 74). The nadir is concordant in all studies occurring at 60 minutes after the meal or in some individuals at 30 minutes (52), with the inhibition prolonged up to 120 minutes and with a less frequent rebound after the nadir (41, 50). Only two studies (40, 44) did not record any variation in total ghrelin levels for a total of 22 subjects, compared to 152 children and adolescents included in the other studies. This discrepancy could depend on the small sample size or on the meal composition, specifically the study of Bizzarri et al (40) conducted with a liquid meal and a low total caloric intake (100 kcal at fasting). On the other hand, the inhibition is present only for high-caloric meals (about 60% of RDA) with maintenance of refractoriness for small caloric meals (about 25% of RDA) in one study (53). The finding that total ghrelin levels are inhibited by meals in obese but not in lean children is of particular interest if we remember that total ghrelin levels are lower in obese compared to normal-weight subjects (12). This inhibition could likely be dependent on higher insulin levels after feeding. It has been postulated that insulin resistance acts diversely at the level of muscle with respect to the gut, being able to correctly inhibit ghrelin because insulin sensitivity is regarded as a prerequisite for sufficient postprandial ghrelin suppression (76). This could explain why ghrelin inhibition after mixed meals in lean children emerges from puberty when hyperinsulinemia is physiologically coupled with the start of puberty. However, because the interrelation between ghrelin and insulin is conflicting, other candidate mediators seem to be involved in the regulation of postprandial ghrelin secretion such as glucose, cholecystokinin, glucagon-like peptide 1, PYY, pancreatic polypeptide, and somatostatin (3–5). Because ghrelin is the only orexigenic peptide, we can speculate that the meal-induced decrease in total ghrelin levels in obese children is a rapid defensive mechanism to reduce appetite and the risk of weight gain, whereas in lean children the anabolic stimulus for growth has the upper hand.

Although data on UAG response to meals in lean and obese children are completely lacking, findings on AG levels in lean and obese adolescents are contradictory. An increase followed by an inhibition was observed in the study by Mittelman et al (53), whereas no changes were seen in that of Misra et al (42) despite similar populations in terms of puberty, sample size, methods of measurement, and meal composition. An unexpected result in the study of Mittelman et al (53) was the transient increase in AG levels in the first 15 minutes after starting the meal. These data are similar to those of total ghrelin by Erdmann et al (77), who found that total ghrelin levels transiently increased in this time period during meals consisting primarily of fat, protein, or vegetables. Both authors proposed a precocious gastric-stimulated ghrelin response to meals, which is only suppressed once insulin secretion is stimulated by carbohydrate absorption. Because pediatric studies have been conducted in adolescents, a gap remains to be filled in prepubertal children.

Two studies evaluated AG and UAG levels after OGTT in both prepubertal and pubertal obese subjects (45, 63), with one of these studies also including lean children and adolescents (45). Four other studies evaluated AG levels in an obese pediatric population (57, 60, 62, 64). Ghrelin forms presented a specific profile with a decrease up to 90 minutes and a maximum inhibition of AG at 60 minutes, followed by a rise back to baseline in most of the studies, which involved a total of 108 patients (45, 62–64) and in agreement with those in adults (78). However, one study reported a precocious surge at 30 minutes with 70 subjects (57), whereas in another involving 20 obese children there was no significant variation (60). Indeed, further studies are needed to clarify the AG profile after glucose ingestion in childhood. The profile of AG was correlated with insulinogenic index at 120 minutes in children (45). One could hypothesize that a progressive decrease in insulin secretion after OGTT allows AG levels to rebound, or the increase in AG concentrations inhibits the later glucose-induced insulin secretion (79, 80). The two studies on UAG levels were both concordant in recording decreased UAG levels up to 120 minutes after OGTT, with a maximum inhibition of UAG at 60 minutes (45, 63) being more pronounced in prepubertal than pubertal individuals (45). Indeed, UAG inhibition was lower in those with a higher insulin resistance and hyperinsulinemia. Because UAG is the most abundant circulating form, these findings fit well with the role of insulin in total ghrelin secretion. It is likely that UAG secretion reflects well total ghrelin secretion also in childhood.

Five studies reported ghrelin variations after meals, OGTT, or clamps in children with PWS. Four of them investigated total ghrelin levels, of which three recorded an inhibition after feeding (40, 66, 67) and one showed a trend to decrease without significance, due to wide variability in a small group of patients (65). Indeed, the reduction in ghrelin levels is concordant in pediatric PWS subjects. It has to be emphasized that the decrease is deeper and with a later nadir at 90–120 minutes than with respect to studies in healthy children. Similarly, the study on AG and UAG observed an inhibition of both ghrelin forms in PWS children (63) with a decrease in AG levels again deeper yet precocious, whereas UAG levels decreased slowly and in a comparable manner to healthy obese children. As such, the postmeal ghrelin inhibition in PWS is conserved, in particular by influencing AG (63). Because this specific profile deteriorates in some studies in PWS adults (81), this would imply that the physiological regulation of appetite/satiety of ghrelin is operative during childhood and progressively deteriorates and vanishes in adulthood as hyperphagia and obesity increase.

Particular attention has to been paid to the methods used to measure ghrelin. Assays and methods are variable across the studies, and many of them did not report how they stabilized plasma samples after collections. All studies on total ghrelin used a single-antibody assay, according to the general assumption that this reflects AG levels well. However, the data of Liu et al (32) call this into question, in particular for studies at fasting. Moreover, most studies on AG used aprotinin or HCl to stabilize the peptide. However, it has been demonstrated that the pretreatment with 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride in addition to EDTA and without plasma acidification is the best method to accurately quantify AG (32, 75).

The findings of this systematic review indicate that ghrelin secretion after feeding is differently regulated during infancy and childhood and is further modulated by weight status. Total and UAG levels are refractory to an inhibition by food intake in the first part of life, maybe due to anabolic processes. When puberty starts or obesity is present, total ghrelin and UAG decrease after a mixed meal. Glucose ingestion is able to inhibit total ghrelin, UAG, and AG levels at any age. AG levels rapidly rise after feeding. Because studies on AG and UAG forms are less prevalent, there is a need for high-quality data in the future. Standardization in the method of sample collection should be encouraged to provide more clear and comparable data.

Acknowledgments

The authors thank Gillian Walker for her technical assistance.

This study was supported by University of Piemonte Orientale “A. Avogadro,” Ministero dell'Università e della Ricerca Scientifica (MIUR, grant n°20082P8CCE, 2008).

Disclosure Summary: None of the authors of this manuscript have any financial interest that has influenced the results or interpretation of the manuscript.

Abbreviations

 
• AG

  acylated ghrelin

 
• AGA

  appropriate for gestational age

 
• AUC

  area under the curve

 
• BMI

  body mass index

 
• CI

  confidence interval

 
• GHS-R

  GH secretagogue receptor

 
• GOAT

  ghrelin O-acyltransferase

 
• HOMA-IR

  homeostasis model assessment for insulin resistance

 
• IGFBP-1

  IGF binding protein-1

 
• IVGTT

  iv glucose tolerance test

 
• LGA

  large for gestational age

 
• OGTT

  oral glucose tolerance test

 
• PWS

  Prader-Willi syndrome

 
• PYY

  peptide YY

 
• SDS

  SD score

 
• SGA

  small for gestational age

 
• UAG

  unacylated ghrelin

 
• WBISI

  whole-body insulin sensitivity index.

References