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GHRELIN LEVELS FROM FETAL LIFE THROUGH EARLY ADULTHOOD:
RELATIONSHIP WITH ENDOCRINE AND METABOLIC AND
ANTHROPOMETRIC MEASURES
LEANDRO SORIANO-GUILLE
´N,MD,VICENTE BARRIOS,PHD, JULIE A. CHOWEN,PHD, IGNACIO SA
´NCHEZ,MD,SANTIAGO VILA,MD,
JOSE
´QUERO,MD,PHD, AND JESU
´SARGENTE,MD,PHD
Objective To establish mean plasma ghrelin levels during fetal life and childhood.
Study design Cord blood was obtained at birth from premature (n = 29) and full-term newborns (n = 124). Fasting blood
samples were taken from 224 normal subjects divided according to Tanner stage and sex. Ponderal index or body mass index
was determined. Ghrelin; insulin-like growth factor (IGF)-I; IGF-II; IGF binding proteins 1, 2, and 3; insulin; glucose; and leptin
levels were measured.
Results Ghrelin levels did not differ between preterm and full-term newborns. Ghrelin increased significantly after birth,
peaking during the first 2 years of life, then decreasing until the end of puberty. Ghrelin levels correlated negatively with
anthropometric variables in full-term newborns and postnatally, but not in preterm newborns. A positive correlation between
ghrelin and IGF binding protein 1 was found.
Conclusions Ghrelin changes significantly throughout development, correlating with anthropometric and metabolic
parameters during extrauterine life. The highest levels of ghrelin are found during early postnatal life, when growth hormone
begins to exert its effects on growth and important changes in food intake occur, suggesting that this hormone may participate in
these processes. (J Pediatr 2004;144:30-5)
The recent discovery of ghrelin, an acylated 28 amino acid peptide,
1
has added new
perspectives to our understanding of the control of food intake, energy balance, and
growth. Ghrelin is primarily secreted by the stomach and duodenum, although
a minor portion of ghrelin synthesis occurs in other sites such as the hypothalamus,
pituitary, and lung. Ghrelin can bind to two different receptors
2
: growth hormone
secretagogue receptor 1a, involved in the control of growth hormone (GH) secretion, and
growth hormone secretagogue receptor 1b, whose function remains unknown. A new cell
type in the human pancreas, lung, and stomach produces ghrelin in the fetus, suggesting an
important role for this peptide in intrauterine life.
3,4
In addition to its GH-releasing
properties,
5
ghrelin stimulates appetite, reduces fat utilization, produces adiposity,
6-8
and
induces hyperglycemia.
9
Moreover, ghrelin is increased after fasting
10
and decreased after
feeding.
11,12
These data suggest that ghrelin plays an important role in feeding and GH
secretion and raises the question whether during gestation and in early extrauterine life,
ghrelin is implicated in the control of growth and metabolism.
It is well established that insulin, insulin-like growth factors (IGFs), and their
binding proteins (IGFBPs) play an important role in the regulation of fetal growth.
13,14
These factors are also good measures for the evaluation of growth during extrauterine life
15
and are altered in nutritional disorders affecting systemic growth.
16,17
Circulating levels of
See related article, p 36.
From the Department of Pediatric
Endocrinology and Research and the
Department of Biochemistry, Univer-
sidad Auto
´noma, Hospital Infantil
Universitario Nin
˜o Jesu´s; the Pediatric
Intensive Care Unit. Universidad
Complutense, Hospital Infantil Univer-
sitario 12 de Octubre; and the
Department of Neonatology, Univer-
sidad Auto
´noma, Hospital Infantil
Universitario La Paz, Madrid, Spain.
Submitted for publication Apr 25, 2003;
revision received July 21, 2003; accepted
Aug 29, 2003.
Reprint requests: Jesu´s Argente, MD,
PhD, Department of Pediatric Endo-
crinology and Research, Hospital In-
fantil Universitario Nin
˜o Jesu´s, Avda
Mene
´ndez Pelayo 65, E-28009 Madrid,
Spain. E-mail: argentefen@terra.es.
0022-3476/$ - see front matter
Copyright ª2004 Elsevier Inc. All rights
reserved.
10.1016/j.jpeds.2003.08.050
AGA Adequate for gestational age
ANOVA Analysis of variance
BMI Body mass index
GH Growth hormone
IGF Insulin-like growth factor
IGFBP Insulin-like growth factor binding protein
LGA Large for gestational age
PI Ponderal index
SGA Small for gestational age
30
leptin, a hormone secreted by adipocytes and the placenta that
acts as a hormonal feedback signal to regulate fat stores
through a hypothalamic mechanism, are positively correlated
with intrauterine growth
18
and are also modulated in
nutritional disorders.
19
Although it is known that ghrelin has important
metabolic effects postnatally, there are few data concerning
cord plasma ghrelin levels in newborns
20
and in normal
controls
21
and their relationship with anthropometric data and
the previously mentioned endocrine factors implicated in
growth and nutritional disorders. To investigate further the
role of ghrelin in growth and the evolution of ghrelin levels
from the fetus through puberty, we analyzed (1) the cord
plasma ghrelin levels in term and preterm newborns, (2) the
plasma ghrelin levels in normal controls, (3) the relationship
between ghrelin and anthropometric data, (4) the rela-
tionships between ghrelin and leptin, insulin, IGF-I, IGF-II,
and IGFBP-1 in newborns, and (5) the relationships bet-
ween ghrelin and leptin, insulin, glucose, IGF-I, IGFBP-1,
IGFBP-2, and IGFBP-3 in normal postnatal subjects.
SUBJECTS AND METHODS
Subjects
NEWBORNS.The study cohort included 29 premature
newborns (13 male and 16 female patients), four <32 weeks’
gestational age with a mean gestational age of 31.1 ± 0.1, and
25 with a period of gestation ranging from 32 to 36 weeks with
a mean gestational age of 35.1 ± 0.3 weeks. We also included
124 full-term newborns (60 male and 64 female patients) with
a mean gestational age of 39.4 ± 0.02 weeks. Only neonates
born by uncomplicated vaginal delivery and without hypoxia
were included. Mothers with pre-eclampsia, hypertension, or
diabetes or who smoked were excluded. Gestational age was
determined from the date of the last menstrual period and was
confirmed by early ultrasound scan.
Cord blood samples were obtained at birth. Venous cord
blood was collected in tubes containing EDTA plus aprotinin
and centrifuged at 48C. Plasma was stored at 808C until
assayed.
To evaluate the intrauterine nutritional state, the
ponderal index (PI: weight [g]/length
3
[cm] 3100) was
calculated. The newborns were then divided into three groups
according to Spanish PI tables
22
: >1 SD (high PI), between
1 and 1 SD (medium PI), and <1 SD (low PI). Birth
weight was measured and the newborns were divided into
adequate for gestational age (AGA: 10th to 90th percentile),
small for gestational age (SGA: < 10th percentile), and large
for gestational age (LGA: 90th percentile) according to
Spanish standard curves.
23
The weight was determined by
using a platform scale with an accuracy of ±10 g, and the
length was measured by using a Holtain infantometer
(Crymych, Wales, UK).
All mothers were informed of the purpose of the study
and gave consent as required by the local human ethics
committee.
Tanner Stages I-V
We also included 224 normal Spanish children divided
into five groups according to Tanner stage: I (n = 87), 45 male
and 42 female patients; II (n = 55), 21 male and 34 female
patients; III (n = 20), eight male and 12 female patients; IV
(n = 22), 10 male and 12 female patients; and V (n = 40), 16
male and 24 female patients. Control subjects were referred to
our division for suspected endocrine abnormalities and were
found to be normal with height between p10 and p90, weight
between p10 and p85, and body mass index (BMI) between
2 and +2 SD according to Spanish standards.
24
Blood
samples were obtained in the morning from fasting subjects
into chilled tubes containing EDTA (1 mg/mL) plus
aprotinin (500 U/mL). The tubes were centrifuged and stored
at 808C until assayed. The BMI was calculated as weight
(kg)/height (m
2
). The BMI SD score was based on normative
data from Spanish children.
24
All subjects were informed of the purpose of the study
and gave consent as required by the local human ethics
committee.
Biochemical Measurements
Plasma ghrelin levels were measured by a commercial
radioimmunoassay (Phoenix Pharmaceutical, Belmont, Calif )
using a polyclonal antibody that recognizes octanoylated and
nonoctanoylated ghrelin and
125
I-ghrelin as a tracer molecule.
The intra-assay and interassay coefficients of variation were
5.0% and 11.2%, respectively. Assay sensitivity was 12 pg/mL.
Serum IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3,
insulin, and leptin were measured as previously reported.
3-5,7
Plasma glucose was measured by the glucose oxidase
method on a Beckman Glucose Analyzer (Fullerton, Calif ).
Statistics
All data are reported as the mean ± SEM. When two
experimental groups were compared, the Student ttest was
applied. For more than two experimental groups, analysis was
performed by analysis of variance (ANOVA), followed by the
Scheffe F test. Correlation analysis was performed to assess the
effect of age and Tanner stage on ghrelin levels. Multiple
regression analysis was performed to determine the overall
relationship of the variables studied, followed by partial
correlation analysis. Stepwise multiple regression analysis was
performed to determine the effect of the other independent
variables on ghrelin. A Pvalue <.05 was chosen as the level of
significance.
RESULTS
Ghrelin Levels and Sex
No differences were found in ghrelin levels between male
and female newborns (male newborns [n = 73], 503 ± 26 pg/
mL vs female newborns [n = 80], 504 ± 24 pg/mL) or between
male and female newborns at any Tanner stage (I, male
newborns, 866 ± 39 pg/mL vs female newborns, 924 ± 35 pg/
Ghrelin Levels from Fetal Life Through Early Adulthood: Relationship with
Endocrine and Metabolic and Anthropometric Measures 31
mL; II, male newborns, 547 ± 27 pg/mL vs female newborns,
490 ± 18 pg/mL; III-IV, male newborns, 444 ± 39 pg/mL vs
female newborns, 401 ± 44 pg/mL; V, male newborns,
350 ± 16 pg/mL vs female newborns, 367 ± 25 pg/mL.
Ghrelin Levels and Age
When mean ghrelin levels were compared in preterm
infants <32 weeks (558 ± 241 pg/mL [n = 4]), preterm
infants with gestational age ranging from 32 to 37 weeks
(468 ± 48 pg/mL [n = 25]), and full-term newborns (514 ± 19
pg/mL [n = 124]), no difference was found. However,
significantly higher ghrelin levels were observed at Tanner
stage I compared with newborns (894 ± 26 pg/mL vs 511 ± 19
pg/mL, P< .001). There was a significant decline in ghrelin
concentrations throughout postnatal development (Figure,
A). At Tanner stage I, ghrelin levels were significantly higher
than at all subsequent stages. There was also a significant
difference between Tanner II and III-IV and between Tanner
II and V (Tanner I, 894 ± 26 pg/mL; Tanner II, 510 ± 15 pg/
mL; Tanner III-IV, 424 ± 29 pg/mL; Tanner V, 370 ± 18).
When Tanner I subjects were analyzed separately, the group
between 1 and 24 months had significantly higher ghrelin
levels compared with newborns and children >24 months
(P< .001; Figure, B). There was no significant difference
between the neonatal groups.
By regression analysis, there was a negative correlation
between age and ghrelin levels (r = 0.73, P< .001) and
between Tanner stage and ghrelin levels (r = 0.67, P< .001).
As expected, there was also a direct correlation between age
and Tanner stage (r = 0.82, P< .001). Because endocrine
studies in children are normally reported according to Tanner
stage, all results are expressed according to Tanner stage.
Anthropometric Data and Ghrelin Levels
In preterm infants, no difference in ghrelin levels was
found between the three groups of ponderal index studied
(<1SD [n = 4], 620 ± 222 pg/mL; 1 to 1 SD [n = 15],
406 ± 71 pg/mL; >1 SD [n = 10], 493 ± 54 pg/mL. In term
newborns, a significant difference was found between infants
with a low ponderal index (<1 SD, 685 ± 64 pg/mL) and
those with a medium ponderal index (1 to 1 SD, 490 ± 27
pg/mL) or a high ponderal index (>1 SD, 484 ± 25 pg/mL;
P< .05 by ANOVA). In term newborns, significant di-
fferences in ghrelin levels were found between infants who
were SGA (n = 10, 621 ± 89 pg/mL) and infants who were
LGA (n = 11, 376 ± 32 pg/mL), and between infants who
were AGA (n = 103, 521 ± 20 pg/mL) and newborns who
were LGA (P< .05 by ANOVA). In preterm newborns, no
significant differences were found (SGA [n = 5], 546 ± 185
pg/mL; AGA [n = 23], 450 ± 247 pg/mL; LGA [n = 1], 417
pg/mL. There was a significant negative correlation between
ghrelin levels and PI in term infants (r = 0.50, P< .05) that
was not present in preterm newborns, and between ghrelin
levels and BMI in normal controls (r = 0.39, P< .001).
During childhood and adolescence, a significant difference
between children with low BMI (<1 SD, 779 ± 59 pg/mL)
and children with normal BMI (1 to 1 SD, 597 ± 24 pg/
mL), and between low BMI and high BMI (>1 SD, 518 ± 56
pg/mL) was found (P< .05 by ANOVA).
Figure. A, Mean ( ± SEM) plasma ghrelin levels in the four groups (Tanner I, II, III-IV, V). B, Comparison of plasma ghrelin levels
between preterm infants <32 weeks, preterm infants with gestational age ranging from 32 to 36 weeks, full-term newborns, and
Tanner stage I divided into groups of <1 month, between 1 and 24 months, and >24 months. *P< .001, ANOVA.
32 Soriano-Guille´n et al The Journal of Pediatrics January 2004
Correlation Among Plasma Glucose, IGF-I, IGF-II,
IGFBP-1, IGFBP-3, Insulin, Leptin, and Ghrelin
Levels
No correlation between ghrelin and insulin, leptin,
IGF-I, or IGF-II was found in newborns (Table I). A
significant correlation between ghrelin and IGFBP-1 in term
(r = 0.52, P< .01) and preterm infants (r = 0.56; P< .01) was
found. During postnatal life, there was a significant correlation
between ghrelin and IGF-I, IGFBP-1, IGFBP-2, IGFBP-3,
insulin, and leptin. Although the last was significant, the
r value was very low. When the analysis was performed
according to Tanner stage, there was a significant correlation
between ghrelin and IGFBP-1 at all Tanner stages (I,
r = 0.50, P< .05; II, r = 0.68, P< .05; III-IV, r = 0.55,
P<.001; V, r = 0.45, P< .05). No correlation between
ghrelin and IGF-I, IGFBP-3, IGFBP-2 and leptin was
observed at any of the Tanner stages (Table I). A negative
correlation between ghrelin and insulin (r = 0.58, P< .01)
was observed only at Tanner stage III-IV (Table II). There
was a significant negative correlation between ghrelin and
glucose levels in all subjects (r = 0.51, P< .001).
DISCUSSION
Controversy exists concerning the prenatal ontogeny
and embryonic role of ghrelin in rats.
25,26
In human beings,
this peptide
27
and the cells that produce ghrelin
3,4
are detected
very early in intrauterine life. Although Chanoine et al
20
analyzed ghrelin levels in term newborns, we have also
included preterm infants. We have found that ghrelin is
detectable in fetal cord blood as early as 30 weeks’ gestational
age and that there is no significant difference in ghrelin levels
between newborns <32 weeks of gestation and newborns >32
weeks of gestation. Unfortunately, only four infants <32
weeks of gestation could be studied, and a large SE was found.
This finding could suggest that this is a period in which
changes in ghrelin levels are taking place, but more data are
needed in early gestation to confirm this possibility. The early
(10 weeks of gestation) and widespread distribution of
ghrelin-producing cells in embryonic tissues, including the
placenta, stomach, lung, and pancreas, suggests a trophic and
morphogenetic role for this peptide.
3,4,27
Whether ghrelin acts
as a trophic or endocrine factor could be related to the receptor
subtype expressed in the tissue. Recent studies suggest that one
receptor subtype may be more important in transmitting the
trophic effects of ghrelin.
2
Hence, it would be of great interest
to examine the expression of this receptor subtype in the
developing fetus. Furthermore, an increased metabolic or
endocrine role for ghrelin could be related to increased
expression of type 1A receptor in endocrine tissues such as
hypothalamus or pituitary during the last period of gestation.
Indeed, there is developmental regulation of this receptor
postnatally,
28
suggesting that this may be one mechanism by
which the response to ghrelin is modulated.
In agreement with Chanoine et al,
20
we observed
a negative correlation between ghrelin and anthropometric
measures in term newborns. However, in contrast, we used PI
rather than weight or BMI because it has been suggested to be
a better indicator of nutritional status in newborn infants. In
preterm infants, there was no correlation between PI and
ghrelin, whereas in term infants, there was a significant
negative correlation. Ghrelin levels were significantly higher
in term infants with a low PI compared with those with a high
PI. A similar observation was made comparing newborns who
were SGA and LGA. When preterm infants were divided into
three groups according to PI or size for gestational age, no
significant difference was found. However, the same tendency,
in which the smaller neonates have higher levels of ghrelin
compared with the neonates who were normal or LGA, was
observed. Taken together, these data suggest that ghrelin may
acquire its role in regulating appetite and metabolism during
the latest stage of gestation. Hence, as occurs in other endo-
crine axes, the premature infant may not have a mature system
of energy balance control. During late gestation, ghrelin may
assume its metabolic role, preparing the late fetus for extra-
uterine life by inducing adiposity,
6
stimulating food intake,
7,8
maintaining glucose levels,
9
and stimulating GH secretion.
10
In extrauterine life, ghrelin levels are higher compared
with newborns, especially during the first 2 years of life. At the
beginning of extrauterine life, GH begins to exert its effect in
growth and development. In addition, important changes in
food intake and metabolism take place. Ghrelin levels then
begin to diminish with age. This finding contrasts with results
Table I. Linear correlation among ghrelin, IGF-I,
IGF-II, IGFBP-1, insulin, leptin levels, and ponderal
index in preterm and term newborns
Preterm Term
Ghrelin vs PI r = 0.08 (NS) r = 0.5
*
Ghrelin vs IGF-I r = 0.12 (NS) r = 0.13 (NS)
Ghrelin vs IGF-II r = 0.15 (NS) r = 0.16 (NS)
Ghrelin vs IGFBP-1 r = 0.56
y
r = 0.52
y
Ghrelin vs insulin r = 0.30 (NS) r = 0.22 (NS)
Ghrelin vs leptin r = 0.3 (NS) r = 0.15 (NS)
IFG-I vs PI r = 0.53
*
r = 0.55
z
IGF-I vs IGF-II r = 0.67
z
r = 0.23
*
IGF-I vs IGFBP-1 r = 0.14 (NS) r = 0.52
z
IGF-I vs insulin r = 0.18 (NS) r = 0.02 (NS)
IGF-I vs leptin r = 0.44
*
r = 0.26
*
IGF-II vs PI r = 0.54
z
r = 0.22
*
IGF-II vs IGFBP-1 r = 0.30 (NS) r = 0.13 (NS)
IGF-II vs insulin r = 0.09 (NS) r = 0.14 (NS)
IGF-II vs leptin r = 0.39
*
r = 0.13 (NS)
IGFBP-1 vs PI r = 0.44
*
r=0.55
z
IGFBP-1 vs insulin r = 0.15 (NS) r = 0.09 (NS)
IGFBP-1 vs leptin r = 0.23 (NS) r = 0.37
z
Insulin vs PI r = 0.19 (NS) r =0.11 (NS)
Insulin vs leptin r = 0.8
z
r = 0.24
*
Leptin vs PI r = 0.06 (NS) r = 0.41
z
*P< .05.
yP< .01.
zP< .001.
Ghrelin Levels from Fetal Life Through Early Adulthood: Relationship with
Endocrine and Metabolic and Anthropometric Measures 33
of a previous report, in which ghrelin levels did not differ
between prepubertal and pubertal children.
21
This discrepancy
may be a result of the small number of subjects included in the
previous study and the fact that the children were not divided
into groups according to Tanner stage.
A high density of ghrelin-binding sites has been
demonstrated in human ovary and testis,
29
and ghrelin has
been shown to exert a strong inhibitory action on several
steroidogenic enzymes.
30
Recent studies
31
show a negative
relationship between androstenedione and ghrelin levels,
suggesting that ghrelin may regulate glandular steroidogen-
esis. Our data show a decrease in ghrelin levels from early
childhood to puberty. Whether ghrelin is involved in pubertal
onset remains to be elucidated.
Although ghrelin levels correlate with parameters of the
GH axis, leptin, and insulin levels throughout development,
this relation is most likely a result of the variation of ghrelin as
well as these factors with age and Tanner stage. Hence, when
analyzed separately at each Tanner stage, no correlation
between these parameters, except IGFBP-1, was found.
There was a positive correlation between ghrelin and
IGFBP-1 levels from the fetus through Tanner stage V. This
positive relationship between ghrelin and IGFBP-1 is also
observed after weight normalization in obese children and
anorexic adolescents. Like ghrelin levels, IGFBP-1 levels
decrease during childhood until adulthood,
15
vary markedly
during the day in relation to the metabolic status in a GH-
independent manner,
14,32
and are elevated in intrauterine
growth retardation.
13
It is thought that IGFBP-1 levels
partially depend on insulin levels,
16,17,32
although we found no
relationship between ghrelin and insulin. Ghrelin and
IGFBP-1 are both secreted in a pulsatile fashion, and the
other measured parameters are not. Synchronization of these
pulses may underlie the correlation found at all ages. Hence,
our data suggest a possible relationship between ghrelin and
IGFBP-1, or their mechanisms of control, but the basis of this
relationship remains to be elucidated.
Ghrelin and its receptor are expressed in pancreatic
bcells,
2,3
suggesting that it could have effects on insulin
secretion. However, the influence of ghrelin on glucose
metabolism and insulin is still controversial.
33-36
We found
a significant correlation between ghrelin and insulin only at
Tanner stages III and IV. The lack of correlation at other
stages cannot be easily explained. It is possible that the
inhibitory action of ghrelin on insulin secretion is dose-
dependent and glucose-dependent and that this peptide does
not affect baseline insulin secretion. The negative relationship
observed between ghrelin and glucose levels during de-
velopment indicates a possible role for ghrelin in glucose
metabolism, although it is not clear whether this role is
mediated through effects on adiposity, through paracrine
effects on insulin secretion, by modulation of insulin signaling
pathways, or by its somatotropic effects.
37
Although leptin levels are positively correlated with PI
18
and BMI,
19
and ghrelin is negatively correlated with PI and
BMI, we found no relationship between ghrelin and leptin
in newborns or during childhood, as has been previously
reported.
38
The presence of ghrelin in cord plasma as early as 30
weeks of gestation suggests a role for this peptide in the
developing fetus, possibly as a trophic factor. During later
stages of gestation, ghrelin may acquire its role in controlling
metabolism and GH secretion, with the highest levels found
during early postnatal life, when growth and metabolism are
accelerated.
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Ghrelin Levels from Fetal Life Through Early Adulthood: Relationship with
Endocrine and Metabolic and Anthropometric Measures 35
