International Journal of Obesity (2015) 39, 1501–1503

© 2015 Macmillan Publishers Limited All rights reserved 0307-0565/15

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PEDIATRIC SHORT COMMUNICATION

Circulating GLP-1 in infants born small-for-gestational-age:
breast-feeding versus formula-feeding
M Díaz1,2, J Bassols3,4, G Sebastiani1,2, A López-Bermejo3,4, L Ibáñez1,2,6 and F de Zegher5,6

Prenatal growth restraint associates with the risk for later diabetes, particularly if such restraint is followed by postnatal formula-
feeding (FOF) rather than breast-feeding (BRF). Circulating incretins can influence the neonatal programming of hypothalamic
setpoints for appetite and energy expenditure, and are thus candidate mediators of the long-term effects exerted by early nutrition.
We have tested this concept by measuring (at birth and at age 4 months) the circulating concentrations of glucagon-like peptide-1
(GLP-1) in BRF infants born appropriate-for-gestational-age (AGA; n = 63) and in small-for-gestational-age (SGA) infants receiving
either BRF (n = 28) or FOF (n = 26). At birth, concentrations of GLP-1 were similar in AGA and SGA infants. At 4 months, pre-feeding
GLP-1 concentrations were higher than at birth; SGA-BRF infants had GLP-1 concentrations similar to those in AGA-BRF infants but
SGA-FOF infants had higher concentrations. In conclusion, nutrition appears to influence the circulating GLP-1 concentrations in
SGA infants and may thereby modulate long-term diabetes risk.

International Journal of Obesity (2015) 39, 1501–1503; doi:10.1038/ijo.2015.117

INTRODUCTION
Fetal growth restraint is associated with risk of becoming adipose
and insulin resistant in childhood 1 and of developing type 2
diabetes in later life.2,3 The mediating mechanisms are poorly
understood, but neonatal breast-feeding (BRF) is thought to
confer long-term protective effects.4 A decade ago, an impaired
effect of the incretin glucagon-like peptide-1 (GLP-1) was
suspected as candidate mechanism, but GLP-1 secretion, as well
as GLP-1 action on insulin secretion, were found to be normal in
young adults with low birth weight.5
Early infancy is a critical window of neurodevelopmental
plasticity, and recent data suggest that neonatally circulating
incretins can influence the hypothalamic setpoints of features
such as appetite and energy expenditure.6–9 We performed a first
test of this novel concept in human infants: longitudinally (at birth
and at 4 months), we have measured the circulating concentra-
tions of GLP-1 in BRF infants born appropriate-for-gestational-age
(AGA) and in small-for-gestational-age (SGA) infants receiving
either BRF or formula-feeding (FOF).
MATERIALS AND METHODS
Study population
The study population consisted of 117 term infants (63 AGA-BRF,
28 SGA-BRF and 26 SGA-FOF) who participated in a previously
described, longitudinal study of the body composition and the
endocrine-metabolic state of SGA infants, as compared with AGA
controls.10 The difference between the original population10 and
the present population ensues from the availability of serum to
assess GLP-1 (at birth and at 4 months) and from the prioritization
of the three subgroups that are nowadays most relevant for
clinical practice (Supplementary Figure 1).
Thus, the inclusion criteria for the present report were:
1. Birth at Hospital Sant Joan de Déu, Barcelona, after an
uncomplicated, term (37–42 weeks), singleton pregnancy (no
maternal hypertension, preeclampsia, gestational diabetes,
alcohol abuse or drug addiction).
2. Birth weight between 2.9 and 3.9 kg for AGA (between − 1 s.d.
and +1 s.d. for gestational age) and between 1.9 and 2.6 kg for
SGA infants (⩽ −2 s.d. for gestational age).
3. Exclusive BRF for 4 months in AGA controls; either exclusive
BRF for 4 months, or exclusive FOF (Enfalac 1, Mead Johnson,
Glenview, IL, USA) in SGA infants.
4. Auxological assessments at birth and at the age of 2 weeks and
at 4 months; endocrine assessments at birth and age 4 months;
body-composition assessments at age of 2 weeks and at
4 months.
5. Enough cord serum available (at birth) and enough serum
available in pre-feeding state at age 4 months to enable
measurement of circulating GLP-1.
6. Written, informed consent in Spanish/Catalan language at birth.
Exclusion criteria were: complications at birth (need for
resuscitation or for parenteral nutrition) and congenital
malformations.
Assessments
As described,10 weight and length were measured by the same
investigator at birth and at 4 months. Weight was measured with a

1
Hospital Sant Joan de Déu, University of Barcelona, Esplugues, Barcelona, Spain; 2Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas
(CIBERDEM), ISCIII, Madrid, Spain; 3Department of Pediatrics, Dr. Josep Trueta Hospital, Girona, Spain; 4Girona Institute for Biomedical Research, Girona, Spain and 5Department of
Development and Regeneration, University of Leuven, Leuven, Belgium. Correspondence: Professor L Ibáñez, Hospital Sant Joan de Déu, University of Barcelona, Passeig de Sant
Joan de Déu, 2, Esplugues, Barcelona 08950, Spain.
E-mail: libanez@hsjdbcn.org
6
These authors contributed equally to this work.
Received 8 April 2015; revised 8 June 2015; accepted 13 June 2015; accepted article preview online 19 June 2015; advance online publication, 14 July 2015
 Circulating GLP-1 in infants
M Díaz et al
1502
beam balance (Seca, Hamburg, Germany) and length with a length RESULTS
board, the mean of three measurements being used for analysis. Figure 1 shows that at birth, the circulating concentrations of
Body composition was assessed by absorptiometry at the age of GLP-1 were readily detectable and similar in AGA and SGA infants.
2 weeks and at 4 months with a Lunar Prodigy, coupled to Lunar GLP-1 concentrations were also similar in girls and boys. In each
software (version 3.4/3.5; Lunar, Madison, WI, USA), adapted for study subgroup, GLP-1 levels at 4 months were higher than at
assessment of infants. Bone mineral content, lean mass, as well as
total, truncal and abdominal fat mass were assessed during
natural sleep; coefficients of variation (CVs) were o3% for lean 80
and fat mass.
p=0.001 *
Blood was sampled in the morning, in pre-feeding state. Neither
a DPP4 inhibitor nor aprotinin were added to the sample. Total p=0.045
serum GLP-1 was assessed by ELISA (Millipore, Billerica, MA, USA). 60 *
The antibody pair in this assay measures GLP-1 (7–36) and (9–36) *

GLP-1 (pmol/L)
and has no significant cross-reactivity with GLP-2, GIP, glucagon or
oxyntomodulin. The intra- and inter-assay CVs were o2% and 40
o10%; the lower detection limit was 1.5 pM. Serum insulin-like
growth factor I concentrations were measured by immunochem-
iluminiscence (IMMULITE 2000, Diagnostic Products, Los Angeles,
CA, USA), the detection limit being 25 ng/mL and the intra- and 20

inter-assay CVs o 10%. Serum high-molecular-weight adiponectin

was assessed by ELISA with intra- and inter-assay CVs o 9%.
0
Statistics and ethics
Statistical analyses were performed with IBM SPSS Statistics 19.0 AGA
Breastfed
SGA
Breastfed
SGA
Formulafed
(IBM SPSS, Chicago, IL, USA). Comparisons were performed by N=63 N=28 N=26
two-tailed t-test or Mann–Whitney test, as appropriate; P o 0.05
was considered statistically significant. Figure 1. Longitudinal, pre-feeding results of circulating GLP-1
concentrations in appropriate- and small-for-gestational-age infants
The original study was approved by the institutional review (AGA and SGA) at birth and at the age of 4 months. Results are
board of Barcelona University, Hospital of Sant Joan de Déu; depicted as medians (horizontal lines) with interquartile ranges
informed written consent was an inclusion criterion. The present (white boxes at birth; gray boxes at 4 months) and percentiles 10
manuscript focuses on GLP-1 because other endocrine, auxological and 90 (lower and upper whiskers). *Po 0.0001 versus birth, for each
and body-composition results have been described.10 study subpopulation.

Table 1. Results of clinical, endocrine and body-composition (by absorptiometry) assessments in infants born either AGA or SGA, and either BRF or
FOF throughout the first 4 postnatal months

At birth At age 4 months Changes between birth and 4 months

AGA BRF SGA BRF SGA FOF AGA BRF SGA BRF SGA FOF AGA BRF SGA BRF SGA FOF
(n = 63) (n = 28) (n = 26) (n = 63) (n = 28) (n = 26) (n = 63) (n = 28) (n = 26)

Birth
Gestational age 39.8 ± 0.2 38.7 ± 0.2* 38.5 ± 0.3* — — — — — —
(weeks)
Birth weight (kg) 3.2 ± 0.1 2.4 ± 0.1* 2.3 ± 0.1* — — — — — —
Birth length (cm) 49.4 ± 0.2 46.0 ± 0.3* 45.8 ± 0.3* — — — — — —
Birth weight Z-score − 0.2 ± 0.1 − 2.1 ± 0.1* − 2.3 ± 0.1* — — — — — —
Birth length Z-score − 0.3 ± 0.1 − 1.8 ± 0.1* − 1.8 ± 0.1* — — — — — —

Clinical featuresa
Age (days) 15 ± 1 17 ± 2 17 ± 1 136 ± 3 139 ± 3 139 ± 3 121 ± 3 122 ± 4 122 ± 3
Length (cm) 50.8 ± 0.3 48.0 ± 0.5* 47.1 ± 0.3* 63.6 ± 0.3 61.4 ± 0.5* 61.1 ± 0.4* 12.8 ± 0.3 13.4 ± 0.5 14.0 ± 0.5
Weight (kg) 3.5 ± 0.1 2.7 ± 0.1* 2.5 ± 0.1* 7.0 ± 0.1 6.3 ± 0.1* 6.3 ± 0.1* 3.6 ± 0.1 3.5 ± 0.2 3.8 ± 0.1
BMI (kg m − 2) 13.7 ± 0.2 12.0 ± 0.3* 11.4 ± 0.3* 17.3 ± 0.2 16.6 ± 0.2* 16.8 ± 0.3 3.6 ± 0.3 4.7 ± 0.3 5.4 ± 0.4*
PI (kg m − 3) 27.0 ± 0.4 25.0 ± 0.5* 24.4 ± 0.7* 27.2 ± 0.3 27.2 ± 0.4 27.6 ± 0.6 0.2 ± 0.4 2.2 ± 0.6* 3.2 ± 0.8*

Endocrinology
IGF-I (ng ml − 1) 54 ± 3 50 ± 13 46 ± 5 45 ± 3 43 ± 3 66 ± 5*,** −9±4 –7 ± 10 21 ± 5*,**
HMW adiponectin 35 ± 2 30 ± 3 26 ± 3* 35 ± 2 32 ± 3 41 ± 4 0±3 2±4 15 ± 4*,**
(mg l − 1)
GLP-1 (pmol l − 1) 18 ± 1 17 ± 2 19 ± 2 34 ± 1 35 ± 2 44 ± 3*,** 16 ± 2 18 ± 2 25 ± 2*,**

Absorptiometrya
BMC (g) 108 ± 2 84 ± 3* 80 ± 3* 198 ± 5 167 ± 6* 180 ± 5* 90 ± 4 83 ± 6 100 ± 5**
Lean mass (g) 2964 ± 53 2468 ± 70* 2408 ± 77* 4274 ± 99 4004 ± 103 4113 ± 110 1310 ± 94 1536 ± 101 1705 ± 113*
Fat mass (g) 740 ± 35 545 ± 54* 401 ± 37*,** 2812 ± 83 2548 ± 97 2352 ± 88* 2072 ± 71 2003 ± 111 1951 ± 86
Truncal fat mass (g) 244 ± 14 171 ± 20* 115 ± 13*,** 1064 ± 39 928 ± 39* 897 ± 39* 820 ± 35 757 ± 44 782 ± 39
Abdominal fat mass (g) 40 ± 2 33 ± 3* 20 ± 2*,** 171 ± 7 155 ± 10 140 ± 8* 131 ± 7 122 ± 9 120 ±9

Abbreviations: AGA, appropriate-for-gestational-age; BMC, bone mineral content; BMI, body mass index; BRF, breast-fed; FOF, formula-fed; GLP-1, glucagon-like
peptide-1; HMW, high molecular weight; IGF-I, insulin-like growth factor I; PI, ponderal index; SGA, small-for-gestational-age. Values are mean ± s.e.m. *Po0.05
versus AGA BRF, **Po0.05 versus SGA BRF. aat age 2 weeks instead of at birth.

International Journal of Obesity (2015) 1501 – 1503 © 2015 Macmillan Publishers Limited

birth. SGA-BRF infants had GLP-1 concentrations at 4 months and
GLP-1 increments between birth and 4 months that were
comparable to those in AGA-BRF controls. However, SGA-FOF
infants had higher GLP-1 concentrations at 4 months and also
higher GLP-1 increments than AGA-BRF controls and SGA-BRF
infants (Table 1 shows the numerical results).
Positive Spearman correlations (P o0.01) were identified
between GLP-1 and insulin-like growth factor I concentrations at
4 months (r = 0.27) and also between changes (0–4 months) of
GLP-1 and HMW adiponectin concentrations (r = 0.25).
DISCUSSION
At birth, circulating GLP-1 concentrations were similar in AGA and
SGA infants, suggesting that the intestinal transit of amniotic fluid
is sufficient (or that no intestinal transit is required) to generate
some release of GLP-1 by intestinal L-cells, and that prenatal
growth and fetal GLP-1 levels are essentially independent of
each other.
At 4 months, in pre-feeding state, the GLP-1 concentrations of
BRF infants were about twice as high as at birth. Our study design
does not allow to infer when exactly those GLP-1 levels surge, but
cross-sectional evidence from newborns aged 4–10 days suggests
that this surge (to supra-adult concentrations) occurs within the
first few days after birth.11
The observation that pre-feeding GLP-1 levels are higher in SGA
infants receiving FOF than in those receiving BRF during early
infancy suggests that neonatal nutrition is a modulator of
circulating GLP-1 in SGA infants and is thus a potential modulator
of hypothalamic setpoints that relate to appetite and energy
expenditure in subsequent life.6–9 For example, higher concentra-
tions of circulating GLP-1 during early infancy may be accom-
panied by lower hypothalamic sensitivity to GLP-1 in later life.
Given that SGA-BRF infants follow a less adipose and more
favorable endocrine-metabolic course than SGA-FOF infants,12
circulating GLP-1 may become a helpful biomarker in the design
of future milk formulas for SGA infants.
The strengths of the present study include the longitudinal
design, the co-availability of body-composition assessments and
of AGA-BRF controls. Study limitations include the relatively small
study population, the absence of sampling times in the neonatal
phase, and the absence of AGA-FOF controls. Future studies are
likely to investigate the potential influence of gut microbiota on
circulating GLP-1 and other incretins in early infancy,13 and thus
perhaps on the neonatal programming of appetite and other
hypothalamic setpoints.
In conclusion, circulating GLP-1 concentrations were normal in
SGA infants at birth, normal in SGA-BRF infants at 4 months but
elevated in SGA-FOF infants. It remains to be studied whether
elevated GLP-1 concentrations in early infancy have long-term
implications, for example, on the hypothalamic control of energy
homeostasis and thus on diabetes risk.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on International Journal of Obesity website (http://www.nature.com/ijo)
© 2015 Macmillan Publishers Limited
Circulating GLP-1 in infants
M Díaz et al
1503
ACKNOWLEDGEMENTS
This Study was supported by the Ministerio de Ciencia e Innovación, Instituto de
Salud Carlos III, by The Fondo Europeo de desarrollo Regional (FEDER), Madrid, Spain
(PI11/0443) and by the research Foundation Sant Joan de Déu (AFR 00020). MD and
LI are clinical investigators of CIBERDEM (www.ciberdem.org). JB is an investigator of
the Miguel Servet Fund from Carlos III National Institute of Health, Spain. AL-B is an
investigator of the 13 Fund for Scientific research (Ministry of Education and Science,
Spain). FdZ is a clinical investigator supported by the Clinical Research Council of the
University Hospital Leuven.
AUTHOR CONTRIBUTIONS
MD contributed to the study design and researched data; JB and SG researched
data; AL-B contributed to discussion; LI contributed to the study design; FdZ
contributed to the study design and wrote the manuscript. All the authors
reviewed/edited the manuscript.
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International Journal of Obesity (2015) 1501 – 1503