International Journal of Obesity (2015) 39, 1501–1503 © 2015 Macmillan Publishers Limited All rights reserved 0307-0565/15 www.nature.com/ijo
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 formulafeeding (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 concentrations 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: [email protected] 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
beam balance (Seca, Hamburg, Germany) and length with a length board, the mean of three measurements being used for analysis. Body composition was assessed by absorptiometry at the age of 2 weeks and at 4 months with a Lunar Prodigy, coupled to Lunar software (version 3.4/3.5; Lunar, Madison, WI, USA), adapted for 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 and fat mass. Blood was sampled in the morning, in pre-feeding state. Neither a DPP4 inhibitor nor aprotinin were added to the sample. Total serum GLP-1 was assessed by ELISA (Millipore, Billerica, MA, USA). The antibody pair in this assay measures GLP-1 (7–36) and (9–36) and has no significant cross-reactivity with GLP-2, GIP, glucagon or oxyntomodulin. The intra- and inter-assay CVs were o2% and o10%; the lower detection limit was 1.5 pM. Serum insulin-like growth factor I concentrations were measured by immunochemiluminiscence (IMMULITE 2000, Diagnostic Products, Los Angeles, CA, USA), the detection limit being 25 ng/mL and the intra- and inter-assay CVs o 10%. Serum high-molecular-weight adiponectin was assessed by ELISA with intra- and inter-assay CVs o 9%.
RESULTS Figure 1 shows that at birth, the circulating concentrations of GLP-1 were readily detectable and similar in AGA and SGA infants. GLP-1 concentrations were also similar in girls and boys. In each study subgroup, GLP-1 levels at 4 months were higher than at
80
p=0.001
*
p=0.045 60
GLP-1 (pmol/L)
1502
* *
40
20
0
Statistics and ethics Statistical analyses were performed with IBM SPSS Statistics 19.0 (IBM SPSS, Chicago, IL, USA). Comparisons were performed by two-tailed t-test or Mann–Whitney test, as appropriate; P o 0.05 was considered statistically significant. The original study was approved by the institutional review board of Barcelona University, Hospital of Sant Joan de Déu; informed written consent was an inclusion criterion. The present manuscript focuses on GLP-1 because other endocrine, auxological and body-composition results have been described.10
AGA Breastfed N=63
SGA Breastfed N=28
SGA Formulafed N=26
Figure 1. Longitudinal, pre-feeding results of circulating GLP-1 concentrations in appropriate- and small-for-gestational-age infants (AGA and SGA) at birth and at the age of 4 months. Results are depicted as medians (horizontal lines) with interquartile ranges (white boxes at birth; gray boxes at 4 months) and percentiles 10 and 90 (lower and upper whiskers). *Po 0.0001 versus birth, for each 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 (n = 63)
SGA BRF (n = 28)
SGA FOF (n = 26)
AGA BRF (n = 63)
SGA BRF (n = 28)
SGA FOF (n = 26)
AGA BRF (n = 63)
SGA BRF (n = 28)
SGA FOF (n = 26)
Birth Gestational age (weeks) Birth weight (kg) Birth length (cm) Birth weight Z-score Birth length Z-score
39.8 ± 0.2
38.7 ± 0.2*
38.5 ± 0.3*
—
—
—
—
—
—
3.2 ± 0.1 49.4 ± 0.2 − 0.2 ± 0.1 − 0.3 ± 0.1
2.4 ± 0.1* 46.0 ± 0.3* − 2.1 ± 0.1* − 1.8 ± 0.1*
2.3 ± 0.1* 45.8 ± 0.3* − 2.3 ± 0.1* − 1.8 ± 0.1*
— — — —
— — — —
— — — —
— — — —
— — — —
— — — —
Clinical featuresa Age (days) Length (cm) Weight (kg) BMI (kg m − 2) PI (kg m − 3)
15 ± 1 50.8 ± 0.3 3.5 ± 0.1 13.7 ± 0.2 27.0 ± 0.4
17 ± 2 48.0 ± 0.5* 2.7 ± 0.1* 12.0 ± 0.3* 25.0 ± 0.5*
17 ± 1 47.1 ± 0.3* 2.5 ± 0.1* 11.4 ± 0.3* 24.4 ± 0.7*
136 ± 3 63.6 ± 0.3 7.0 ± 0.1 17.3 ± 0.2 27.2 ± 0.3
139 ± 3 61.4 ± 0.5* 6.3 ± 0.1* 16.6 ± 0.2* 27.2 ± 0.4
139 ± 3 61.1 ± 0.4* 6.3 ± 0.1* 16.8 ± 0.3 27.6 ± 0.6
121 ± 3 12.8 ± 0.3 3.6 ± 0.1 3.6 ± 0.3 0.2 ± 0.4
122 ± 4 13.4 ± 0.5 3.5 ± 0.2 4.7 ± 0.3 2.2 ± 0.6*
122 ± 3 14.0 ± 0.5 3.8 ± 0.1 5.4 ± 0.4* 3.2 ± 0.8*
54 ± 3 35 ± 2
50 ± 13 30 ± 3
46 ± 5 26 ± 3*
45 ± 3 35 ± 2
43 ± 3 32 ± 3
66 ± 5*,** 41 ± 4
−9±4 0±3
–7 ± 10 2±4
21 ± 5*,** 15 ± 4*,**
18 ± 1
17 ± 2
19 ± 2
34 ± 1
35 ± 2
44 ± 3*,**
16 ± 2
18 ± 2
25 ± 2*,**
108 ± 2 2964 ± 53 740 ± 35 244 ± 14 40 ± 2
84 ± 3* 2468 ± 70* 545 ± 54* 171 ± 20* 33 ± 3*
198 ± 5 4274 ± 99 2812 ± 83 1064 ± 39 171 ± 7
167 ± 6* 4004 ± 103 2548 ± 97 928 ± 39* 155 ± 10
90 ± 4 1310 ± 94 2072 ± 71 820 ± 35 131 ± 7
83 ± 6 1536 ± 101 2003 ± 111 757 ± 44 122 ± 9
Endocrinology IGF-I (ng ml − 1) HMW adiponectin (mg l − 1) GLP-1 (pmol l − 1) Absorptiometrya BMC (g) Lean mass (g) Fat mass (g) Truncal fat mass (g) Abdominal fat mass (g)
80 ± 3* 2408 ± 77* 401 ± 37*,** 115 ± 13*,** 20 ± 2*,**
180 ± 5* 4113 ± 110 2352 ± 88* 897 ± 39* 140 ± 8*
100 ± 5** 1705 ± 113* 1951 ± 86 782 ± 39 120 ±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
Circulating GLP-1 in infants M Díaz et al
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).
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 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 concentrations of circulating GLP-1 during early infancy may be accompanied 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.
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.
REFERENCES 1 Ibáñez L, Ong K, Dunger DB, de Zegher F. Early development of adiposity and insulin resistance after catch-up weight gain in small-for-gestational-age children. J Clin Endocrinol Metab 2006; 91: 2153–2158. 2 Hales CN, Barker DJP, Clark PMS, Cox LJ, Fall C, Osmond C et al. Foetal and infant growth and impaired glucose tolerance at age 64. BMJ 1991; 303: 1019–1022. 3 Whincup PH, Kaye SJ, Owen CG, Huxley R, Cook DG, Anazawa S et al. Birth weight and risk of type 2 diabetes: a systematic review. JAMA 2008; 300: 2886–2897. 4 Schou JH, Pilgaard K, Vilsbøll T, Jensen CB, Deacon CF, Holst JJ et al. Normal secretion and action of the gut incretin hormones glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide in young men with low birth weight. J Clin Endocrinol Metab 2005; 90: 4912–4919. 5 Vaag A. Low birth weight and early weight gain in the metabolic syndrome: consequences for infant nutrition. Int J Gynaecol Obstet 2009; 104: S32–S34. 6 Secher A, Jelsing J, Baquero AF, Hecksher-Sørensen J, Cowley MA, Dalbøge LS et al. The arcuate nucleus mediates GLP-1 receptor agonist liraglutide-dependent weight loss. J Clin Invest 2014; 124: 4473–4488. 7 Beiroa D, Imbernon M, Gallego R, Senra A, Herranz D, Villarroya F et al. GLP-1 agonism stimulates brown adipose tissue thermogenesis and browning through hypothalamic AMPK. Diabetes 2014; 63: 3346–3358. 8 Steculorum SM, Collden G, Coupe B, Croizier S, Lockie S, Andrews ZB et al. Neonatal ghrelin programs development of hypothalamic feeding circuits. J Clin Invest 2015; 125: 846–858. 9 Tong J, D'Alessio D. Ghrelin and hypothalamic development: too little and too much of a good thing. J Clin Invest 2015; 125: 490–492. 10 de Zegher F, Sebastiani G, Diaz M, Sánchez-Infantes D, Lopez-Bermejo A, Ibáñez L. Body composition and circulating high-molecular-weight adiponectin and IGF-I in infants born small for gestational age: breast- versus formula-feeding. Diabetes 2012; 61: 1969–1973. 11 Padidela R, Patterson M, Sharief N, Ghatei M, Hussain K. Elevated basal and postfeed glucagon-like peptide 1 (GLP-1) concentrations in the neonatal period. Eur J Endocrinol 2009; 160: 53–58. 12 de Zegher F, Sebastiani G, Diaz M, Gómez-Roig MD, López-Bermejo A, Ibáñez L. Breast-feeding vs formula-feeding for infants born small-for-gestational-age: divergent effects on fat mass and on circulating IGF-I and high-molecular-weight adiponectin in late infancy. J Clin Endocrinol Metab 2013; 98: 1242–1247. 13 Hwang I, Park YJ, Kim YR, Kim YN, Ka S, Lee HY et al. Alteration of gut microbiota by vancomycin and bacitracin improves insulin resistance via glucagon-like peptide 1 in diet-induced obesity. FASEB J 2015; 29: 2397–2411.
Supplementary Information accompanies this paper on International Journal of Obesity website (http://www.nature.com/ijo)
© 2015 Macmillan Publishers Limited
International Journal of Obesity (2015) 1501 – 1503
1503
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 formulafeeding (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 concentrations 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: [email protected] 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
beam balance (Seca, Hamburg, Germany) and length with a length board, the mean of three measurements being used for analysis. Body composition was assessed by absorptiometry at the age of 2 weeks and at 4 months with a Lunar Prodigy, coupled to Lunar software (version 3.4/3.5; Lunar, Madison, WI, USA), adapted for 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 and fat mass. Blood was sampled in the morning, in pre-feeding state. Neither a DPP4 inhibitor nor aprotinin were added to the sample. Total serum GLP-1 was assessed by ELISA (Millipore, Billerica, MA, USA). The antibody pair in this assay measures GLP-1 (7–36) and (9–36) and has no significant cross-reactivity with GLP-2, GIP, glucagon or oxyntomodulin. The intra- and inter-assay CVs were o2% and o10%; the lower detection limit was 1.5 pM. Serum insulin-like growth factor I concentrations were measured by immunochemiluminiscence (IMMULITE 2000, Diagnostic Products, Los Angeles, CA, USA), the detection limit being 25 ng/mL and the intra- and inter-assay CVs o 10%. Serum high-molecular-weight adiponectin was assessed by ELISA with intra- and inter-assay CVs o 9%.
RESULTS Figure 1 shows that at birth, the circulating concentrations of GLP-1 were readily detectable and similar in AGA and SGA infants. GLP-1 concentrations were also similar in girls and boys. In each study subgroup, GLP-1 levels at 4 months were higher than at
80
p=0.001
*
p=0.045 60
GLP-1 (pmol/L)
1502
* *
40
20
0
Statistics and ethics Statistical analyses were performed with IBM SPSS Statistics 19.0 (IBM SPSS, Chicago, IL, USA). Comparisons were performed by two-tailed t-test or Mann–Whitney test, as appropriate; P o 0.05 was considered statistically significant. The original study was approved by the institutional review board of Barcelona University, Hospital of Sant Joan de Déu; informed written consent was an inclusion criterion. The present manuscript focuses on GLP-1 because other endocrine, auxological and body-composition results have been described.10
AGA Breastfed N=63
SGA Breastfed N=28
SGA Formulafed N=26
Figure 1. Longitudinal, pre-feeding results of circulating GLP-1 concentrations in appropriate- and small-for-gestational-age infants (AGA and SGA) at birth and at the age of 4 months. Results are depicted as medians (horizontal lines) with interquartile ranges (white boxes at birth; gray boxes at 4 months) and percentiles 10 and 90 (lower and upper whiskers). *Po 0.0001 versus birth, for each 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 (n = 63)
SGA BRF (n = 28)
SGA FOF (n = 26)
AGA BRF (n = 63)
SGA BRF (n = 28)
SGA FOF (n = 26)
AGA BRF (n = 63)
SGA BRF (n = 28)
SGA FOF (n = 26)
Birth Gestational age (weeks) Birth weight (kg) Birth length (cm) Birth weight Z-score Birth length Z-score
39.8 ± 0.2
38.7 ± 0.2*
38.5 ± 0.3*
—
—
—
—
—
—
3.2 ± 0.1 49.4 ± 0.2 − 0.2 ± 0.1 − 0.3 ± 0.1
2.4 ± 0.1* 46.0 ± 0.3* − 2.1 ± 0.1* − 1.8 ± 0.1*
2.3 ± 0.1* 45.8 ± 0.3* − 2.3 ± 0.1* − 1.8 ± 0.1*
— — — —
— — — —
— — — —
— — — —
— — — —
— — — —
Clinical featuresa Age (days) Length (cm) Weight (kg) BMI (kg m − 2) PI (kg m − 3)
15 ± 1 50.8 ± 0.3 3.5 ± 0.1 13.7 ± 0.2 27.0 ± 0.4
17 ± 2 48.0 ± 0.5* 2.7 ± 0.1* 12.0 ± 0.3* 25.0 ± 0.5*
17 ± 1 47.1 ± 0.3* 2.5 ± 0.1* 11.4 ± 0.3* 24.4 ± 0.7*
136 ± 3 63.6 ± 0.3 7.0 ± 0.1 17.3 ± 0.2 27.2 ± 0.3
139 ± 3 61.4 ± 0.5* 6.3 ± 0.1* 16.6 ± 0.2* 27.2 ± 0.4
139 ± 3 61.1 ± 0.4* 6.3 ± 0.1* 16.8 ± 0.3 27.6 ± 0.6
121 ± 3 12.8 ± 0.3 3.6 ± 0.1 3.6 ± 0.3 0.2 ± 0.4
122 ± 4 13.4 ± 0.5 3.5 ± 0.2 4.7 ± 0.3 2.2 ± 0.6*
122 ± 3 14.0 ± 0.5 3.8 ± 0.1 5.4 ± 0.4* 3.2 ± 0.8*
54 ± 3 35 ± 2
50 ± 13 30 ± 3
46 ± 5 26 ± 3*
45 ± 3 35 ± 2
43 ± 3 32 ± 3
66 ± 5*,** 41 ± 4
−9±4 0±3
–7 ± 10 2±4
21 ± 5*,** 15 ± 4*,**
18 ± 1
17 ± 2
19 ± 2
34 ± 1
35 ± 2
44 ± 3*,**
16 ± 2
18 ± 2
25 ± 2*,**
108 ± 2 2964 ± 53 740 ± 35 244 ± 14 40 ± 2
84 ± 3* 2468 ± 70* 545 ± 54* 171 ± 20* 33 ± 3*
198 ± 5 4274 ± 99 2812 ± 83 1064 ± 39 171 ± 7
167 ± 6* 4004 ± 103 2548 ± 97 928 ± 39* 155 ± 10
90 ± 4 1310 ± 94 2072 ± 71 820 ± 35 131 ± 7
83 ± 6 1536 ± 101 2003 ± 111 757 ± 44 122 ± 9
Endocrinology IGF-I (ng ml − 1) HMW adiponectin (mg l − 1) GLP-1 (pmol l − 1) Absorptiometrya BMC (g) Lean mass (g) Fat mass (g) Truncal fat mass (g) Abdominal fat mass (g)
80 ± 3* 2408 ± 77* 401 ± 37*,** 115 ± 13*,** 20 ± 2*,**
180 ± 5* 4113 ± 110 2352 ± 88* 897 ± 39* 140 ± 8*
100 ± 5** 1705 ± 113* 1951 ± 86 782 ± 39 120 ±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
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Circulating GLP-1 in infants M Díaz et al
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).
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 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 concentrations of circulating GLP-1 during early infancy may be accompanied 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.
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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