Download A Physiological Role of Breast Milk Leptin in Body Weight

Human Physiology
A Physiological Role of Breast Milk Leptin in
Body Weight Control in Developing Infants
Olga Miralles, Juana Sánchez, Andreu Palou, and Catalina Picó
Abstract
MIRALLES, OLGA, JUANA SÁNCHEZ, ANDREU
PALOU, AND CATALINA PICÓ. A physiological role of
breast milk leptin in body weight control in developing
infants. Obesity. 2006;14:1371–1377.
Objective: Leptin, a hormone that regulates food intake and
energy metabolism, is present in breast milk. The aim of this
study was to determine whether milk leptin concentration is
correlated with maternal circulating leptin and BMI and
with body weight gain of infants.
Research Methods and Procedures: A group of 28 nonobese women (BMI between 16.3 and 27.3 kg/m2) who
breast-fed their infants for at least 6 months and their infants
were studied. Venous blood and milk samples were obtained from mothers at 1, 3, 6, and 9 months of lactation,
and leptin concentration was determined. Infant body
weight and height were followed until 2 years of age.
Results: During the whole lactation period, milk leptin
concentration correlated positively with maternal plasma
leptin concentration and with maternal BMI. In addition,
milk leptin concentration at 1 month of lactation was negatively correlated with infant BMI at 18 and 24 months of
age. A better negative correlation was also found between
log milk leptin concentration at 1 and at 3 months of
lactation and infant BMI from 12 to 24 months of age.
Discussion: We concluded that, in a group of non-obese
mothers, infant body weight during the first 2 years may be
influenced by milk leptin concentration during the first
stages of lactation. Thus, moderate milk-borne maternal
leptin appears to provide moderate protection to infants
Received for review December 1, 2005.
Accepted in final form June 5, 2006.
The costs of publication of this article were defrayed, in part, by the payment of page
charges. This article must, therefore, be hereby marked “advertisement” in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Biochemistry, Molecular Biology, Nutrition. and Biotechnology (Nutrigenomics). Department of Fundamental Biology and Health Sciences, University of the Balearic Islands,
Palma de Mallorca, Spain.
Address correspondence to A. Palou, Departamento de Biologı́a Fundamental y Ciencias de
la Salud, Universitat de les Illes Balears, Cra. Valldemossa Km 7.5, E-07122-Palma de
Mallorca, Spain.
E-mail: [email protected]
Copyright © 2006 NAASO
from an excess of weight gain. These results seem to point
out that milk leptin is an important factor that could explain,
at least partially, the major risk of obesity of formula-fed
infants with respect to breast-fed infants.
Key words: leptin, milk, breast-feeding, infants, metabolic imprinting
Introduction
Obesity and overweight are the most frequent nutritional
disorders in children and adolescents in industrialized countries, and there is a continuing increase in their prevalence
(1). Numerous studies have found an association between
being overweight as a child and being overweight in adulthood (2,3) with the risk of associated health complications
such as cardiovascular disease and diabetes (2,4). Given that
obesity is associated with considerable morbidity, is increasing in prevalence, and is often recalcitrant to therapy
(5,6), the identification of strategies for its prevention is of
crucial relevance.
Concerning obesity prevention, the notion that nutrition
during the early phases of human development can predispose or program individuals to adult disease has aroused
considerable interest, particularly since the last decade. The
fetal origins hypothesis of Barker et al. (7) proposes that
poor fetal nutrition causes adaptations that program future
propensity to obesity and other related diseases. Much evidence supports this idea, such as the emblematic example
of the Dutch famine (8,9). Adults who were conceived
during the acute famine that ravaged the western part of
Holland during the last 6 months of World War II, whose
mothers experienced poor nutrition in the 1st and 2nd trimesters of their pregnancies, were more likely to be obese
than their peers whose mothers did not experience poor
nutrition. In contrast to the contribution of the fetal period,
the contribution of the early postnatal environment, particularly early nutrition during lactation, has received less
attention. Different studies have shown a clear association
between overnutrition during infancy and later obesity
(10,11). In this sense, a vigorous breast-feeding style, with
high energy intake, has been associated with greater adiposity in the first 6 years of age (12,13). In addition, several
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Milk Leptin and Infant Body Weight, Miralles et al.
studies have shown that breast-feeding, compared with formula feeding, is associated with a lower risk of later obesity
(14 –18), although some studies do not support this hypothesis (19,20). The duration of lactation has also been inversely associated with obesity and overweight, although
generally to a lesser extent (14 –17). Breast milk is known to
contain many bioactive hormones and peptides, which may
play important roles in neonatal health and development
(21,22). However, which breast milk components are involved are, as yet, unknown.
Leptin is an anorexigenic hormone (23) that plays an
important role in the central regulation of energy balance,
decreasing food intake and increasing energy expenditure
(24,25). Leptin is produced mainly by the adipose tissue but
also by other tissues such as placenta (26,27), stomach
(28 –30), and mammary epithelium (31), and is also present
in maternal milk (32,33). In rats, it has been shown that
leptin present in maternal milk can be absorbed by the
immature stomach of nursing rats (32,34,35) and transferred
to the infant rat circulation (32). Leptin supplied from
maternal milk is the main source of leptin in the stomach
during the first one-half of the suckling period, whereas
endogenous production of leptin by the gastric mucosa
increases at the end of the suckling period and with the
change of diet to solid food (34). We have shown recently
that oral administration of leptin, with doses close to the
physiological concentration present in milk, can reduce
food intake in suckling rats (35). Thus, exogenous leptin
supplied by maternal milk could regulate short-term feeding
in neonates and exert other biological effects, at a time in
which both the adipose tissue and the appetite regulatory
systems are immature (35).
In humans, the role of milk leptin in lactating infants is
not known. However, it is known that leptin concentration
in human milk varies significantly between people (32,33),
and some authors have found a positive correlation between
leptin concentration in milk and maternal plasma leptin
concentration and adiposity (33,36), although contrary data
have been published (37). It is not known whether the
amount of leptin supplied with maternal milk or the lack of
supply when using infant formulas (38) may have significant effects on infant development and whether this may
have further implications in the prevention of or propensity
toward obesity in adulthood. The present study was aimed at
determining, in a group of non-obese mothers who breastfed their infants during a period of at least 6 months,
whether milk leptin concentration is correlated with maternal plasma leptin concentration and BMI and with body
weight gain of infants during the first 2 years.
Research Methods and Procedures
Subjects
Twenty-eight healthy lactating women ages 23 to 37
years (31.19 ⫾ 0.68 years) with a normal BMI of 21.6 ⫾ 0.5
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kg/m2 (range, 16.3 to 27.3 kg/m2) who planned to breastfeed their full-term newborn infants for a long period (at
least 6 months) were enrolled in the study. Maternal anthropometric parameters, such as height and body weight before
gestation and during the lactation period and the evolution
of infant body weight and length during the whole lactation
period and until 2 years of age, were registered. BMI was
determined as weight in kilograms divided by height in
meters squared. The protocol followed in this study was
reviewed and approved by the Ethical Committee of our
Regional Government, and written informed consent was
obtained for all participants.
Sample Collection
During the whole lactation period, venous blood and milk
samples were obtained from mothers at 1 (⫾3 days) and 3,
6, and 9 (⫾1 week) months. To minimize variation in leptin
concentrations in plasma due to time of day and time
relative to eating a meal, all blood samples were obtained in
the morning at 9 to 10 AM after an 11- to 12-hour fast. Blood
samples were collected in EDTA tubes and then centrifuged
at 850 g for 10 minutes at 4 °C to collect the plasma. Milk
samples were collected manually in the morning immediately after suckling, when the infant had self-terminated
suckling due to satiation. Previously published results have
shown no significant difference between leptin levels of
breast milk samples obtained in the initial and terminal
phases of suckling (37). Breast milk and plasma samples
were stored frozen at ⫺20 °C until analysis.
Leptin Assay
Leptin concentration in maternal plasma and in whole
milk was determined by a commercially available enzymelinked immunosorbent assay kit (human leptin; R&D Systems, Minneapolis, MN).
Concerning milk leptin analysis, we previously validated
the assay and tested different described protocols for sample
preparation (31,33,38) and different dilutions with the assay
buffer to ensure the suitability of the enzyme-linked immunosorbent assay kit for these samples: whole milk samples
(vigorously vortexed to ensure sample uniformity);
skimmed milk samples, prepared by centrifuging whole
milk at 8000 g for 15 minutes at 4 °C to separate milk fat
(31); sonicated milk, prepared by 5-second bursts with
20-second cooling intervals using a Misonix sonicator (33);
and lipase-treated milk [obtained by adding 3 ␮L of pancreatic lipase (Sigma, Madrid, Spain) and 6 ␮L of 1 M
sodium bicarbonate to 600 ␮L of whole milk samples and
incubating them at 37 °C for 1 hour, according to (38)] to
degrade triacylglycerides, which could interfere with the
immunoassay. In addition, samples were supplemented with
125 ng/L human leptin standard before analysis, and recovery of added leptin was calculated. No significant differences were observed with these different protocols, except
Milk Leptin and Infant Body Weight, Miralles et al.
Table 1. Characteristics of lactating mothers and their infants
Mothers
Age (years)
BMI (kg/m2)
Plasma leptin concentration at 1 month (ng/mL)
Milk leptin concentration at 1 month (ng/mL)
Infants
Sex (female/male)
Weight at birth (kg)
Weight at 2 years (kg)
BMI at 2 years (kg/m2)
31.2 ⫾ 0.7 (23 to 37)
21.6 ⫾ 0.5 (16.3 to 27.3)
12.8 ⫾ 1.7 (6.7 to 37.5)
0.156 ⫾ 0.039 (ND to 0.853)
16/12
3.20 ⫾ 0.07 (2.33 to 3.97)
12.3 ⫾ 0.3 (9.3 to 15.6)
16.1 ⫾ 0.3 (13.8 to 20.4)
Data are shown as means ⫾ standard error (range). ND, non-detected values. Maternal BMI value considered was before pregnancy.
by using sonicated samples, where leptin concentration detected was lower; thus, we decided to use whole milk
samples diluted 1:1 with the assay buffer. The recovery of
added leptin using this protocol was 103.1 ⫾ 1.4% (n ⫽ 4).
Statistical Analysis
Simple correlations were assessed by Pearson’s correlation coefficients. Student’s t test was used to compare mean
values of leptin concentration in maternal plasma and in
breast milk. In all cases, threshold of significance was
defined as p ⬍ 0.05.
Results
The characteristics of mothers and infants are shown in
Table 1. The mothers included were not obese (BMI before
gestation was between 16.3 and 27.3 kg/m2, and the mean
was 21.6 ⫾ 0.5 kg/m2), only three of them were slightly
over a BMI of 25 (25.3, 25.8, and 27.3 kg/m2), and their
infants were born full-term (gestation age, 39.7 ⫾ 0.2
weeks). All infants were breast-fed for at least 6 months
(and exclusively for at least 4 months).
Mean values of breast milk and maternal plasma leptin
concentrations are shown in Table 1. In agreement with the
literature (32,33), in our study, the concentration of leptin in
breast milk was significantly lower than in maternal plasma
(p ⬍ 0.001, Student’s t test) during the whole lactation
period. There was a positive correlation between leptin
concentration in milk and in maternal plasma at each of the
measured time-points (1, 3, 6, and 9 months), which are
shown plotted all together in Figure 1A (r ⫽ 0.519, p ⬍
0.001). In addition, maternal BMI correlated positively with
plasma leptin concentration (Figure 1B; r ⫽ 0.598, p ⬍
0.001) and with milk leptin concentration (Figure 1C; r ⫽
0.387, p ⬍ 0.001) at each of the measured time-points. It is
remarkable that a stronger correlation was found between
maternal log BMI and both maternal plasma and milk log
leptin concentration (r ⫽ 0.696 and 0.607, respectively)
(data not shown).
Milk leptin concentration at 1 month of lactation, but not
at any other time-point studied, was negatively correlated
with infant BMI at 18 and 24 months of age (r ⫽ ⫺0.493,
p ⬍ 0.05; r ⫽ ⫺0.456, p ⬍ 0.05, respectively; see Figure
2A for correlation at 24 months of age). Additionally, a
stronger correlation was found between the log-transformed
milk leptin concentration at 1 and 3 months of lactation and
infant BMI from 12 to 24 months of age; see Figure 2B
showing the correlation between log milk leptin concentration at 1 month of lactation and infant BMI at 24 months of
age. Correlation values at 1 month of lactation were r ⫽
⫺0.434, p ⬍ 0.05; r ⫽ ⫺0.547, p ⬍ 0.01; and r ⫽ ⫺0.601,
p ⬍ 0.01; at 12, 18, and 24 months of life, respectively;
correlation values at 3 months of lactation were r ⫽
⫺0.486, p ⬍ 0.05; r ⫽ ⫺0.451, p ⬍ 0.05; and r ⫽ ⫺0.505,
p ⬍ 0.01; at 12, 18, and 24 months of life, respectively.
No significant correlations were found between milk
leptin concentration and infant body weight and body
weight gain at all ages reported, when all data were considered. It should be noted that most milk leptin concentrations
ranged from undetected values (⬍0.0078 ng/mL in two
subjects) to 0.35 ng/mL; however, there were two higher
values at 1 month of lactation (0.629 and 0.853 ng/mL)
that were above that range. Without considering these two
high values, milk leptin concentration at 1 month of lactation, but not at any other time-point studied, was negatively
correlated with infant body weight and with body weight
gain at all ages reported (1, 3, 6, 9, 12, 18, and 24 months
of life) (see correlations for some representative ages in
Figure 3).
Discussion
To our knowledge, this study provides the first evidence
showing that leptin present in breast milk may regulate body
weight gain during infancy in humans and, thus, supports
the hypothesis that leptin is one of the bioactive components
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Milk Leptin and Infant Body Weight, Miralles et al.
Figure 1: Correlations between leptin concentration in milk and in
maternal plasma (A), leptin concentration in maternal plasma and
maternal BMI (B), and leptin concentration in milk and maternal
BMI (C) throughout the whole lactation period. Plasma and milk
samples were collected from 28 mothers at 1, 3, 6, and 9 months.
Leptin concentrations were determined as described in “Research
Methods and Procedures.” Simple correlations were assessed by
Pearson’s correlation coefficients. The r and p values for correlations are indicated.
present in milk that could be responsible for the role of
breast-feeding in lowering the risk of childhood obesity.
Leptin concentration in milk varies widely among people
(32,33). Some authors have found a positive correlation
between maternal BMI or adiposity and plasma leptin concentration (33,36), although contrary results have also been
published (37). We have studied milk leptin concentration
during the lactating period in a group of non-obese mothers
and also have found a wide variation, ranging from values
under the detection limit of the assay (⬍0.0078 ng/mL) to
0.853 ng/mL. In addition, we have found that milk leptin
concentration positively correlates with both maternal
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Figure 2: Correlations between milk leptin concentration (A) or
log milk leptin concentration (B) at 1 month of lactation and infant
BMI at 2 years of age. Milk leptin concentration was determined
as described in “Research Methods and Procedures.” Simple correlations were assessed by Pearson’s correlation coefficients. The
r and p values for correlations are indicated.
plasma leptin concentration and BMI at any time-point of
the lactation period studied. Even considering methodological differences, values obtained in this group of non-obese
mothers are lower than values published for women with
higher BMI values (33). Thus, the amount of leptin supplied
to an infant through breast milk depends on the mother’s
adiposity. Lean mothers with very low plasma leptin concentrations are producing milk with little or even no significant leptin, similar in this sense to infant formula, which
does not have leptin as an ingredient (38). Conversely, only
breast-fed infants nursed by mothers with relatively significant adiposity are exposed to significant amounts of leptin
in milk.
Although some studies do not support this hypothesis
(19,20), there is increasing epidemiological evidence suggesting that breast-feeding compared with infant formula
confers protection against obesity in later life (14 –18), but
which components are responsible is currently unknown.
Milk Leptin and Infant Body Weight, Miralles et al.
Figure 3: Correlations between milk leptin concentration at 1 month of lactation and infant body weight (left) or body weight gain from
birth (right) at 1 (top), 12 (middle), and 24 (bottom) months of age. Milk leptin concentration was determined as described in “Research
Methods and Procedures.” Simple correlations were assessed by Pearson’s correlation coefficients. The r and p values for correlations are
indicated.
Human milk contains many hormones and growth factors
(21), the function of some of which is still unknown in
neonatal development. The known role of leptin in energy
balance (24), its presence in human milk (32,33), and its
absence in infant formula (38) make leptin a good candidate
to be considered. However, a correlation between milk
leptin concentration and infant body weight had not yet
been demonstrated. We show here that, at least in non-obese
nursing women, both leptin concentration (at 1 month of
lactation) and log milk leptin concentration (mainly at 1
month of lactation but also at 3 months) are negatively
correlated with infant BMI at 2 years of age. This correlation was more pronounced considering log milk leptin concentration but lineal correlation was also significant at least
in this margin of BMI. This would indicate that differences
in milk leptin concentration within the lower range may
have a big impact on infant BMI; differences within the
range of higher milk leptin concentrations may have less
repercussion on infant BMI.
Results also show that the first stages of lactation, a
period in which the gastric mucosa is still immature and the
absorption of intact leptin may be facilitated (38), are of
critical relevance for the action of leptin. Moderate milkborne maternal leptin mainly during these first stages of
neonatal development can cause an adaptation that programs future obesity prevention. Interestingly, the two highest values of infant BMI at 2 years of age (20.4 and 18.1
kg/m2), which are over the 85th percentile, come from two
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Milk Leptin and Infant Body Weight, Miralles et al.
infants who were fed by two mothers whose milk leptin
concentration was under the assay detection limit (⬍0.0078
ng/mL).
These results in infant humans agree with our previous
observations in neonate rats. We described that leptin orally
taken by neonate rats can reach target cells through the
immature stomach (34). In addition, leptin orally administered to neonate rats, in a dose close to physiological concentration present in milk, is absorbed by the stomach and
reaches circulation, resulting in lower food intake (35). Milk
leptin absorption by the stomach of neonate rats has been
particularly observed during the first one-half of the lactation period, when stomach production of leptin is still low
(34). During the second one-half of this period and paralleling the maturation process of the gastric mucosa, the
absorption of exogenous leptin decreased, whereas endogenous production by gastric mucosa increased (34). The fact
that leptin absorption by the immature stomach occurs, at
least in rats, mainly during the first stages of the lactation
period could explain the importance of the amount of leptin
supplied during this period on body weight control in later
life.
Leptin receptor is expressed in the vagal nerve innervation of the stomach (39); thus, oral leptin could act either
through the activation of afferent vagal nerves (leptin in the
gastric lumen) or centrally (leptin in the systemic circulation). Both pathways could participate in the short-term
regulation of food intake during the neonatal period (35).
Previous studies have not found a significant association
between milk leptin concentration and infant adiposity
(36,37). Several factors such as the time-point of sampling
during the lactation period, the shorter period of follow-up
of infant weight, and the inclusion of obese women and
obese infants in those studies can make the difference. In
fact, family heredity, involving genetic and shared environmental components, which are a strong determinant of
childhood obesity (40), could be masking the effects of
leptin. In our study, we have enrolled a more homogeneous
group of non-obese mothers. In addition, we have not considered the effect of high milk-borne maternal leptin, e.g.,
characteristic of obese or overweight women; however, the
two cases of maternal milk with abnormally high leptin
concentration in relation to their BMI did not seem to
provide further protection against body weight gain.
In conclusion, to our knowledge, this is the first time that,
in a population of non-obese mothers, it is shown that infant
body weight may be influenced by milk leptin concentration
until 2 years of age. Moderate amounts of leptin supplied by
normal weighted mothers through the milk seem to provide
moderate protection to infants from an excess of weight
gain. This protective effect could be sharpened in adulthood. The lower protection observed in very lean mothers
seems to point out that milk leptin is an important factor that
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could explain, at least in part, the major risk of overweight
and obesity of formula-fed infants during adulthood with
respect to breast-fed infants.
Acknowledgments
This work was supported by the Spanish Government
(Grants G03/028, BFI2003-04439, and AGL2004-07496/
ALI). We acknowledge the women and infants who participated in the study and also the Balearic Association for
Breast-feeding, and the midwives C. Artigues and M. Mantxola, who supported mothers during the lactation period.
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