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Institut National de la Recherche Agronomique, Station de Recherches Porcines, 35590 Saint Gilles, France
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • preadipocyte • milk • colostrum • primary culture • pigs
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Rogers et al. 1997
). Despite the large amount of
information about the composition of maternal milk, the function of
many compounds thought to be beneficial remains to be elucidated. In
addition to nutrients, colostrum and milk contain a large group of
biologically active components including hormones and growth factors
that are known to stimulate cellular growth and differentiation
(Grosvenor et al. 1993
). In many species, the
concentrations of these factors are higher in colostrum than in mature
milk (Donovan et al. 1994
, Simmen et al. 1990
, Westrom et al. 1987
), whereas formula milk
is usually totally devoid of these factors (Nagashima et al. 1990
). A number of studies suggest a role for milk-borne
growth factors in the development of the neonatal gastrointestinal
tract (Donovan and Odle 1994
, Grosvenor et al. 1993
, Odle et al. 1996
). Very little is known
about the effects on other tissues. In humans, some clinical studies
have suggested that the early infant diet may influence adipose tissue
development, whereas other studies have failed to show such an effect
(Dewey et al. 1993
, Dewey 1998
,
Rogers et al. 1997
). Therefore, this experiment was
undertaken to determine whether factors present in maternal milk can
influence the development of adipose tissue. The study was carried out
in neonatal pigs, thus allowing careful control of food intake, genetic
origin and environment. The potential for proliferation and
differentiation of preadipocytes derived from maternal milk- and
formula-fed pigs was evaluated in primary cultures of
stromal-vascular (s-v) cells. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The protocol was conducted in accordance with the national regulation
for the human care and use of animals in research (certificate of
authorization to experiment on living animals n° 7676 delivered by
the French Department of Agriculture to I. Louveau). Six litters of
Large White-Landrace x Piétrain pigs from the INRA herd
were used. Within each litter, two pigs of similar body weight were
allotted to one of two groups. One pig was bottle-fed sow colostrum
then mature milk
(SM)3
and its littermate was bottle-fed sow’s milk replacement formula
(F) (Table 1
) for a 7-d period. Colostrum and mature
milk that were allocated to SM pigs were obtained from several sows by
manual expression during or soon after parturition (C0), at ~12 h
after parturition (C12) and at d 6 of lactation (M). Sow’s milk
replacement formula was a commercial piglet formula (Toniporc, Agralco,
France). It was supplemented (FI) with purified porcine immunoglobulins
G (Isogamma-Pig, Aprocat, Barcelona, Spain) during the first 20 h
to provide adequate immunity. The first meal was given ~6 h after
birth. Piglets of the two groups were bottle-fed isoenergetically
every 2 h from 0700 to 2300 h and once during the night at
0300 h. Milk intake was 2.50 ± 0.16 and 2.45 ± 0.21
MJ/d for SM and F pigs, respectively. Milk quantity allocated to SM and
F pigs was adjusted daily from daily measurement of body weight. Ninety
minutes after the last meal, pigs were weighed, anesthetized with the
use of halothane and exsanguinated. Dorsal subcutaneous adipose tissue
was aseptically removed immediately after death for cell preparation.
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One cell preparation per pig was made. Cells were isolated and cultured
as described previously (De Clercq et al. 1997
) with
minor modifications. Adipose tissue (6 g) was minced and digested in a
digestion buffer (2 mL/g of tissue) consisting of HEPES phosphate
buffer, 20 g/L bovine serum albumin (BSA) and 2 g/L collagenase II and
XI (800 U/mg; Sigma, St-Quentin Fallavier, France) in a shaking
water bath for 45 min at 37°C. After successive filtration through
200-, 100- and 25-µm nylon meshes, two aliquots (10
µL) of cell suspension were stained with Trypan blue
stain and counted on a hemocytometer to estimate the concentration of
s-v cells. Cells in 0.5 mL of plating medium were inoculated into
24-well plates (2 cm2/well) at densities of 2.5 x 104 and 7.5 x 104 cells/cm2
to study proliferation and differentiation, respectively. They were
maintained in a humidified 5% CO2 atmosphere for 24 h
at 37°C. Plating medium consisted of Dulbecco’s modified Eagle’s
medium (DMEM; Gibco, Cergy Pontoise, France) supplemented with 10%
fetal bovine serum, 100 kU/L penicillin, 100 mg/L
streptomycin, 0.25 mg/L fungizone, 0.25 mg/L nystatine, 4 mmol/L
L-glutamine, 2.6 nmol/L insulin and 100 nmol/L cortisol.
Cells were then cultured in a serum-supplemented medium to study
proliferation or in a serum-free medium to study differentiation.
The serum-supplemented medium contained the same components as the
plating medium except for serum (2.5% porcine serum). The
serum-free medium consisted of DMEM/Ham’s Nutrient Mixture F-12
(1:1) (DMEM/F12, Gibco), 100 kU/L penicillin, 100 mg/L
streptomycin, 0.25 mg/L fungizone, 0.25 mg/L nystatine, 50
µmol/L ß-mercaptoethanol, 0.1 nmol/L ascorbic acid,
2.5 mmol/L L-glutamine, 10 mg/L transferrin, 8 nmol/L
insulin, 100 nmol/L cortisol and 0.2 nmol/L triiodothyronine. Cells
were cultured for 10 d and the media were changed every other day.
Proliferation assay.
Thymidine incorporation was used as a measure of DNA synthesis to estimate cell proliferation. On d 3 of culture, [3H]-thymidine (1 TBq/mmol, Amersham, Les Ulis, France) was added (37 kBq/well) into the medium and the incubation was continued for 24 h. On d 4, cells were washed three times with DMEM containing unlabeled thymidine. Cells were detached with 0.5% trypsin solution and placed in vials with scintillation cocktail. Radioactivity was counted using a ß-scintillation counter.
Morphology.
Cells were fixed for at least 2 h in Bouin fixative, washed with water and stained for lipid with oil red-O and for nuclei with Hemalun Mayer stain. Wells were covered with aquamount and the proportion of differentiated cells was estimated by direct counting using a microscope (magnification, X200). Five different areas per well, 1.05 mm2 each, were counted. Cells were considered differentiated when the nucleus was surrounded by lipid droplets.
Enzyme analyses.
For analysis of lipoprotein lipase (LPL; EC 3.1.1.34) activity,
serum-free media were eliminated and cells were incubated with 5
mmol/L veronal buffer, pH 7.4, containing 0.5 mmol/L MgCl2,
1 mol/L glycerol, 1.8 mmol/L CaCl2, 20 mmol/L mannitol and
heparin (20 kU/L) for 30 min at 37°C. After sonication, homogenates
were stored at -70°C until use. LPL was assayed with glycerol
tri[9,10(n)-3H]-oleate (0.88 TBq/mmol, Amersham) as
substrate (Vannier et al. 1985
). For analysis of malic
enzyme (ME; EC 1.1.1.40) activity, serum-free media were replaced
with a 0.25 mol/L sucrose solution. After sonication, homogenates were
stored at -70°C until use. ME activity was determined according to
the method of Hsu and Lardy (1969)
. Protein
concentrations of homogenates were determined using the bicinchoninic
acid assay (Pierce, Rockford, IL) with BSA as a standard.
Statistical analysis.
All data were expressed as means ± SEM Data were
analyzed by ANOVA using the generalized linear model of SAS (1996)
. The
model included the effect of feeding treatment, and animal nested
within feeding treatment. When appropriate, multiple comparison of the
means was performed using Duncan’s test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Body weights and growth rates did not differ (P > 0.1) between SM and F pigs. At birth, body weights were 1494 ± 105 and 1504 ± 135 g for SM and F pigs, respectively. At 7 d of age, body weights were 2443 ± 174 and 2430 ± 210 g for SM and F pigs, respectively.
Proliferation of stromal-vascular cells.
The number of s-v cells isolated from 1 g of adipose tissue
was 40% higher (P < 0.01) in F than SM pigs
(Fig. 1A
). In addition, [3H]-thymidine incorporation
into s-v cells cultured in serum-supplemented medium was 60%
higher (P < 0.05) in F than SM pigs (Fig. 1B
). Such a difference was also observed for cells cultured
in serum-free medium, although the proliferation rate was ~95%
lower (data not shown) under these conditions.
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Differentiated adipocytes appeared on d 3. Cells acquired numerous
lipid droplets of variable size (Fig. 2
). The percentage of differentiated cells was markedly lower
(P < 0.01) in F than in SM pigs on d 6 and 9 of
culture (Fig. 3A
). On d 9 of culture, 75 ± 6% of the s-v cells were
differentiated into mature adipocytes in SM pigs, whereas only 51
± 7% of s-v cells were differentiated in F pigs
(P < 0.05). LPL, an early marker enzyme of adipose
differentiation, appeared between d 1 and 3 of culture (Fig. 3B
). The activity of this enzyme was lower (P
< 0.05) in s-v cells from F than in those from SM pigs on d 3
and 6 of culture. The activity of ME, a late marker of adipose
differentiation, appeared on d 3 of culture. This activity did not
differ (P > 0.1) between SM and F pigs (data not
shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, these
findings cannot be compared with our observations because of the
approach used. In this study, some components of milk may have been
modified by the gastrointestinal tract or may not have been absorbed
intact. Although proliferation and differentiation are difficult to
evaluate in vitro, the finding of differences between the two groups of
pigs reflects a difference in the intrinsic potential because cells
were cultured under identical environmental conditions. Therefore, this
experiment indicates that the potential of preadipocyte development can
be modified during the early postnatal period as shown previously in
rats (Dugail et al. 1986
).
The mechanisms underlying the different effects of maternal milk and
formula milk remain to be elucidated. There are several possible
explanations. As suggested by Dewey et al. (1993)
, the
difference may be attributed to energy intake. However, this hypothesis
can be ruled out because pigs were fed isoenergetically and exhibited
similar body weight gain. One likely hypothesis is the involvement of
growth factors or hormones that are present in milk (Grosvenor et al. 1993
) but not in formula and that affect preadipocyte
development (Grégoire et al. 1998
, Suryawan and Hu 1995
). Among the factors present in high concentrations
in colostrum, insulin and insulin-like growth factor-I (IGF-I),
which have been investigated for this role, may be potential
candidates. The involvement of insulin is possible because we (data not
shown) and others (Baumrucker et al. 1994
) have shown
that plasma insulin is increased transiently by intake of milk formula.
It remains to be determined whether this change is sufficient to affect
cell development. Given the finding that IGF-I decreases
proliferation under our cell culture conditions (Gerfault et al. 1999
), the lower cell proliferation in SM than F pigs is
consistent with a higher level of plasma IGF-I in SM pigs. However,
the possible involvement of IGF-I must be clarified. First,
IGF-I has also been shown to increase both proliferation and
differentiation of porcine s-v cells in vitro (Ramsay et al. 1989
). Second, the suggestion that a higher plasma IGF-I
concentration in SM pigs results from the absorption of maternal milk
IGF-I into the circulation needs to be verified or clarified. One
study has shown that feeding colostrum increases circulating IGF-I
in newborn pigs (Wester et al. 1998
), whereas other
studies (Donovan et al. 1997
, Houle et al. 1997
) did not show such an effect. In addition, the effects of
other growth factors or hormones that could be absorbed from the
gastrointestinal tract or secreted peripherally in response to
colostrum feeding cannot be ruled out.
The differences observed between maternal- and formula-fed pigs
could also be attributed to the nutrients. The amount of protein,
lactose and lipid, as well as fatty acid composition differed between
the SM and F diets. Although the role of protein and lactose in adipose
tissue development remains unknown, the involvement of lipid and fatty
acids is more likely. It has been hypothesized recently that lipids
present in whole human milk stimulate 3T3-L1 preadipocyte
differentiation (Lyle et al. 1998
). It has been also
demonstrated that some fatty acids can increase the expression of
adipocyte-specific genes (Amri et al. 1991
) and
stimulate proliferation and differentiation of preadipocytes in vitro
(Ailhaud et al. 1996
).
The important question is whether long-term adipose tissue
development is affected as reported in rats (Gaben-Cogneville et al. 1981
). After 10 d of food deprivation, stromal cell
proliferation was strongly decreased and the cellular change could not
be reversed by restoration of normal feeding. In humans, the
relationship between infant adipose tissue cellularity and subsequent
development remains unclear. It has been reported that childhood
obesity at all ages is often characterized by increased fat cell number
(Brook et al. 1972
, Hirsch 1975
). On the
other hand, a recent study indicates that obesity in children <3 y of
age is not an important predictor of adult obesity (Whitaker et al. 1997
). From the present findings, two hypotheses can be
proposed if the increase in s-v cell number represents an increase
in preadipocyte number. First, the finding of lower cell
differentiation in formula-fed pigs may result in a reduction of
subsequent adipose tissue development. A more likely hypothesis is that
the lower cell differentiation observed in F pigs may represent a delay
in differentiation related to active proliferation. The observations
that adipocyte diameters were smaller and that the estimated number of
adipocytes tended (P = 0.10) to be higher in F than SM
pigs (data not shown) support this statement. In this context, the
higher number of s-v cells in formula-fed pigs may lead to an
increase in subsequent adiposity. The finding of both a higher cell
proliferation in neonatal Meishan pigs and a higher subsequent
adiposity than conventional pigs (Gerfault et al. 1999
)
is consistent with this hypothesis. In addition, overfeeding of rats
during the neonatal period induces excess fat storage, which is
associated with an increase in cell number in the stromal-vascular
fraction (Dugail et al. 1985
).
In conclusion, this study shows that in neonatal pigs, formula milk increases both the number of s-v cells in adipose tissue and their capacity to proliferate, whereas it decreases their potential to differentiate in comparison to maternal milk. The underlying mechanisms by which the effects are mediated remain to be determined. Further studies are also required to evaluate the effect of these diets on long-term adipose tissue development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BSA, bovine serum albumin;
C0, colostrum obtained during or soon after parturition; C12, colostrum
obtained at 12 h after parturition; DMEM, Dulbecco’s modified
Eagle’s medium; F, formula milk bottle-fed pigs; FI, formula milk
supplemented with porcine immunoglobulins G; IGF-I, insulin-like
growth factor-I; LPL, lipoprotein lipase; M, milk obtained at d 6 of
lactation; ME, malic enzyme; SM, sow’s milk bottle-fed pigs;
s-v, stromal-vascular.
Manuscript received September 10, 1999. Revision accepted January 18, 2000.
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