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* Research Centre for the Early Origins of Adult Health, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia; and
Obesity and Metabolic Division, Rowett Research Institute, Bucksburn, Aberdeen, Scotland, UK
1Correspondence: Sansom Research Institute, University of South Australia, SA 5005, Australia. E-mail: caroline.mcmillen{at}unisa.edu.au
ABSTRACT
Individuals exposed to an increased nutrient supply before birth have a high risk of becoming obese children and adults. It has been proposed that exposure of the fetus to high maternal nutrient intake results in permanent changes within the central appetite regulatory network. No studies, however, have investigated the impact of increased maternal nutrition on the appetite regulatory network in species in which this network develops before birth, as in the human. In the present study, pregnant ewes were fed a diet which provided 100% (control, n =8) or 
160% (well-fed, n=8) of metabolizable energy requirements. Ewes were allowed to lamb spontaneously, and lambs were sacrificed at 30 days of postnatal age. All fat depots were dissected and weighed, and expression of the appetite-regulating neuropeptides and the leptin receptor (OBRb) were determined by in situ hybridization. Lambs of well-fed ewes had higher glucose (Glc) concentrations during early postnatal life (F=5.93, P<0.01) and a higher relative subcutaneous (s.c.) fat mass at 30 days of age (34.9±4.7 g/kg vs. 22.8±3.3 g/kg; P<0.05). The hypothalamic expression of pro-opiomelanocortin was higher in lambs of well-fed ewes (0.48±0.09 vs. 0.28±0.04, P<0.05). In lambs of overnourished mothers, but not in controls, the expression of OBRb was inversely related to total relative fat mass (r2=0.50, P=0.05, n=8), and the direct relationship between the expression of the central appetite inhibitor CART and fat mass was lost. The expression of neuropeptide Y and AGRP was inversely related to total relative fat mass (NPY, r2=0.28, P<0.05; agouti-related peptide, r2=0.39, P<0.01). These findings suggest that exposure to increased nutrition before birth alters the responses of the central appetite regulatory system to signals of increased adiposity after birth.—Muhlhausler, B. S., Adam, C. L., Findlay, P. A., Duffield, J. A., and McMillen, I. C. Increased maternal nutrition alters development of the appetite-regulating network in the brain.
Key Words: nutrition • programming • energy balance • obesity
EPIDEMIOLOGICAL STUDIES have shown that infants exposed to an increased supply of nutrients before birth are at increased risk of becoming overweight or obese in later life (1
, 2)
. In pregnancies complicated by maternal diabetes or Glc intolerance, maternal hyperglycemia results in increased fetal plasma Glc and insulin concentrations, and offspring from these pregnancies are at increased risk of later obesity (1
, 3)
. It has therefore been proposed that exposure to excess nutrient supply during critical windows of fetal development may result in permanent changes within either the appetite regulatory system and/or the adipocyte to program an increase in body fat mass in later life (4
, 5)
.
In the adult, Glc, insulin, and leptin act through a range of mechanisms to alter hypothalamic expression of the orexigenic neuropeptides, neuropeptide Y (NPY) and agouti-related peptide (AGRP), and the anorexigenic precursor, pro-opiomelanocortin (POMC), and neuropeptide, cocaine-and amphetamine-regulated transcript (CART), and thereby regulate appetite and energy balance (6
7
8)
.
In the rat, NPY-containing projections develop between the arcuate nucleus (ARC), the dorsomedial nucleus (DMN) and paraventricular nucleus (PVN) of the hypothalamus during the first two weeks of life (9)
. This early postnatal period is also a critical period for the programming of appetite and body fat mass. Overnutrition during this time induces permanent alterations in the hypothalamic neurons that express the appetite-regulating neuropeptides, leading to persistent hyperphagia and associated obesity (4)
. It has also been demonstrated that leptin exposure in the neonatal period alters the development of neuronal connections between the ARC and hypothalamic nuclei, which mediate changes in feeding behavior (10)
. Although the appetite regulatory system develops postnatally in the rat, this neural network develops before birth in precocial species, such as the human and sheep (11
, 12)
. We have also recently demonstrated that the appetite regulatory network in the fetal hypothalamus is responsive to changes in fetal nutrient supply (13
, 14)
and that intrafetal Glc infusion increases hypothalamic expression of POMC in the late gestation fetal sheep (14)
. It is not known, however, whether an increased nutrient supply before birth results in altered expression of ‘appetite regulatory’ neuropeptides in the hypothalamus or responsiveness of these neuropeptides to nutrient signals in postnatal life.
Our aim, therefore, was to determine whether an increase in maternal nutrition in late pregnancy increased fat deposition in postnatal life and resulted in changes in the expression of the leptin receptor (OBRb) and of the appetite regulatory neuropeptides NPY and AGRP (appetite stimulators) and POMC and CART (appetite inhibitors) within the hypothalamus of the postnatal lamb at 30d of age.
MATERIALS AND METHODS
Animals and feeding regimen
All procedures were approved by the University of Adelaide Animal Ethics Committee. Merino ewes were mated and pregnancy confirmed by ultrasound scanning in early gestation. From 90 days gestation, ewes were individually housed and were acclimatized to a control diet, which consisted of 1 kg lucerne chaff (85% dry matter, metabolizable energy (ME) content=8.3 MJ/kg) and 300 g concentrated pellets containing: straw, cereal, hay, clover, barley, oats, lupins, almond shells, oat husks, and limestone (89% dry matter, ME content=11.6 MJ/kg; Ridley Agriproducts Sheep Nut Ration, Murray Bridge, SA). The diet was calculated to provide 100% of the energy requirements for the maintenance of a pregnant ewe bearing a singleton fetus or twin fetuses as appropriate, as specified by the Ministry of Agriculture, Fisheries and Food, UK (15)
. At 115 days gestation, that is, before the commencement of the rapid fetal growth phase in late gestation (16)
, ewes were randomly assigned to either a control (n=8) or well-fed group (n=8). Between 115 and 124 days gestation, control ewes were provided with 14.0 ± 0.4 g of lucerne chaff and 6.5 ± 0.4 g of pelleted concentrate per kg bodyweight, and well-fed ewes were provided with 22.1 ± 0.8 g lucerne chaff and 10.4 ± 0.7 g pelleted concentrate/kg body wt each day. The feed allowance of all ewes was proportionately increased by 10% every 10 days in order to meet the increasing substrate demands of the growing fetus in late gestation (15)
. All feed refusals were recorded and weighed daily and used to calculate the ME intake of all ewes as a percentage of their maintenance energy requirements.
Before lambing, food was provided to each ewe in two equal portions each day, one at 9:00 AM and one at 3:00 PM, with water provided ad libitum. After lambing, all ewes were provided with 1 kg Lucerne chaff and 500 g of pelleted concentrate once daily. If all feed was consumed before 3:00 PM, an additional 1 kg of chaff was provided. After birth, each ewe and her lamb(s) were housed in an individual pen in an indoor housing facility, which was maintained at a constant ambient temperature of between 20 and 22°C and a 12-h light:12-h dark light cycle.
Growth rate and blood sampling
Lambs were born spontaneously at term. The day of birth was designated as day 1. Birth wt (kg), crown rump length (cm), abdominal circumference (cm), and shoulder height (cm) were recorded within 6 h of birth.
Body wt (kg), crown rump length (cm), abdominal circumference (cm), and shoulder height were recorded each day for the first 5 days of life and at least once every 2 days thereafter until day 30. All measurements were obtained between 8:00 and 11:00 AM. Venous blood samples were collected in chilled tubes between 8:00 AM and 1:00 PM after a 2 h fast on days 1–5 and every 3 day thereafter until postnatal day 30. The first blood sample was collected within 24 h of birth. All blood samples were centrifuged at 1500 g for 10 min, and plasma was separated into aliquots and stored at –20°C.
Food intake
The food (milk) intake of lambs was measured using a modification of the weigh-suckle-weigh (WSW) protocol as described by Hinch (17)
on days 2, 5, 8, 11, 14, 20, and 23 of postnatal life. Briefly, lambs were fasted for at least 2 h, a diaper was applied in order to control for weight changes due to urination or defecation, and the lambs were then allowed to suckle freely for 2 h. Total time spent at the teat, the number of suckling bouts, and the length of each bout was also recorded by continuous observation during this time. All feeding experiments were conducted between 8:00 AM and 12:00 PM and were conducted by a single observer. The lambs were weighed at the end of the 2-h suckling period and the wt gain during the 2-h period was measured.
Total food (milk) intake was determined by calculating the weight gain of the lambs during each 2-h period. Relative food (milk) intake was determined by normalizing the total food (milk) intake to the weight of the lamb at the start of the 2-h suckling period.
Postmortem and tissue collection
At 30 days of age, lambs were killed with an overdose of pentobarbital sodium (Virbac, Peakhurst, NSW, Australia). A sample of s.c. adipose tissue from the fore and hind region was immediately collected and snap frozen in liquid N2. All organs were dissected out and individually weighed. All adipose tissue from the perirenal, omental, pericardial, pelvic, epididymal/parametrial and axillary fat depots was carefully excised and weighed. The whole brain was removed, frozen in isopentane on dry ice, and stored at –80°C for subsequent determination of hypothalamic neuropeptide expression by in situ hybridization.
Plasma Glc
Plasma Glc concentrations were measured by enzymatic analysis using hexokinase and Glc-6-phosphate dehydrogenase to measure the formation of NADH photometrically at 340 nM (COBAS MIRA automated analysis system, Roche Diagnostica, Basel, Switzerland). The sensitivity of the assay was 0.5 mmol/l and the intra- and inter assay coefficients of variation were both <5%.
Plasma insulin
Plasma insulin concentrations were measured using a RIA (Rat insulin kit, Linco Research, MO, USA), which was validated for use with sheep plasma. This assay has previously been shown to have a cross-reactivity of 100% with sheep insulin and no detectable cross-reactivity with related proteins (C-peptide, glucagon, somatostatin, pancreatic polypeptide or insulin-like growth factor-1) (Linco Research). The recovery of insulin from lamb plasma was 96.5 ± 3.7%. When insulin was measured in increasing volumes of lamb plasma, the displacement curve was parallel to the assay standard curve. Samples (10 µl) were assayed in duplicate and added to borosilicate glass tubes with 100 µl of hydrated 125I-Insulin and guinea-pig anti-rat insulin antibody and incubated overnight at 4°C. Precipitating reagent (1 ml) was added, and tubes were centrifuged for 25 min at 2000 g, then aspirated and total counts measured by gamma counter. The sensitivity of the assay was 0.01 ng/ml and the intra- and interassay coefficients of variance were both <10%.
Plasma nonesterified fatty acids
Plasma nonesterified fatty acids (NEFAs) were measured by an in vitro enzymatic colorimetric method (Wako Pure Chemicals Industries, Osaka, Japan). The method relies on the acylation of coenzyme A by the fatty acids in the presence of added acyl-coenzyme A synthetase (ACS). The acyl-coenzyme A thus produced is oxidized by added acyl-coenzyme A oxidase (ACOD) with the generation of hydrogen peroxide. Hydrogen peroxide, in the presence of peroxidase (POD) permits the oxidative condensation of 3-methyl-N-ethyl-N-(ß-hydroxyethyl)-aniline (MEHA) with 4-aminoantipyrine to form a purple-colored adduct, which can be measured colorimetrically at 550 nM (COBAS MIRA automated analysis system, Roche Diagnostica, Basel, Switzerland). The sensitivity of the assay was 0.25 mEq/l and the intra- and interassay coefficients of variation were both <10%.
Plasma leptin
Plasma leptin concentrations were measured using a competitive bovine leptin ELISA, which has previously been validated for sheep plasma (18)
. Briefly, an ELISA plate was preincubated with recombinant bovine leptin in 50 µl of 0.1 M bicarbonate buffer and blocked with 200 µl of 5% skim milk in ELISA buffer. Chicken antirecombinant bovine leptin antiserum (50 µl) was added to the wells, followed by the addition of samples (100 µl) in duplicate. After an overnight incubation at 37°C, a biotinylated phosphatase-streptavidin conjugate (Amrad Biotech, Boronia, Vic, Australia) was added, incubated for 1 h, and the plate was developed with p-nitrophenylphosphate disodium salt hexahydrate. The sensitivity of the assay was 0.5 ng/ml and the intra- and inter assay CVs were <16%.
Hypothalamic gene expression
Coronal cryostat sections (20 µm) through fetal hypothalami were collected from the mamillary body (caudal boundary) to the optic chiasm (rostral boundary). Frozen whole brains in which the ME (median eminence of the hypothalamus) was absent or in which morphological integrity had been disrupted in the freezing process were identified before sectioning and were excluded. High-quality coronal sections were subsequently obtained for 17 lambs (control, n=9; well-fed, n=8). Sections were thaw-mounted onto slides double-coated with gelatin and poly-L-lysine and stored at –80°C until analysis of mRNA expression.
A riboprobe complementary to fragments of the intracellular domain of OBRb was generated from cloned sheep cDNA, as described previously (19)
. The NPY riboprobe was generated from a rat cDNA (20)
. The CART riboprobe was generated from a cloned sheep cDNA, as described previously (21)
. AGRP and POMC probes were generated from cloned Siberian hamster cDNA (22)
. All probes were previously validated for use with sheep brain tissues (23
, 24)
.
Expression of mRNA for POMC, CART, NPY, AGRP, and OBRb in the lamb hypothalamus was detected by in situ hybridization using methods described in detail elsewhere (24
, 25)
. Briefly, sections were fixed in 4% paraformaldehyde, acetylated, dehydrated through an ethanol gradient, and hybridized overnight at 55°C using [35S]-labeled cDNA probes (5–10x106 cpm/ml). On the following day, the sections were treated with RNaseA (Ambion, TX, USA) to remove unhybridized probe and desalted with a high stringency wash (30 min) in 0.1 x saline-sodium citrate at 55°C (23)
. The slides were air-dried at room temperature and apposed to Hyperfilm B-max (Amersham Pharmacia Biotech, Castle Hill, NSW, Australia) for 7 days (CART) or 14 days (NPY, OBRb, POMC, AGRP). The slides from all experimental animals for each probe were analyzed in a single incubation run. The resulting autoradiographic films were scanned at high resolution. All images on a film were captured in a single image using a high-resolution densitometer (Bio-Rad Laboratories, Richmond, CA, USA). The localization of specific hybridization signals was confirmed in the resulting images using computerized densitometry measurements. The area of positive signal was traced manually for each image and the integrated intensity (vol xmm2) of the hybridization signal was then computed using Quantity One Image Analysis software (Bio-Rad Laboratories, Richmond, CA, USA). The background signal was subtracted from each image in order to control for variations in background signal intensity across the film. For each probe, the sensitivity of measurement was confirmed using standard curves generated from 14C autoradiographic microscales (Amersham Pharmacia Biotech). For each probe, up to nine sections spanning the caudal, medial, and rostral extent of the hypothalamus were examined from each lamb. The results were averaged to give a single value for each experimental animal. All reagents were obtained from Sigma (Sydney, Australia) unless otherwise stated. Because of technical difficulties, it was not possible to determine hypothalamic POMC and NPY expression in one experimental animal in the Control group and one experimental animal did not express OBRb in the VMN.
Calculations
Daily fractional growth rate (FGR) was determined using a measure of weight gained per day (bodyweightx–body weightx–1/bodyweightx–1) where x refers to any day between day 1 and 30 after birth. Relative mass of perirenal and s.c. fat was calculated as grams of fat relative to kilogram of total body weight at day 30.
The daily ME intake for all ewes in Control and Well-Fed groups was used to calculate the ME intake of each ewe as a percentage of maintenance energy requirements (MER) (15)
(%MER =ME intake (MJ/day)/maintenance energy requirements (MJ)). The mean maternal intake expressed as the %MER across the whole period of late gestation (115 days to lambing) was then calculated for each ewe. The maternal energy intake during late gestation was also determined for each of the three separate 10-day windows in late gestation corresponding to the three time periods during which ME intake was increased by 10% (115–124 days, 125–134 days, and 135 days gestation to lambing).
Statistical analyses
The control group consisted of 6 males and 6 females (8 twins, 4 singletons), and the Well-Fed group consisted of 3 males and 6 females (2 twins, 7 singletons). Data are presented as the mean ± SEM.
Multifactorial ANOVA was used to determine the main effects of maternal nutritional treatment, sex, and fetal number and their interaction on birth weight, feeding behavior, fat mass, and neuropeptide expression. There was no effect of fetal number on any of the measures and it was also the case that averaging values from each twin pair and including the average as a single data point in the analyses also had no significant impact on the results. Data from each twin and singleton lamb were therefore included as individual data points in subsequent analyses.
The effect of increased maternal nutrition on fetal plasma Glc, insulin, NEFA, and leptin concentrations, as well as on lamb milk intake, time spent suckling, and suckling bouts was determined by multifactorial ANOVA with repeated measures with group, sex, and postnatal age (weeks) as the specified factors, using Statistical Package for Social Scientists. A Duncan’s post hoc test was used to identify significant differences between values. Two-way ANOVA was used to determine the main effects of maternal nutritional treatment (Control or Well-Fed) and sex and their interaction on birth weight, fat mass, and hypothalamic neuropeptide and OBRb expressions. Simple linear regression analyses were used to determine relationships between postnatal measures of fat mass, feed intake, and hypothalamic gene expression and mean maternal intake expressed as %MER across the whole period of late gestation (115 days to lambing). To define whether there are critical gestational periods, during which increased maternal nutrition programs, postnatal fat mass, and appetite occur, relationships were also determined between these variables and the mean maternal %MER intake during each of 3 separate gestational time periods (115–124 days, 125–134 days, and 135 days to lambing). Plasma Glc, insulin, and leptin concentrations across postnatal week 1–4 were averaged for correlation analyses unless stated otherwise. Partial correlation analysis was used to control for the effects of maternal intake or mean plasma Glc levels where appropriate. A probability of 5% (P<0.05) was taken as the concentration of significance in all analyses.
RESULTS
The effect of maternal ME intake during late gestation on fetal and postnatal growth
Maternal ME intake was significantly higher in well-fed compared to control ewes across late gestation (control, 91.9±2.2%MER; well-fed, 133.4±3.5%MER, P<0.01). There was no difference in birth wt, fractional growth rate or body wt at postnatal day 30 between the well-fed and control groups or between male and female lambs. The mean birth weights for twin and singleton lambs were not different in either the control or well-fed groups (singleton, 5.2±0.3 kg; twin, 4.8±0.2 kg). Relative s.c., but not perirenal, fat mass was significantly higher in the well-fed lambs compared to control group (Fig. 1
A, B) and relative total fat mass tended (P=0.06) to be higher in the well-fed lambs (control: male, 45.9±8.3 g/kg; female 59.8±9.0 g/kg; well-fed: male: 58.2±1.9 g/kg; female, 83.9±8.0 g/kg). Relative total and perirenal fat mass were significantly higher (P<0.05) in females compared to males, and this occurred independent of maternal nutritional treatment or of the number of lambs carried by the pregnant ewe (Fig. 1B
).
|
Impact of increased maternal nutrition on lamb milk intake
Relative milk intake (total milk intake by the lamb expressed as a percentage of lamb’s current body wt) was significantly higher in the well-fed group during postnatal weeks 1–3, but was not different from the control group in week 4 (Fig. 2
). There was no significant difference in relative milk intake during weeks 1–3 between twin and singleton lambs (singleton: 2.0±0.3%; twin, 1.5±0.2%).
|
Lamb plasma Glc, NEFA, insulin, and leptin concentrations
Plasma Glc concentrations were significantly higher in lambs of well-fed ewes during the first 4 wk of postnatal life (F=5.93, P<0.05) (Fig. 3
A). There was no difference between the control and well-fed groups or between twin and singleton or male and female lambs in plasma NEFA, insulin, or leptin concentrations during the first 30 days (Fig. 3B--D
). There was a direct relationship between plasma Glc and insulin concentrations on postnatal day 30 when data from all lambs were combined (plasma insulin=0.67 plasma Glc – 2.8; r2=0.45; P<0.01, n=14). Total relative fat mass was directly related to mean plasma Glc concentrations during the first 30 days of postnatal life (total fat=30.1 plasma Glc –117.0; r2=0.50; P<0.05, n=21). There was also a direct relationship between plasma leptin concentrations on postnatal day 30 and total relative fat mass when data from all lambs were combined (plasma leptin=0.04 fat mass+0.61, r2=0.24; P<0.03, n=19).
|
The impact of increased maternal nutrition on the expression of hypothalamic neuropeptides in 30-day lambs
OBRb
OBRb mRNA was expressed in both the ARC and VMN of the lamb hypothalamus (Fig. 4
A, B). The intensity of expression of OBRb mRNA was higher in the VMN compared with the ARC in both the control and well-fed groups (Fig. 4C
). There was no difference between the control and well-fed groups in the amount of expression of OBRb mRNA in either the ARC or the VMN and was not influenced by sex or by the number of lambs carried by the pregnant ewe.
|
In the well-fed group, the expression of OBRb in the VMN was positively related to the mean maternal energy intake (%MER) between 135 days gestation and lambing (OBRbVMN=0.01 maternal %MER–0.50, r2=0.64, P<0.03, n=7). OBRb expression in the VMN was not related to any measures of relative adiposity in either the control or well-fed groups. In the well-fed, but not control group, however, expression of OBRb in the ARC was inversely related to the relative total fat mass (OBRbARC=–0.005 total fat+0.71, r2=0.50, P=0.05, n=8) (Fig. 5
A).
|
POMC and CART
The expression of POMC mRNA in the ARC of the 30-day lamb hypothalamus was significantly higher in the well-fed group compared to controls, independent of sex or the number of lambs carried by the pregnant ewe (Fig. 6
A–C). The expression of POMC mRNA was not related to plasma concentrations of insulin, NEFA, leptin, or Glc (P=0.08) during the first 4 postnatal weeks in either the Control or Well-Fed groups. POMC mRNA expression was correlated with maternal energy intake (%MER) at 135 days gestation to lambing (POMC=0.004 maternal %MER–0.03; r2=0.28; P=0.03, n=16). However, this relationship was lost when the effect of plasma Glc concentrations during the first 4 postnatal weeks were controlled for.
|
There was no difference in the concentration of CART mRNA expression in the hypothalamus of lambs in the control and well-fed groups and no effect of sex or the number of lambs carried by the pregnant ewe. In the control group, CART mRNA expression was positively related to the relative total fat mass (CART=0.01 total fat+0.10; r2=0.69, P <0.01, n=9, Fig. 5B
) and to plasma Glc (CART=0.46 plasma Glc–1.9; r2=0.76, P<0.005, n=9) and NEFA (CART=1.03 plasma NEFA– 0.07; r2=0.60, P<0.02, n=9) concentrations across the first 30 days. In controls, CART mRNA expression was also directly related to plasma leptin concentrations on postnatal day 30 (CART=0.10 plasma leptin+0.46, r2=0.54, P<0.04, n=9), but this relationship was no longer significant after the effects of total relative fat mass were controlled for in the analysis. In contrast to the control group, however, there were no relationships between CART mRNA expression and plasma Glc, leptin, or total fat mass in the Well-Fed group.
NPY and AGRP
There was no difference in the hypothalamic gene expression for the appetite-stimulating neuropeptides, NPY and AGRP, on postnatal day 30 between the control and well-fed groups (Fig. 6D
). AGRP expression was significantly lower in females compared to males, independent of maternal nutrition or the number of lambs carried by the pregnant ewe (P<0.05). There was a positive relationship between the expression of AGRP and NPY mRNAs when data from all lambs were combined (AGRP=0.87 NPY–0.03, r2=0.39, P<0.02, n=16). When data from the control and well-fed groups were combined, there were significant inverse relationships between relative total fat mass and both NPY mRNA expression (total fat: NPY =–0.005 total fat+0.89; r2=0.28, P<0.05, n=16) and AGRP mRNA expression (total fat: AGRP =–0.007 total fat+0.95; r2=0.39, P<0.01, n=17) (Fig. 7
A, B). These relationships persisted after controlling for the effects of either plasma Glc concentrations or maternal energy intake (%MER) in late pregnancy.
|
DISCUSSION
We have shown that a 40% increase in maternal nutrient intake in late pregnancy results in an increase in relative milk intake and plasma Glc concentrations in lambs during early postnatal life and in an increased s.c. adiposity at 30 days of age. Increased maternal nutrition in late pregnancy also resulted in changes in the relationships between current nutritional status or fat mass, and the hypothalamic expression of appetite-regulating neuropeptides.
Maternal overnutrition and pre- and postnatal growth
In the present study, a 40% increase in maternal nutrition in late pregnancy had no effect on either birth weight or the final body weight of lambs at 30 days of age. These results are consistent with our previous findings (26)
but are different from those of Budge and colleagues who reported a significant increase in fetal weight at 144 days gestation when ewes were fed at 150% of maintenance levels between 80 and 144 days of pregnancy (27)
. These differences are likely due to the timing of the respective nutritional protocols, since early (i.e., pre-110-day gestation) increases in maternal nutrition have been shown to increase placental size (28)
.
Maternal overnutrition and milk intake
In the present study, lambs of well-fed ewes had a higher milk intake relative to their current body weight across the first 3 postnatal weeks, which may be due to a combination of an increase in maternal milk production in the well-fed group and/or an increased appetite drive in the lambs during this period. In late pregnancy, a large proportion of maternal substrates are directed toward mammary development (29)
, and it is therefore possible that the increased availability of maternal substrate resulted in increased milk production and/or changes in milk composition. Given that the suckling phase of the weigh-suckle-weigh protocol was always preceded by a 2-h fast, an alternate possibility is that the appetite-regulating network in lambs of well-fed ewes is more sensitive to signals of reduced energy supply. Although relative milk intake in the well-fed group was higher during the first 3 wk of postnatal life, this effect did not persist into week 4. In the present study, well-fed ewes were returned to a maintenance diet after lambing, and thus any effects of increased maternal nutrition on milk production or composition or on lamb appetite may not carry over into this final postnatal week.
The relative mass of s.c. adipose tissue at 30 days was greater in lambs of well-fed ewes, and this increase was directly related to the circulating Glc concentrations in early life. Although females tended to have a higher relative fat mass overall, the effects of increased maternal nutrition on postnatal fat deposition were similar for both sexes. These results are consistent with human clinical data, which show that adiposity is increased in infants of diabetic or Glc-intolerant mothers (2
, 3
,30
, 31)
. Thus the effects of increased maternal nutrition during pregnancy on fat deposition in the offspring may be mediated through an increase in plasma Glc concentrations during the early postnatal period.
Increased maternal nutrition and the appetite-regulating neuropeptides
The long form of the leptin receptor OBRb was expressed in both the ARC and VMN of the lamb hypothalamus. The expression of OBRb was higher in the VMN, a region of the hypothalamus that has an important role in the regulation of thermogenesis (32)
, compared to the ARC, a hypothalamic region that plays a major role in appetite regulation (33)
. This pattern of OBRb localization in the lamb hypothalamus is similar to the pattern of localization of OBRb in the fetal, but not adult, sheep hypothalamus (14
, 34)
. In the well-fed group there was also an inverse relationship between OBRb expression in the ARC and relative fat mass, such that increased adiposity was associated with reduced expression of the leptin receptor in the ARC. This suggests that, in lambs of overnourished ewes, an increase in adipose tissue mass is associated with a down-regulation of the leptin receptor in the arcuate nucleus, which would therefore result in a decreased sensitivity to the actions of leptin in this group. It would therefore appear that exposure to prenatal overnutrition may alter the subsequent responses of the central neural network to an increase in fat mass in postnatal life. A reduced sensitivity of ARC neurons to signals of body fat mass, such as circulating leptin, may therefore contribute, at least in part, to an increase in relative fat mass in lambs of well-fed ewes.
In the present study, a period of increased maternal nutrition in late pregnancy resulted in a specific increase in the expression of the appetite-inhibiting neuropeptide precursor, POMC, in the lamb hypothalamus. The level of POMC expression in the lamb hypothalamus was also directly related to maternal nutrient intake in late pregnancy, but this relationship did not persist after controlling for the effects of plasma Glc. Thus the effect of increased maternal nutrition on POMC expression may have been a direct consequence of the higher circulating Glc concentrations in the lambs of the well-fed ewes. This is consistent with recent evidence that there are subpopulations of POMC-containing neurons in the mouse hypothalamus that are Glc-responsive, responding to increases in extracellular Glc with increased activity (35
, 36)
. Although hypothalamic POMC expression was higher in the well-fed group, there was no evidence of an associated decrease in feed intake in this group during the first 30 days of postnatal development. It is possible, however, that the lower relative milk intake of lambs of well-fed ewes in postnatal week 4, relative to the first 3 wk after birth may have been a consequence of the higher hypothalamic POMC expression in this group.
Exposure to increased maternal nutrition in late pregnancy also resulted in a change in the relationship between expression of CART mRNA in the hypothalamic arcuate nucleus and the relative body fat mass. Although CART mRNA expression was positively correlated with relative adiposity and plasma leptin concentrations in control lambs, these relationships were not present in lambs of well-fed ewes. It would therefore appear that the sensitivity of the CART-expressing neurons in the ARC to signals of increased nutrient supply and body fat mass may be reduced in lambs exposed to an increased supply of nutrients before birth, which would have important implications for the subsequent regulation of energy balance homeostasis It has been shown that the density of neuronal connections between hypothalamic nuclei, and therefore the subsequent function of the neural network regulating appetite, is strongly influenced by the availability of leptin during hypothalamic development (10)
. One possibility, therefore, is that the differential regulation of CART in the control and well-fed groups is a consequence of programmed changes to the hypothalamic architecture. An alternative possibility is that the reduced sensitivity of CART expression to increased adiposity was a consequence of down-regulation of leptin receptor on CART-containing hypothalamic neurons. The results of the present study are also consistent with studies that have shown that the responsiveness of neurons within the ARC to peripheral signals of increased nutrient supply (insulin and leptin) is reduced in the hypothalamus of adult rats exposed to overnutrition in early postnatal life (37
38
39
40)
.
There were no differences in the expression of either NPY or AGRP mRNA between the control and well-fed groups in the present study. NPY and AGRP expression were each inversely related to lamb adiposity and these relationships persisted after controlling for the effects of maternal nutrient intake in late pregnancy. It would therefore appear that the NPY/AGRP systems are highly sensitive to increases in nutrition and energy status and that signals of current fat stores, rather than nutrition during the prenatal period, may be the more important determinants of NPY and AGRP expression in early postnatal life. These results are consistent with those of Plagemann and colleagues (4)
, who also found no decrease in either the number or density of NPY-positive neurons in the hypothalamic VMN or ARC in young adult rats overnourished in early postnatal life.
In summary, we have demonstrated that an increase in maternal nutrition in late pregnancy increases food intake, Glc concentrations, and the relative mass of s.c. adipose tissue in the postnatal lamb in the first month of postnatal life. Importantly, in the well-fed lambs, expression of the leptin receptor in the hypothalamic arcuate nucleus decreased as fat mass increased, and the direct relationship between the expression of the central appetite inhibitor, CART, and fat mass was lost. We speculate that the failure of lambs of well-fed mothers to up-regulate hypothalamic anorexigenic pathways in response to increases in adiposity may be a consequence of a central resistance to the actions of leptin. These changes may represent one potential mechanism whereby exposure to an increased nutrient supply before birth may lead to a subsequent increase in childhood and adult obesity.
ACKNOWLEDGMENTS
We gratefully acknowledge the support of Laura O’Carroll and Animal Technician staff of the University of Adelaide for assistance with animal protocols. This research was funded by an NHMRC Program grant.
Received for publication October 6, 2005. Accepted for publication January 3, 2006.
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