help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-0555
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
144/11/4947    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mühlhäusler, B. S.
Right arrow Articles by McMillen, I. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mühlhäusler, B. S.
Right arrow Articles by McMillen, I. C.
Endocrinology Vol. 144, No. 11 4947-4954
Copyright © 2003 by The Endocrine Society

Determinants of Fetal Leptin Synthesis, Fat Mass, and Circulating Leptin Concentrations in Well-Nourished Ewes in Late Pregnancy

B. S. Mühlhäusler, C. T. Roberts, B. S. J. Yuen, E. Marrocco, H. Budge, M. E. Symonds, J. R. McFarlane, K. G. Kauter, P. Stagg, J. K. Pearse and I. C. McMillen

Departments of Physiology (B.S.M., B.S.J.Y., E.M., P.S., J.K.P., I.C.M.) and Obstetrics and Gynecology (C.T.R.), University of Adelaide, South Australia 5000, Australia; Academic Division of Child Health, School of Human Development, University Hospital (H.B., M.E.S.), Nottingham, United Kingdom NG7 2UH; and Department of Physiology, University of New England (J.R.M., K.G.K.), New South Wales 2351, Australia

Address all correspondence and requests for reprints to: Prof. I. C. McMillen, Physiology, School of Molecular and Biomedical Science, University of Adelaide, South Australia 5000, Australia. E-mail: caroline.mcmillen{at}adelaide.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have investigated the factors regulating leptin synthesis, fat deposition, and circulating leptin concentrations in fetuses of well nourished ewes in late pregnancy. Vascular catheters were surgically inserted in 17 pregnant ewes and their fetuses at 103–120 d gestation (term = 147 ± 3 d). Ewes were fed a diet providing either 100% (control; n = 9) or approximately 155% (well fed; n = 8) of the maintenance energy requirements and fetal perirenal and interscapular fat depots were collected at 139–141 d gestation. There was a significant relationship between the relative mass of fetal unilocular fat and fetal glucose (relative mass of unilocular fat, 1.14; fetal glucose, +0.16; r = 0.50; P < 0.04; n = 17), but not insulin, concentrations in the control and well-fed groups. In contrast to the controls, there was a positive relationship between the relative abundance of leptin mRNA and fetal insulin, but not glucose, concentrations in fetal perirenal adipose tissue in the well-fed group. A moderate increase in maternal nutrition also resulted in a strong reciprocal relationship between uncoupling protein 1 and leptin expression in fetal perirenal adipose tissue in late gestation (well-fed group: uncoupling protein 1 mRNA:18S rRNA, -0.51; leptin mRNA:ß-actin mRNA, +1.53; r = 0.80; P < 0.02; n = 8). These studies provide evidence that fetal glucose and insulin differentially regulate fetal fat deposition and leptin mRNA expression within the fetal perirenal fat depot in the well nourished animal during late gestation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN ADULT MAMMALS, the 16-kDa protein hormone, leptin, is principally synthesized and secreted by adipose tissue and acts as a circulating signal of fat mass (1). Plasma leptin concentrations are directly related to body fat content and to the prevailing level of nutrient intake (1, 2, 3) In the human infant, there is a positive relationship between cord blood concentrations of leptin at delivery and birth weight or neonatal adiposity (4, 5, 6, 7). Furthermore, in pregnancies complicated by maternal diabetes, the fetus is hyperglycemic and hyperinsulinemic, and cord blood leptin concentrations are increased in parallel with increases in infant adiposity (8, 9). It has also been shown that a high maternal prepregnancy weight, maternal diabetes mellitus, gestational diabetes, or even mildly impaired glucose tolerance during pregnancy are all risk factors for the development of obesity or glucose intolerance in the offspring (10, 11, 12, 13). Furthermore, serum leptin is elevated early in the development of childhood-onset obesity, and obese children have a high serum leptin even when normalized to fat mass (14). There is therefore considerable interest in the mechanisms by which an increase in fetal nutrient supply initiates changes in the development of the adipocyte and leptin signaling system that may underlie the association between enhanced fetal nutrition and postnatal obesity.

In animals such as the sheep and pig in which fat is deposited before birth, leptin is synthesized in fetal adipose tissue and is present in the fetal circulation through late gestation (15, 16, 17, 18, 19, 20). In the sheep, fetal adipose tissue is comprised of multilocular adipocytes (19, 21, 22). Although some of these adipocytes may contain one dominant lipid locule along with other smaller locules, all of them possess an abundance of mitochondria and express uncoupling protein 1 (UCP1) (23, 24). These characteristics are typical of thermogenic or brown adipocytes (25). Interestingly, in a cohort of well nourished sheep, we found that fetal plasma leptin concentrations were directly related to the relative mass of the lipid contained within the dominant lipid locules (unilocular lipid) present in perirenal and interscapular adipose tissues, which are the major internal fat depots in the sheep fetus (19). There is relatively little known, however, about the factors that determine relative unilocular fat mass or regulate the leptin synthetic capacity of fetal adipose tissue. In the adult human, rat, and cow, circulating leptin concentrations are directly related to the size of the unilocular white adipocytes, i.e. large fat cells are associated with higher circulating leptin concentrations (26, 27, 28). We therefore determined whether the relationship that exists between lipid locule size and circulating leptin after birth also exists before birth in the two major fat depots present in the fetus, the perirenal and interscapular depots. We also investigated the relative roles of circulating glucose and insulin in determining fetal adiposity and the relationship between prevailing fetal glucose and insulin concentrations and the expression of leptin and UCP1 mRNA in fetal perirenal adipose tissue in a cohort of pregnant ewes fed at or above maintenance energy requirements.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and surgery
All procedures were approved by the University of Adelaide animal ethics committee. Singleton pregnancies were confirmed in 17 adult Merino ewes by ultrasound scanning in early gestation. Surgery was then performed on these ewes between 103–120 d gestation (term = 147 ± 3 d) using aseptic techniques. General anesthesia was induced by iv injection of sodium thiopentone (1.25 g, iv; Pentothal, Rhone Merieux, Pinkenba, Australia) and was maintained with 2.5–4% halothane (Fluothane, ICI, Melbourne, Australia) in oxygen. Vascular catheters were implanted in a jugular vein and carotid artery of the ewe and fetus and in the amniotic cavity, as previously described (29). During surgery, im injections of antibiotics (2 ml; 250 mg/ml Procaine penicillin, 250 mg/ml dihydrostreptomycin, and 20 mg/ml procaine hydrochloride (Lyppards, Adelaide, Australia) or 0.1 ml/kg terramycin 100 and 100 mg/ml oxytetracycline hydrochloride (Pfizer, Sydney, Australia)] were administered to each ewe and fetus. All catheters were filled with heparinized saline, and the fetal catheters were exteriorized through an incision in the ewe’s flank. Before and after surgery the ewes were housed in individual pens in animal holding rooms with a 12-h light, 12-h dark cycle. Ewes were allowed at least 3 d to recover from surgery before experimentation.

Feeding regimen
Ewes were fed twice daily at 0900 and 1600 h, with water provided ad libitum. Between 103 and 114 d gestation, ewes were fed a diet consisting of Lucerne chaff [85% dry matter; metabolizable energy (ME) content, 8.3 MJ/kg] and concentrated pellets containing straw, cereal, hay, clover, barley, oats, lupins, almond shells, oat husks, and limestone (90% dry matter; ME content, 8.0 MJ/kg; Johnsons and Sons, Kapunda, Australia) (19). The diet was calculated to provide 100% of the energy requirements for the maintenance of a pregnant ewe bearing a singleton fetus, as specified by the Ministry of Agriculture, Fisheries, and Food, United Kingdom (30).

At 115 d gestation, i.e. before commencement of the rapid fetal growth phase in late gestation (31), ewes were randomly assigned to either a control (n = 9) or a well-fed group (n = 8). Between 115 and 124 d gestation, Control ewes were provided with 19.0 ± 1.1 g Lucerne chaff and 4.7 ± 0.3 g pelleted concentrate/kg body weight, and well-fed ewes were provided with 29.6 ± 2.6 g Lucerne chaff and 7.4 ± 0.8 g pelleted concentrate/g body weight each day. All feed refusals were weighed and recorded daily. The total ME intake for the well-fed group (0.26 ± 0.02 MJ/kg·d) was about 55% greater than the total ME intake in the control group (0.17 ± 0.01 MJ/kg·d). The feed allowance of all ewes was proportionately increased by 15% every 10 d to meet the increasing substrate demands of the growing fetus in late gestation (30).

Blood sampling regimen, post mortem, and tissue collection
Between 116 and 139 d gestation, maternal (5.0 ml) and fetal (3.0 ml) arterial blood samples were collected three times per week (on alternate days) before morning feeding at 0900 h. Blood samples were centrifuged at 1500 x g for 10 min at 4 C, and plasma was stored at -20 C for subsequent determination of glucose, insulin, and leptin concentrations as previously described (19). Fetal arterial blood (0.5 ml) was also collected three times per week for determination of fetal blood gases (PO2, PCO2, oxygen saturation, pH, hematocrit, and hemoglobin) using an ABL 520 analyzer (Radiometer, Copenhagen, Denmark) to ensure that fetal arterial blood gas status was within the normal range for fetal sheep in this gestational age range (19). Between 139 and 141 d gestation all ewes were euthanized with an overdose of sodium pentobarbitone (Virbac Pty. Ltd., Peakhurst, Australia). All fetuses (control, three females and six males; well fed, five females and three males) were alive at postmortem. Fetal sheep were delivered by hysterotomy, weighed, and killed by decapitation. All adipose tissue from the perirenal and interscapular sites was dissected and weighed. A sample of adipose tissue from each site was fixed in 4% paraformaldehyde in 0.2 M phosphate buffer for subsequent processing and histological analyses. A second sample from each site was snap-frozen in liquid N2 and stored at -80 C for subsequent determination of leptin and UCP1 mRNA by RT-PCR and Northern blot, respectively.

Glucose and insulin assays
Maternal and fetal plasma glucose concentrations were determined by enzymatic analysis using the COBAS MIRA automated analysis system (Roche, Basel, Switzerland), previously validated for sheep plasma (32). The intra- and interassay coefficients of variation were both less than 5%.

Insulin concentrations were determined in fetal plasma samples using an RIA kit (Phadaseph RIA kit, Pharmacia & Upjohn, Uppsala, Sweden) previously validated for sheep plasma (32). The detection range of the assay was 1.5–240 µU insulin/ml. The inter- and intraassay coefficients of variation were both less than 20%.

Leptin assay
A competitive ELISA, previously validated for sheep plasma (33), was used to determine plasma leptin concentrations in fetal plasma samples. The sensitivity of the assay was 0.5 ng/ml, and the inter- and intraassay coefficients of variation were both less than 10%.

RNA extraction
Total RNA was extracted from perirenal adipose tissue (PAT) samples as previously described (15). Briefly, approximately 100 mg adipose tissue were homogenized with 1 ml Tri-Reagent (Sigma-Aldrich Corp., St. Louis, MO) and allowed to stand at room temperature for 5 min. 1-Bromo-3-chloro-propane (100 µl) was added, and the samples were incubated at room temperature for 8 min and then centrifuged at 3500 x g for 15 min at 4 C. An aliquot of the aqueous layer (~600 µl) was recovered, and RNA was precipitated by the addition of isopropanol (500 µl), followed by a 6-min incubation at room temperature with subsequent centrifugation at 3500 x g for 10 min at 4 C. The pellet was washed in 70% ethanol, air-dried, and resuspended in sterile water (20 µl). The spectrophotometric absorbance at 260 and 280 nm was determined for each sample, and the 260:280 nm absorbance ratio was always greater than 1.6. The RNA yield was 0.81 ± 0.45 µg RNA/mg adipose tissue. The integrity of RNA preparations was evaluated by agarose gel electrophoresis, followed by ethidium bromide staining and identification of ribosomal RNA under transillumination.

Leptin and ß-actin mRNA
Ovine leptin and ß-actin cDNA were amplified by RT-PCR as previously described (15, 23, 34). Briefly, 2 µg total RNA were reverse transcribed with random hexamer oligonucleotides (Invitrogen, Adelaide, Australia) and SuperScript reverse transcriptase (Invitrogen, Adelaide, Australia). The reverse transcribed product (5 µl) was used as a template for the amplification of a fragment of ovine leptin cDNA (183 bp) and ovine ß-actin cDNA (349 bp) using Taq DNA polymerase (Biotech International, Bently, Australia) according to the manufacturer’s protocol. The PCR parameters were 26 cycles of 60 sec at 94 C, 15 sec at 53 C, and 60 sec at 72 C (PCR Express, Hybaid, Teddington, UK). The primers for the amplification of the leptin cDNA fragment were 5'-GAC ATC TCA CAC ACG CAG-3' and 5'-GAG GTT CTC CAG GTC ATT-3' (GeneWorks, Adelaide, Australia), and the primers for amplification of the ß-actin fragment were 5'-TGG ATG GTG GGT ATA TGG GTC-3' and 5'-TAG ATG GGC ACA GTG TGG GT-3'. Both RT-PCR products were sequenced to confirm their identity (15). Each PCR product (8 µl) was electrophoresed through a 2.0% (wt/vol) agarose gel, stained with ethidium bromide, transilluminated with UV radiation, photographed using a digital camera, and quantified using the 1D Image Analysis Software Electrophoresis Documentation and Analysis System 120 (Kodak dS Ditigal Science, Eastman Kodak, Rochester, NY). The RT-PCR was performed in duplicate on RNA from each adipose tissue sample. The relative abundance of leptin mRNA was calculated by referencing the intensity of the leptin amplicon to the intensity of the ß-actin amplicon for each fetus.

UCP1 mRNA and 18S rRNA
An oligonucleotide radiolabeling kit (Amersham Pharmacia Biotech, Piscataway, NJ) was used to end-label 1 ng UCP1 oligonucleotide (Geneworks, Adelaide, Australia) with [32P]deoxy-ATP (4000 Ci/mmol; GRA-32U, Geneworks) according to the manufacturer’s instructions. The UCP1 oligonucleotide, 5'-CGG ACT TTG GCG GTG TCC AGC GGG AAG GTG AT-3', was complementary to nucleotides 267–298 of the 1194-nucleotide cDNA of rat UCP1 (GenBank accession no. NM 012682) (35, 36). An oligonucleotide complementary to nucleotides 151–180 of rat 18S ribosomal RNA (37) was also end-labeled with [{gamma}-32P]ATP (38).

Samples of total RNA (10 or 20 µg) from PAT were electrophoresed through a 1.5% agarose gel containing 2.2 M formaldehyde and 1x Northern buffer [containing 0.1 M 3-(N-morpholino) propanesulfonic acid] at pH 7.0, 40 mM sodium acetate, and 5 mM EDTA (EDTAate·2H20 disodium salt, pH 8.0). Total RNA was transferred overnight at room temperature onto a {zeta}-Probe nylon membrane (Bio-Rad Laboratories, Richmond, CA) by capillary transfer in 10x saline sodium citrate (SSC). Membranes were then washed twice in fresh 10x SSC and baked at 80 C for 50 min. Before probing, the blots were prehybridized with 100 µg/ml heat-denatured salmon sperm DNA at 52 C for 2 h in 5x SSC, 20 mM NaH2PO4, 7% SDS, and 5x Denhardt’s solution at pH 7.2 (38). The UCP1 oligonucleotide probe was labeled with [{gamma}-32P]ATP, purified through a Sephadex column, and added to the hybridization solution. The probe was allowed to hybridize for 14–16 h at 52 C. Membranes were then washed in 1x SSC/0.1% SDS at 52 C for 30 min and were washed again in fresh 1x SSC/0.1% SDS for an additional 10 min at 52 C. Membranes were exposed to a phosphorimaging screen for 2.5 d (Fuji-BAS MP2040, Fuji Photo Film Co. Ltd., Tokyo, Japan) and visualized using a Fuji-BAS 1000 phosphorimager (Fuji Photo Film Co. Ltd.). The hybridization signal was quantified with Fuji-MacBAS software (version 2.21). Membranes were then washed in stripping solution containing 0.1x SSC and 0.5% SDS at 85 C for 30 min before being rehybridized with a probe for 18S rRNA. The procedure was performed in duplicate for each adipose tissue sample. The relative abundance of UCP1 mRNA was calculated by referencing the intensity of the UCP1 mRNA band to the intensity of the 18S rRNA band for each fetus.

Volume density of unilocular and multilocular adipose tissues
Samples of perirenal and interscapular adipose tissue were fixed in 4% paraformaldehyde in 0.2 M phosphate buffer at 4 C for a maximum of 3 d. Tissues were then washed in 0.01 M PBS and immersed in 70% ethanol for 24 h before being processed and embedded in paraffin wax. Sections were cut (4 µm), stained with hematoxylin and eosin, and examined using an Olympus BH2 microscope (20x objective and 2.5x NFK photo eyepiece; Olympus, New Hyde Park, NY). Standard point-counting techniques were used with Video Image Analysis using Video Pro software (Leading Edge, Adelaide, Australia) to determine the volume density of unilocular tissue in the perirenal and interscapular fat depots as described previously (19, 23). Regions of adipose tissue containing lipid locules with a cross-sectional area greater than 10 µm2 (21, 22) were classified as the unilocular component, whereas the remaining regions were classified as the multilocular component of the depot (Fig. 1Go). For each adipose tissue site, 1 section was selected for each animal, and systematic random sampling was used to select 10 fields (360 points) for each section. The volume density (Vd) of unilocular and multilocular adipose tissue was calculated as described previously (19, 23, 39) using the formula: Vd = N/T, where N is the number of points falling on the unilocular or multilocular component, and T is the total number of points counted.



View larger version (142K):
[in this window]
[in a new window]
  		FIG. 1. Light micrograph of a typical sample of fetal PAT at 139–141 d gestation, stained with hematoxylin and eosin. U, Dominant lipid locule comprising the unilocular adipose tissue; M, smaller locules comprising the multilocular adipose tissue. Scale bar, 50 µm.

 
Cross-sectional area and density of lipid locules
In each field of view, the perimeter of each of the lipid locules with a cross-sectional area greater than 10 µm2 was manually traced, and the Area-Pro software package (Video Pro Image Analysis, Leading Edge) was used to determine the enclosed area. The program was validated by the measurement of a known area using a hemocytometer. The mean cross-sectional areas of the dominant lipid locules within the perirenal and interscapular fat depots represented the average cross-sectional area of all lipid locules falling completely within each of 10 fields of view within a section. The total numbers of lipid locules for which areas were determined in each fetus were 258 ± 25 and 285 ± 20 in the perirenal and interscapular depots, respectively. The distribution of the size ranges (<400, 401–800, or >800 µm2) of lipid locules in the perirenal and interscapular adipose depots was then determined for 17 experimental animals. The total number of lipid locules in the 10 selected fields and the area of each field were determined for each animal and used to calculate the number of lipid locules per square millimeter (i.e. the density of the lipid locules) in each of the perirenal and interscapular adipose depots for these 14 fetuses (6 control and 8 well fed).

Statistical analysis
Data are presented as the mean ± SEM. The masses of the unilocular and multilocular components for each fat depot were calculated by multiplying the mass of each fat depot by the volume density of the unilocular or multilocular component of that depot. Total unilocular and multilocular fat masses were calculated as the sum of the respective interscapular and perirenal fat masses. The relative unilocular and multilocular fat mass (grams per kilogram) were each calculated by dividing the total unilocular or multilocular fat mass by fetal weight.

The effect of an increase in maternal nutrition on measures of fat composition and relative leptin mRNA and UCP1 mRNA abundance in fetal PAT was determined by t test. Plasma concentrations of glucose and insulin between 115 and 139 d gestation were averaged to obtain a mean gestational value for each ewe and fetus. Simple linear regression analyses were then used to determine relationships between mean fetal glucose or insulin concentrations and measures of fetal adiposity. Relationships between the mean fetal plasma concentrations of leptin in the last 5 d before the postmortem and fetal unilocular fat mass or mean lipid locule size were determined using linear regression analysis.

The total leptin synthetic capacity of fetal PAT was estimated as the product of relative abundance of leptin mRNA, the yield of RNA (micrograms per milligram of tissue), and total PAT mass. This was then divided by fetal body weight to give an estimate of the total leptin synthetic capacity of PAT per kilogram of fetal body weight.

Simple linear regression analyses were used to determine relationships between mean fetal glucose or insulin concentrations and the leptin/ß-actin mRNA or UCP1/18S rRNA ratios. The independent effects of leptin synthetic capacity and mean lipid locule size in PAT on plasma leptin concentrations were analyzed by partial correlation and multiple linear regression analyses. A probability of 5% (P < 0.05) was taken as the level of significance in all analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Maternal and fetal plasma glucose and insulin concentrations
Maternal plasma glucose concentrations were significantly increased in the well-fed group compared with the control ewes (Table 1Go). Fetal plasma glucose and insulin concentrations were also significantly higher in the well-fed group compared with controls (Table 1Go).


View this table:
[in this window]
[in a new window]
  		TABLE 1. The effect of an increase in maternal nutrient intake on plasma glucose, insulin, and leptin concentrations

 
Size and density of lipid locules in fetal perirenal and interscapular fat depots
In both the control and well-fed groups, the perirenal depot made up a greater (P < 0.05) proportion of the total fetal fat mass (control, 91.4 ± 3.6%; well-fed, 86.3 ± 4.7%; n = 17) compared with the interscapular fat depot (control, 8.6 ± 3.6%; well-fed, 13.7 ± 4.7%; n = 17). There was no effect of an increase in maternal nutrition on the mass of the perirenal, interscapular, total, or relative fetal fat mass (Table 2Go). There was also no difference in the size distribution, mean size, or density of the lipid locules in either the perirenal or interscapular depots between the control and well-fed groups (Table 2Go and Fig. 2Go, A and B). There was a positive relationship, however, between the proportion of each fetal fat depot that was comprised of unilocular adipose tissue and the mean size of the lipid locules within the fat depot [PAT = 0.04(mean lipid locule size) + 22.3; r = 0.67; P < 0.005; n = 17; interscapular fat depot: proportion of unilocular tissue = 0.11(mean lipid locule size) - 13.7; r = 0.84; P < 0.01; n = 10; Fig. 2Go, C and D]. There was no relationship between the proportion of fetal perirenal or interscapular adipose tissue comprised of unilocular tissue and the density of lipid locules per square millimeter within each fat depot.


View this table:
[in this window]
[in a new window]
  		TABLE 2. The effect of increasing maternal nutrient intake on fetal fat deposition and morphology

 


View larger version (25K):
[in this window]
[in a new window]
  		FIG. 2. A–D, The size distribution of lipid locules in the perirenal (A) and interscapular (B) depots in the control ({blacksquare}) and well-fed ({square}) groups. There was a significant correlation between the proportion of unilocular adipose tissue and the mean size of the lipid locules in both perirenal (r = 0.67; P < 0.005; C) and interscapular (r = 0.84; P < 0.01; D) depot in the control ({bullet}; n = 9) and well-fed ({circ}; n = 8) groups.

 
Glucose, insulin, and unilocular fat mass
The relative total mass of the unilocular adipose tissue component in the PAT and interscapular fat depots was directly related to fetal glucose concentrations [relative mass of unilocular fat = 1.1(fetal glucose) + 0.2; r = 0.50; P < 0.04, n = 17; Fig. 3Go]. Within the fetal PAT, but not interscapular depot, there was also a direct relationship between the mean size of the lipid locules and fetal glucose concentrations [mean lipid locule size = 249 (fetal glucose) + 112; r = 0.65; P < 0.005; n = 17]. There was no relationship, however, between the relative mass or the mean size of the lipid locules and fetal insulin concentrations in either the PAT or the interscapular depot.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 3. The relationship between fetal plasma glucose concentrations and the relative mass of unilocular adipose tissue (grams per kilogram) in PAT in the control ({bullet}; n = 9) and well-fed ({circ}; n = 8) groups. There was a significant correlation between fetal plasma glucose concentrations and the relative mass of unilocular fat (grams per kilogram) in the perirenal depot in both the control and well-fed groups (r = 0.50; P < 0.05).

 
Leptin and UCP 1 mRNA expression in fetal perirenal adipose tissue
There was no difference between the control and well-fed groups in the relative abundance of leptin mRNA/ß-actin mRNA or UCP1 mRNA/18S rRNA in PAT (Table 2Go). In the well-fed, but not the control, group, there was a positive relationship between leptin mRNA expression in PAT and fetal insulin concentrations [leptin mRNA: ß-actin mRNA = 0.07(fetal insulin) + 0.95; r = 0.80; P < 0.02; n = 8; Fig. 4AGo], and in the well-fed animals, there was also an inverse relationship between the relative abundance of UCP1 and leptin mRNA [UCP1 mRNA: 18S rRNA = -0.51(leptin mRNA/ß-actin mRNA) + 1.53; r = 0.80; P < 0.02; n = 8; Fig. 4BGo]. There was no significant relationship between leptin or UCP1 mRNA expression and fetal plasma insulin or glucose concentrations in the control animals.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 4. A and B, The relationship between the relative abundance of leptin mRNA/ß-actin mRNA in PAT and fetal plasma insulin concentrations at 139 d gestation (microunits per milliliter; r = 0.80; P < 0.02; n = 8; A) and between UCP1mRNA/18S rRNA and leptin mRNA/ß-actin mRNA in PAT (r = -0.80; P < 0.02; n = 8; B) in the well-fed group.

 
Leptin and lipid locule size
There was no difference in the mean fetal plasma concentrations of leptin between the well-fed and control groups (Table 1Go). Fetal leptin concentrations were positively related to mean size, but not density, of the lipid locules in the PAT [fetal leptin = 0.008 (mean lipid locule size) + 0.05; r = 0.69; P < 0.005; n = 16], but not in the interscapular adipose depot. Fifty-five percent of the variation in the mean fetal plasma concentrations of leptin was explained by a combination of the estimated total leptin synthetic capacity of PAT and the mean size of the lipid locules within this fetal fat depot in both the well-fed and control groups [fetal leptin = 0.28 (estimated leptin synthetic capacity) + 0.009 (mean lipid locule size) - 1.87; r = 0.74; P< 0.01; n = 16].


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that there is a significant relationship between the relative mass of unilocular fat and fetal glucose, but not insulin, concentrations in ewes fed at or above maintenance energy requirements and that an increase in unilocular fat mass is related to an increase in the mean size of the dominant lipid locules within the fat depot. A moderate increase in maternal and, hence, fetal nutrient supply resulted in a strong reciprocal relationship between leptin and UCP1 expression in fetal fat in late gestation, and we have shown that about half the variation in circulating leptin concentrations in the fetus can be explained by a combination of the estimated total leptin synthetic capacity of fetal perirenal adipose tissue and the mean size of the lipid locules within this fetal fat depot.

Glucose and fetal fat deposition
In a previous study Stevens and Alexander (40) reported that infusion of glucose in fetal sheep for about 30 d in late gestation resulted in an approximately 3-fold increase in plasma glucose concentrations and an increase in the total and relative mass of fetal perirenal and sc adipose tissue (40). In the present study we have determined the impact of a more moderate (~1.25-fold) increase in fetal plasma glucose concentrations on fetal fat depots. We found evidence that an increase in fetal glucose, within the range of 1.1–2.6 mmol/liter, resulted in an increase in the mean size of the lipid locules in fetal fat depots and in the total or relative mass of unilocular adipose tissue in the perirenal and interscapular depots. Interestingly, there was no correlation between fetal insulin concentrations and the mean size, number, or mass of the lipid locules in the fetal fat depots. Thus, glucose appears to be the principal determinant of lipid storage in fetal adipose depots under normal physiological conditions in fetuses of well nourished ewes. It also appears that the uptake of substrate into fetal adipose cells occurs primarily via an insulin-independent mechanism. This is consistent with previous reports that the abundance of the insulin-independent glucose transporter (GLUT1) is greater than that of the insulin-dependent (GLUT4) glucose transporter in adipose tissue of the sheep fetus in late gestation (41, 42). In contrast, insulin appears to play a major role in lipid synthesis in the brown adipocytes of fetal rats through actions via the Insulin receptor substrate 1 pathway and GLUT4 (43, 44). The relative roles of insulin and glucose in the regulation of lipid synthesis within brown adipose tissue may also be dependent on the extent to which the tissue is exposed to a range of circulating factors, such as glucocorticoids, T3, and IGFs, during critical stages of adipose tissue development.

Leptin and UCP mRNA expression in fetal adipose tissue
In well-fed animals, there was a strong positive relationship between leptin mRNA expression and insulin, but not glucose, concentrations. Insulin stimulates leptin expression in white and brown adipose tissue in adults (45, 46, 47), and it has also been reported that intrafetal infusion of insulin for 24 h resulted in high fetal insulin concentrations (50–180 µU/ml) and an increase in leptin mRNA expression in the perirenal adipose tissue of the sheep fetus (17). Thus, it appears that as fetal insulin concentrations increase beyond the moderate elevation achieved by an increase in maternal nutrition (10–15 µU/ml), leptin expression in fetal adipose tissue is significantly increased.

In the adult, an increase in nutrient intake is associated with an increase in UCP1 mRNA and protein in brown adipose tissue (48, 49). Budge and colleagues (50) also previously reported that an increase in maternal nutrient intake to 50% above maintenance requirements during late pregnancy increased the abundance of UCP1 protein and the total thermogenic capacity of the fat depot at 144 d gestation. The impact of the increase in maternal nutrition on fetal plasma glucose and insulin concentrations was not measured in this latter study, and it is possible that the increase in fetal nutrient supply achieved exceeded that in the present study and resulted in a recruitment of sympathetic stimulation of UCP1 expression in fetal adipose tissue. It appears, however, from the present study that a moderate increase in maternal nutrition differentially affects leptin and UCP1 mRNA expression, resulting in a clear reciprocal relationship between their expression in adipose tissue in late gestation.

Leptin synthesis, adiposity, and circulating leptin
In the adult, circulating leptin concentrations are related to both body fat content and current nutritional status (2, 3). Delavaud and colleagues (3) reported that in the adult ewe, body fat content explains approximately 34% of the variation, and current nutritional status explains 17% of the variation in circulating leptin concentrations (3). In the present study the relative mass of unilocular fat or the mean size of the lipid locule in the fetal adipose cells also explained about 40–50% of the variation in plasma leptin concentrations. This is consistent with the results of studies in a range of species demonstrating that there is a positive relationship between circulating leptin concentrations and the size of the unilocular adipocytes in adult fat depots (26, 27, 28). The results of our study indicate that the relationship between circulating leptin and the lipid storage capacity of the adipocyte is established from before birth. Interestingly, a greater proportion (~55%) of the variation in circulating leptin concentrations was explained by the combination of the estimated total leptin synthetic capacity of the fetal fat depots and the mean size of the lipid locules than by mean locule size alone.

Summary
We have demonstrated that glucose, rather than insulin, is a major determinant of the mean size of the lipid locules in unilocular adipose cells in the major internal fat depot of the sheep fetus in late gestation. We have also shown for the first time that there is a positive relationship between the size of lipid locules in the unilocular adipose cells of the perirenal depot and circulating leptin concentrations. An increase in maternal, and hence fetal, nutrition resulted in a change in the relationship between leptin expression and insulin, such that the variation in leptin mRNA expression was directly related to the variation in fetal insulin concentrations. Interestingly, an increase in fetal nutrient supply is also associated with the emergence of a strong reciprocal relationship between UCP1 and leptin mRNA expression in fetal adipose tissue in late gestation. Finally, both the leptin synthetic capacity of the adipose tissue and the mean size of the lipid locules contribute to the variation in circulating leptin concentrations in late gestation.


   Acknowledgments
 
We are grateful to Anne Jurisevic and Frank Carbone for their expert assistance with the sheep surgery, and to Laura O’Carroll for her invaluable assistance with experimental animal protocols.


   Footnotes
 
This work was supported by the National Health and Medical Research Council of Australia.

Abbreviations: GLUT, Glucose transporter; ME, metabolizable energy; PAT, perirenal adipose tissue; SSC, saline sodium citrate; UCP1, uncoupling protein 1.

Received May 5, 2003.

Accepted for publication July 9, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ahima RS, Flier JS 2000 Leptin. Annu Rev Physiol 62:413–437[CrossRef][Medline]
  2. Blache D, Tellam RL, Chagas LM, Blackberry MA, Vercoe PE, Martin GB 2000 Level of nutrition affects leptin concentrations in plasma and cerebrospinal fluid in sheep. J Endocrinol 165:625–637[Abstract]
  3. Delavaud C, Bocquier F, Chilliard Y, Keisler DH, Gertler A, Kann G 2000 Plasma leptin determination in ruminants: effect of nutritional status and body fatness on plasma leptin concentration assessed by a specific RIA in sheep. J Endocrinol 165:519–526[Abstract]
  4. Jaquet D, Leger J, Levy-Marchal C, Oury JF, Czernichow P 1998 Ontogeny of leptin in human fetuses and newborns: effect of intrauterine growth retardation on serum leptin concentrations. J Clin Endocrinol Metabol 83:1243–1246[Abstract/Free Full Text]
  5. Koistinen HA, Koivisto VA, Andersson S, Karonen SL, Kontula K, Oksanen L, Teramo KA 1997 Leptin concentration in cord blood correlates with intrauterine growth. J Clin Endocrinol Metab 82:3328–3330[Abstract/Free Full Text]
  6. Matsuda J, Yokoto I, Mitsuru I, Murakami T, Naito E, Ito M 1997 Serum leptin concentrations in cord blood: relationship to birth weight and gender. J Clin Endocrinol Metab 82:1642–1644[Abstract/Free Full Text]
  7. Shekhawat PS, Garland JS, Shivpuri C, Mick GJ, Sasidharin P, Pelz CJ, McCormick KL 1998 Neonatal cord blood leptin: its relationship to body weight, body mass index, maternal diabetes and steroids. Pediatr Res 43:338–343[Medline]
  8. Tapanainen P, Leinonen E, Ruokonen A, Knip M 2001 Leptin concentrations are elevated in newborn infants of diabetic mothers. Horm Res 55:185–190[CrossRef][Medline]
  9. Cetin I, Morpurgo PS, Radaelli T, Taricco E, Cortelazzi D, Bellotti M, Pardi G, Beck-Peccoz P 2000 Fetal plasma leptin concentrations: relationship with different fetal growth patterns from 19 weeks up to term. Pediatr Res 48:646–651[Medline]
  10. Plagemann A, Harder T, Kohlhoff R, Rhode W, Dorner G 1997 Overweight and obesity in infants of mothers with long-term insulin-dependent diabetes or gestational diabetes. Int J Obes Rel Metabol Disord 21:451–456[CrossRef][Medline]
  11. Whitaker RC, Dietz WH 1990 Role of the prenatal environment in the development of obesity. J Pediatr 132:768–776
  12. Silverman BL, Rizzo T, Green OC, Cho NH, Winter RJ, Ogata ES, Richards GE, Metzger BE 1991 Long-term prospective evaluation of offspring of diabetic mothers. Diabetes 40(Suppl 2):121–125
  13. Parsons TJ, Power C, Manor O 2001 Fetal and early life growth and body mass index from birth to early adulthood in 1958 British cohort: longitudinal study. Br Med J 323:1331–1335[Abstract/Free Full Text]
  14. Lahlou N, Landais P, De Boissieu D, Bougneres PF 1997 Circulating leptin in normal children and during the dynamic phase of juvenile obesity: relation to body fatness, energy metabolism, caloric intake, and sexual dimorphism. Diabetes 46:989–983[Abstract]
  15. Yuen BSJ, McMillen IC, Symonds ME, Owens PC 1999 Abundance of leptin mRNA in fetal adipose tissue is related to fetal body weight. J Endocrinol 163:R11–R14
  16. Ehrhardt RA, Bell AW, Boisclair YR 2002 Spatial and developmental regulation of leptin in fetal sheep. Am J Physiol 282:R1628–R1635
  17. Devaskar SU, Anthony RV, Hay Jr WW 2002 Ontogeny and insulin regulation of fetal ovine white adipose tissue leptin expression. Am J Physiol 282:R431–R418
  18. Forhead AJ, Thomas L, Crabtree J, Hoggard N, Gardner DS, Giussani DA, Fowden AL 2002 Plasma leptin concentration in fetal sheep during late gestation: ontogeny and effect of glucocorticoids. Endocrinology 143:1166–1173[Abstract/Free Full Text]
  19. Muhlhausler BS, Roberts CT, McFarlane JR, Kauter KG, McMillen IC 2002 Fetal leptin is a signal of fat mass independent of maternal nutrition in ewes fed at or above maintenance energy requirements. Biol Reprod 67:493–499[Abstract/Free Full Text]
  20. Chen X, Lin J, Hausman DB, Martin RJ, Dean RG, Hausman GJ 2000 Alterations in fetal adipose tissue leptin expression correlate with the development of adipose tissue. Biol Neonate 78:41–47[CrossRef][Medline]
  21. Gemmell RT, Alexander G, Bell AW 1972 Morphology of adipose cells in lambs at birth and during subsequent transition of brown to white adipose cells in cold and in warm conditions. Am J Anat 133:143–163[CrossRef][Medline]
  22. Gemmell RT, Alexander G 1978 Ultrastructural development of adipose tissue in foetal sheep. Aust J Biol Sci 31:505–515[Medline]
  23. Yuen BSJ, Owens PC, Muhlhausler BS, Roberts CK, Symonds ME, Keisler DH, McFarlane JR, Kauter K, Evens Y, McMillen IC 2003 Leptin alters the structural and functional characteristics of adipose tissue before birth. FASEB J 17:1102–1104[Abstract/Free Full Text]
  24. Symonds ME, Stephenson T 1999 Maternal nutrition and endocrine programming of fetal adipose tissue development. Biochem Soc Trans 27:97–103[Medline]
  25. Cinti S 2001 The adipose organ: morphological perspectives of adipose tissues. Proc Nutr Soc 60:319–328[Medline]
  26. Lonnqvist F, Nordfors L, Jansson M, Thorne A, Schalling M, Arner P 1997 Leptin secretion from adipose tissue in women. J Clin Invest 99:2398–2404[Medline]
  27. Zhang Y, Guo K, Diaz PA, Heo M, Leibel RL 2001 Determinants of leptin gene expression in fat depots of lean mice. Am J Physiol 282:R226–R234
  28. Chilliard Y, Bonnet M, Delavaud C, Faulconnier Y, Leroux C, Djiane J, Bocquier F 2001 Leptin in ruminants. Gene expression in adipose tissue and mammary gland, and regulation of plasma concentration. Dom Anim Endocrinol 21:271–295[CrossRef][Medline]
  29. Edwards LJ, McMillen IC 2001 Maternal undernutrition increases arterial blood pressure in the sheep fetus during late gestation. J Physiol 533:561–570[Abstract/Free Full Text]
  30. Aldermann GA, Morgan DE, Harvard A, Edwards RE, Todd JR 1975 Energy allowances and feeding systems for ruminants. In: Gough HC, ed. Ministry of Agriculture, Fisheries and Food: technical bulletin 33. London: Her Majesty’s Stationery Office; 1–79
  31. Fowden A 1995 Nutrient requirements for normal fetal growth and metabolism. In: Hanson M, Spencer J, Rodeck C, eds. Fetus and neonate: physiology and clinical applications. 1st ed. Cambridge: Cambridge University Press; 31–56
  32. Edwards LJ, Symonds ME, Warnes KE, Owens JA, Butler TG, Jurisevic A, McMillen IC 2001 Responses of the fetal pituitary adrenal axis to acute and chronic hypoglycemia during late gestation in the sheep. Endocrinology 142:1778–1785[Abstract/Free Full Text]
  33. Kauter K, Ball M, Kearney P, Tellam R, McFarlane JR 2000 Adrenaline, insulin and glucagon do not have acute effects on plasma leptin levels in sheep: development and characterisation of an ovine leptin ELISA. J Endocrinol 166:127–135[Abstract]
  34. Yuen BSJ, Owens PC, McFarlane JR, Symonds ME, Kauter K, Edwards LJ, McMillen IC 2002 Circulating leptin concentrations are positively related to leptin messenger RNA expression in the adipose tissue of fetal sheep in the pregnant ewe fed at or below maintenance energy requirements during late gestation. Biol Reprod 67:911–916[Abstract/Free Full Text]
  35. Bouillaud F, Weissenbach J, Riquier D 1986 Complete cDNA-derived amino acid sequence of rat brown uncoupling proteins. J Biochem 261:1487–1490
  36. Schermer SJ, Bird JA, Lomax MA, Sheperd DA, Symonds ME 1996 Effect of fetal thyroidectomy on brown adipose tissue and thermoregulation in newborn lambs. Reprod Fertil Dev 8:995–1002[CrossRef][Medline]
  37. Chan YL, Gutell R, Noller HF, Wool IG 1984 The nucleotide sequence of a rat 18 S ribosomal ribonucleic acid gene and a proposal for the secondary structure of 18 S ribosomal ribonucleic acid. J Biol Chem 259:224–230[Abstract/Free Full Text]
  38. Adams MB, Ross JT, Butler TG, McMillen IC 1999 Glucocorticoids decrease phenylethanolamine N-methyltransferase mRNA expression in the immature foetal sheep adrenal. J Neuroendocrinol 11:569–575[CrossRef][Medline]
  39. Roberts CT, Sohlstrom A, Kind KL, Earl RA, Khong TY, Robinson JS, Owens PC, Owens JA 2001 Maternal food restriction reduces the exchange surface area and increases the barrier thickness of the placenta in the guinea-pig. Placenta 22:177–185[CrossRef][Medline]
  40. Stevens D, Alexander G, Bell AW 1990 Effect of prolonged glucose infusion into fetal sheep on body growth, fat deposition and gestation length. J Dev Physiol 13:277–281[Medline]
  41. Das UG, Schroeder RE, Hay Jr WW, Devaskar SU 1999 Time-dependent and tissue-specific effects of circulating glucose on fetal ovine glucose transporters. Am J Physiol 276:R809–R817
  42. Anderson MS, Flowers-Ziegler J, Das UG, Hay Jr WW, Devaskar SU 2001 Glucose transporter protein responses to selective hyperglycemia or hyperinsulinemia in fetal sheep. Am J Physiol 281:R1545–R1552
  43. Valverde AM, Lorenzo M, Navarro P, Benito M 1997 Phosphatidylinositol 3-kinase is a requirement for insulin-like growth factor I-induced differentiation, but not for mitogenesis, in fetal brown adipocytes. Mol Endocrinol 11:595–607[Abstract/Free Full Text]
  44. Hernandez A, Tereul T, Lorenzo M 2003 Insulin and dexamethasone induce GLUT4 gene expression in foetal brown adipocytes: synergistic effect through CCAAT/enhancer binding protein {alpha}. Biochem J 372:617–624[CrossRef][Medline]
  45. Saladin R, De Vos P, Guerre-Millo M, Leturque A, Girard J, Staels B, Auwerx J 1995 Transient increase in obese gene expression after food intake or insulin administration. Nature 377:527–529[CrossRef][Medline]
  46. Sivitz WI, Bailey HL, Donohoue P 1996 Rat adipose ob mRNA levels in states of altered circulating glucose and insulin. Biochem Biophys Res Commun 220:520–525[CrossRef][Medline]
  47. MacDougald OA, Hwang CS, Fan H, Lane MD 1995 Regulated expression of the obese gene product (leptin) in white adipose tissue and 3T3–L1 adipocytes. Proc Natl Acad Sci USA 92:9034–9037[Abstract/Free Full Text]
  48. Margareto J, Marti A, Martinez JA 2001 Changes in uncoupling protein 1 mRNA expression levels in brown adipose tissue and skeletal muscle after feeding a high-energy diet and relationships with leptin, glucose and PPAR{gamma}. J Nutr Biochem 12:130–137[CrossRef][Medline]
  49. Nicholls DG, Locke RM 1984 Thermogenic mechanisms in brown fat. Physiol Rev 64:1–64[Free Full Text]
  50. Budge H, Bispham J, Dandrea J, Evans E, Heasman L, Ingleton PM, Sullivan C, Wilson V, Stephenson T, Symonds ME 2000 Effect of maternal nutrition on brown adipose tissue and its prolactin receptor status in the fetal lamb. Pediatr Res 47:781–786[Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
D. M. O'Connor, D. Blache, N. Hoggard, E. Brookes, F. B. P. Wooding, A. L. Fowden, and A. J. Forhead
Developmental Control of Plasma Leptin and Adipose Leptin Messenger Ribonucleic Acid in the Ovine Fetus during Late Gestation: Role of Glucocorticoids and Thyroid Hormones
Endocrinology, August 1, 2007; 148(8): 3750 - 3757.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
B. S. Muhlhausler, J. A. Duffield, and I. C. McMillen
Increased Maternal Nutrition Stimulates Peroxisome Proliferator Activated Receptor-{gamma}, Adiponectin, and Leptin Messenger Ribonucleic Acid Expression in Adipose Tissue before Birth
Endocrinology, February 1, 2007; 148(2): 878 - 885.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
I C McMillen, L J Edwards, J Duffield, and B S Muhlhausler
Regulation of leptin synthesis and secretion before birth: implications for the early programming of adult obesity.
Reproduction, March 1, 2006; 131(3): 415 - 427.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. Bispham, D. S. Gardner, M. G. Gnanalingham, T. Stephenson, M. E. Symonds, and H. Budge
Maternal Nutritional Programming of Fetal Adipose Tissue Development: Differential Effects on Messenger Ribonucleic Acid Abundance for Uncoupling Proteins and Peroxisome Proliferator-Activated and Prolactin Receptors
Endocrinology, September 1, 2005; 146(9): 3943 - 3949.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
I. C. McMillen, C. L. Adam, and B. S. Muhlhausler
Early origins of obesity: programming the appetite regulatory system
J. Physiol., May 15, 2005; 565(1): 9 - 17.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
B. S. Muhlhausler, C. L. Adam, E. M. Marrocco, P. A. Findlay, C. T. Roberts, J. R. McFarlane, K. G. Kauter, and I. C. McMillen
Impact of glucose infusion on the structural and functional characteristics of adipose tissue and on hypothalamic gene expression for appetite regulatory neuropeptides in the sheep fetus during late gestation
J. Physiol., May 15, 2005; 565(1): 185 - 195.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
I. C. Mcmillen and J. S. Robinson
Developmental Origins of the Metabolic Syndrome: Prediction, Plasticity, and Programming
Physiol Rev, April 1, 2005; 85(2): 571 - 633.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
L. J. Edwards, J. R. McFarlane, K. G. Kauter, and I. C. McMillen
Impact of periconceptional nutrition on maternal and fetal leptin and fetal adiposity in singleton and twin pregnancies
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2005; 288(1): R39 - R45.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
K. L. Kind, C. T. Roberts, A. I. Sohlstrom, A. Katsman, P. M. Clifton, J. S. Robinson, and J. A. Owens
Chronic maternal feed restriction impairs growth but increases adiposity of the fetal guinea pig
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2005; 288(1): R119 - R126.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
144/11/4947    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mühlhäusler, B. S.
Right arrow Articles by McMillen, I. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mühlhäusler, B. S.
Right arrow Articles by McMillen, I. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH