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Endocrinology Vol. 148, No. 2 878-885
Copyright © 2007 by The Endocrine Society

Increased Maternal Nutrition Stimulates Peroxisome Proliferator Activated Receptor-{gamma}, Adiponectin, and Leptin Messenger Ribonucleic Acid Expression in Adipose Tissue before Birth

B. S. Muhlhausler, J. A. Duffield and I. C. McMillen

Early Origins of Adult Health Research Group (B.S.M., I.C.M.), The Sansom Research Institute, School of Pharmacy and Medical Science, University of South Australia, Adelaide 5000, South Australia, Australia; and the School of Molecular and Biomedical Science (B.S.M., J.A.D., I.C.M.), The University of Adelaide, Adelaide 5005, South Australia, Australia

Address all correspondence and requests for reprints to: Professor I. C. McMillen, Sansom Research Institute, University of South Australia, Adelaide 5000, South Australia, Australia. E-mail: Caroline.McMillen{at}unisa.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During fetal life, adipose tissue is predominantly comprised of brown or thermogenic adipocytes and there is a transition to white, lipid-storing adipocytes after birth concomitant with the onset of suckling. In pregnancies complicated by gestational diabetes, the fetus is hyperglycemic, has an increased fat mass, and is at increased risk of obesity in later life. In the present study, we have investigated the hypothesis that exposure to increased maternal nutrition during late gestation results in increased expression of genes that regulate adipogenesis and lipogenesis in perirenal fat in fetal sheep. Pregnant ewes were fed either at or approximately 55% above maintenance energy requirements during late pregnancy and quantitative RT-PCR was used to measure peroxisome proliferator-activated receptor {gamma}, lipoprotein lipase, glycerol-3-phosphate dehydrogenase, adiponectin, and leptin mRNA expression. We report that exposure to metabolic and hormonal signals of increased nutrition before birth results in an increase in the expression of the adipogenic factor, peroxisome proliferator-activated receptor {gamma}, and in lipoprotein lipase, adiponectin, and leptin mRNA expression in fetal perirenal fat. We propose that an increase in maternal, and hence fetal, nutrition results in a precocial increase in adipogenic, lipogenic, and adipokine gene expression in adipose tissue and that these changes may be important in the development of obesity in later life.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN PREGNANCIES COMPLICATED by maternal diabetes, the fetus is hyperglycemic and hyperinsulinemic, and cord blood leptin concentrations increase in parallel with increased infant adiposity (1, 2). In addition, maternal diabetes mellitus, gestational diabetes, or even mild impairments of glucose tolerance during pregnancy are risk factors for the development of obesity and type 2 diabetes in the offspring (3, 4). It has therefore been proposed that exposure to excess fetal glucose during critical windows of development may result in changes in the biology of the adipocyte, which results in increased adiposity in adult life (5, 6). In studies in sheep, in which fat is deposited before birth, like in humans, we have also demonstrated that intrafetal infusion of glucose for periods of up to 10 d results in a significant increase in the mean size of the lipid locules present in the perirenal adipose tissue, the major fat depot present in the fetal lamb (7). Increasing maternal nutrition in late pregnancy also results in increased fetal glucose concentrations and the emergence of a relationship between circulating glucose concentrations and the size of the lipid locules in the fetal perirenal adipose tissue (8). Interestingly, lambs born to ewes that received increased nutrition during late pregnancy also had a higher relative subcutaneous fat mass when compared with control lambs at 1 month of age (9).

Before birth, most of the visceral fat depots are comprised of brown adipose tissue, which is characterized by an abundance of mitochondria and high levels of expression of the uncoupling protein, UCP-1. After birth, exposure to the relatively increased nutritional environment of the neonate results in a transition from the brown or thermogenic type of adipose tissue to the white or lipid storage form (10, 11, 12). One possibility is that exposure to increased maternal nutrition results in an increase in the expression of genes that regulate adipogenesis and lipogenesis within the major fetal adipose tissue depots, which may then result in accelerated or premature transition of adipose depots from a predominantly thermogenic to a lipid storage function. CCAAT/enhancer-binding protein {alpha} and peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) serve as pleiotropic transcriptional activators that coordinately induce expression of a suite of adipocyte specific genes resulting in the differentiation of adipose cells (13). PPAR{gamma} also regulates adipose tissue mass through stimulation of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (G3PDH) and is also involved in the regulation of the adipokines, including leptin and the insulin-sensitizing hormone, adiponectin (14). In human neonates, plasma concentrations of adiponectin are significantly higher than in adults and are positively correlated with neonatal adiposity, and it has been suggested that adiponectin may have a role in promoting fat deposition in early postnatal life (15).

In the present study, we have investigated the hypothesis that an increase in maternal nutrition results in an increased expression of adipogenic (PPAR{gamma}), lipogenic (LPL and G3PDH), and adipokine (adiponectin and leptin) genes in fetal perirenal adipose tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and surgery
All procedures were approved by the Adelaide University Animal Ethics Committee. Singleton pregnancies were confirmed in 14 adult Merino ewes by ultrasound scanning in early gestation. Surgery was then performed on these ewes between 103 and 113 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, Queensland, Australia) and maintained with 2.5–4% halothane (Fluothane; ICI, Melbourne, Victoria, 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 (16). During surgery, im injections of antibiotics (2 ml procaine penicillin 250 mg/ml, dihydrostreptomycin 250 mg/ml, procaine hydrochloride 20 mg/ml; Lyppards, Adelaide, South Australia, Australia; or 0.1 ml/kg Terramycin 100, 100 mg/ml oxytetracycline hydrochloride; Pfizer, New South Wales, Australia) were administered to each ewe and fetus. All catheters were filled with heparinized saline and the fetal catheters 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 regime
Each ewe was weighed on admission to the animal housing facility (103–106 d gestation) and was 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, South Australia, Australia) (17). 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 (18).

At 115 d gestation, ewes were randomly assigned to either a control (n = 6) or well-fed group (n = 8). Maternal weight at 103–106 d gestation was not different between the control and well-fed groups (control group, 52.3 ± 1.6 kg, well-fed group, 51.7 ± 2.4 kg). Between 115 and 124 d gestation, control ewes were provided with 19.0 ± 1.1 g of Lucerne chaff and 4.7 ± 0.3 g of pelleted concentrate per 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 per kg 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 approximately 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 (18).

Blood sampling regime, postmortem, 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 g for 10 min at 4 C and plasma stored at –20 C for subsequent determination of glucose, insulin, and leptin concentrations. 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. Between 139 and 141 d gestation, all ewes were killed with an overdose of sodium pentobarbitone (Virbac Pty Ltd., Peakhurst, New South Wales, Australia). All fetuses 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. The perirenal adipose depot (PAT) makes up between 85% and 95% of total fetal fat mass at 139–141 d gestation (8) and we have previously reported that maternal overnutrition does not result in an increase in either the total or relative mass of perirenal fat in well-fed ewes or in the morphometric characteristics of fetal perirenal fat (see Fig. 1Go for representative micrographs of perirenal fat from control and well-fed animals) (8). A sample of perirenal adipose tissue was frozen in liquid N2 and stored at –80 C.


Figure 1
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  		FIG. 1. Representative micrographs showing the histological features of perirenal adipose tissue in control and well-fed fetuses at 139–141 d gestation. Scale bar, 50 µm.

 
Glucose and insulin assays
Maternal and fetal plasma glucose concentrations were determined by enzymatic analysis using the COBAS MIRA automated analysis system (Roche Diagnostica, Basel, Switzerland) previously validated for sheep plasma (16). The intra- and interassay coefficients of variation were both less than 5%. Insulin concentrations were determined in fetal plasma samples using a RIA kit (Phadaseph RIA kit; Pharmacia & Upjohn, Uppsala, Sweden) previously validated for sheep plasma (16). 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 (19), 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
RNA from fetal PAT was isolated using Trizol reagent (Invitrogen Australia Pty Ltd., Mount Waverley, Australia) and chloroform. RNA was treated for genomic DNA contamination using Ambion deoxyribonuclease I (DNase I) and after enzyme deactivation, the RNA was run through a secondary purification process using the RNeasy Mini Kit (QIAGEN Pty Ltd. Australia, Doncaster, Australia). The quality and concentration of the RNA was determined by measuring the absorbance at 260 and 280 nm, and RNA integrity was confirmed by agarose gel electrophoresis. cDNA was then synthesized using the purified RNA (~5 µg), Superscript 3 reverse transcriptase (Invitrogen Australia Pty Ltd.) and random hexamers.

Quantitative RT-PCR (qRT-PCR)
The relative expression of PPAR{gamma}, adiponectin, LPL, G3PDH, and leptin mRNA transcripts were determined by qRT-PCR using the Sybr Green system in an ABI Prism 7300 Sequence Detection System (PE Applied Biosystems, Foster City, CA). Primers were designed with the aid of Primer Express (PE Applied Biosystems) software and, when possible, one primer of each pair was positioned over a splice site to prevent amplification of residual genomic DNA. For each transcript, RT-PCR was performed using the appropriate primers (Table 1Go), and to ensure the amplification of a single amplicon of the expected size, the product was viewed on an ethidium bromide stained electrophoresis gel. Each amplicon, which was designed to be approximately 200 bp in length, was sequenced to ensure the authenticity of the DNA product and qRT-PCR melt curve analysis was performed to demonstrate amplicon homogeneity. Controls containing no reverse transcriptase were also used. For the qRT-PCR measurements, the primer concentrations were consistent for all genes and the amplification efficiency of all primers was 0.997–0.999. A constant amount of cDNA (1 µl) was used for each qRT-PCR measurement, and at least three technical replicates were performed in duplicate for each gene.


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  		TABLE 1. Oligonucleotide primer sequences for acidic ribosomal protein large subunit P0 (RPLP0), PPAR{gamma}, leptin, adiponectin, LPL, and G3PDH

 
Each qRT-PCR well (10 µl total volume) contained 6 µl 2x Sybr Green Master Mix (PE Applied Biosystems), 1 µl of each primer giving a final concentration of 450 or 900 nM, 2.0 µl of molecular grade H2O, and 1.0 µl of a 50 ng/µl dilution of the stock template. The cycling conditions consisted of 40 cycles of 95 C for 15 min and 60 C for 1 min. At the end of each run, dissociation melt curves were obtained.

The abundance of each mRNA transcript was measured and expression relative to that of acidic ribosomal protein P0 (RPLP0) was calculated using Q-gene qRT-PCR analysis software, which provides a quantitative measure of the relative abundance of a specific transcript in different tissues by the comparative threshold cycle (Ct) method, which takes into account any differences in the amplification efficiencies of the target and reference genes. The Ct value was taken as the lowest statistically significant (>10 SD) increase in fluorescence above the background signal in an amplification reaction.

Statistical analyses
Data are presented as the mean ± SEM. The effect of increased maternal nutrition on mean fetal plasma glucose, insulin, and leptin concentrations in the last 5 d before postmortem was determined by Student’s t test.

The effect of maternal overnutrition on expression of genes in fetal PAT was also determined using Student’s t test. Relationships among plasma glucose, insulin, and leptin concentrations and the relative expression of adipogenic, lipogenic, and adipokine genes in fetal PAT were determined using simple linear regression analysis. Stepwise linear regression was used to determine the strongest predictor of relative gene expression in analysis that included maternal and fetal body weight, fetal glucose, insulin, and leptin concentrations in late gestation. Partial correlation analysis was used to control for the effects of mean plasma glucose levels or PPAR{gamma} mRNA abundance when appropriate. 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 overnutrition and plasma glucose and insulin concentrations
Maternal plasma glucose concentrations in the last 5 d before postmortem were significantly increased in the well-fed ewes when compared with control ewes (well-fed group, 3.6 ± 0.3 mmol/liter; control group, 2.9 ± 0.1 mmol/liter; P < 0.02) (Fig. 2AGo). Fetal plasma glucose (well-fed group, 1.9 ± 0.11 mmol/liter; control group, 1.4 ± 0.14 mmol/liter; P < 0.02) and insulin (well-fed group, 8.8 ± 0.9 µU/ml; control group, 6.8 ± 0.4 µU/ml; P < 0.05) concentrations in the last 5 d before postmortem were also significantly higher in the well-fed group when compared with controls (Fig. 2Go, B and C).


Figure 2
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  		FIG. 2. Mean maternal plasma glucose concentrations (A) and fetal plasma glucose (B) and insulin (C) concentrations at 135–141 d gestation in the control (open histograms) and well-fed (closed histograms) groups. Asterisks (*) denote a significant effect of increased maternal nutrition (P < 0.05).

 
Maternal overnutrition and PPAR{gamma} and adiponectin mRNA expression in fetal PAT
The relative expression of both PPAR{gamma} and adiponectin mRNA in fetal PAT were higher in the well-fed group compared with controls (Figs. 3AGo and 4AGo). There was a positive relationship between PPAR{gamma} mRNA expression in fetal PAT and mean fetal plasma glucose concentrations during the 5 d before postmortem (PPAR{gamma} mRNA = 0.63 glucose – 0.9, r2 = 0.70, P < 0.001, n = 14) (Fig. 3BGo), and this relationship persisted when the effects of plasma insulin were controlled for in the analysis. There was also a positive relationship between PPAR{gamma} expression in fetal PAT and fetal insulin concentrations; however, this relationship was no longer significant when the effect of fetal plasma glucose was controlled for in the analysis. Adiponectin mRNA expression was directly related to PPAR{gamma} mRNA expression in fetal PAT (adiponectin mRNA = 1.5 PPAR{gamma} mRNA + 0.30, r2 = 0.58, P < 0.005, n = 14) (Fig. 4BGo).


Figure 3
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  		FIG. 3. A, The relative expression of PPAR{gamma} mRNA in fetal perirenal fat in control (open histograms) and well-fed (closed histograms) groups at 139–141 d gestation. B, The relationship between PPAR{gamma} mRNA expression in fetal perirenal fat at 139–141 d gestation and plasma glucose concentrations in the 5 d before postmortem in control (open symbols) and well-fed (closed symbols) groups (PPAR{gamma} mRNA = 0.63 glucose – 0.9, r2 = 0.69, P < 0.001, n = 14). Asterisks (*) denote a significant effect of increased maternal nutrition (P < 0.05).

 

Figure 4
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  		FIG. 4. A, The relative expression of adiponectin mRNA in fetal perirenal fat in control (open histograms) and well-fed (closed histograms) groups at 139–141 d gestation. B, The relationship between adiponectin mRNA and PPAR{gamma} mRNA expression in fetal perirenal fat in control (open symbols) and well-fed (closed symbols) groups at 139–141 d gestation (adiponectin mRNA = 1.5 PPAR{gamma} mRNA + 0.30, r2 = 0.58, P < 0.005, n = 14). Asterisks (*) denote a significant effect of increased maternal nutrition (P < 0.05).

 
Maternal overnutrition and LPL and G3PDH expression
The relative expression of LPL mRNA, but not G3PDH mRNA, in fetal PAT was higher in the well-fed group compared with controls (Table 2Go). The relative expression of LPL and G3PDH mRNA in fetal PAT were each directly related to the expression of PPAR{gamma} mRNA (LPL mRNA = 1.7 PPAR{gamma} mRNA + 0.02, r2 = 0.51, P < 0.01, n = 14; G3PDH mRNA = 0.46 PPAR{gamma} mRNA + 0.07, r2 = 0.34, P < 0.05, n = 14) (Fig. 5Go, A and B). The relative expression of LPL and G3PDH mRNA in fetal PAT were also directly related to plasma glucose concentrations in the last 5 d before postmortem (LPL mRNA = 1.2 glucose – 0.32, r2 = 0.40, P < 0.02, n = 13; G3PDH mRNA = 0.37 glucose – 0.13, r2 = 0.36, P < 0.03, n = 13). These relationships did not persist, however, when the effects of PPAR{gamma} mRNA expression were controlled for in the analysis.


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  		TABLE 2. The normalized expression of G3PDH and LPL mRNA in perirenal adipose tissue at 139–141 d gestation in the control and well-fed groups

 

Figure 5
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  		FIG. 5. The relationship between (A) LPL mRNA and (B) G3PDH mRNA and PPAR{gamma} mRNA in fetal perirenal fat in control (open symbols) and well-fed (closed symbols) groups at 139–141 d gestation.

 
Maternal overnutrition and leptin mRNA
The expression of leptin mRNA in fetal PAT was significantly higher in the well-fed group compared with the control group (Fig. 6Go). There was a direct relationship between plasma insulin, but not glucose, concentrations during the last 5 d before tissue collection and leptin mRNA expression in PAT (leptin mRNA = 0.003 insulin + 0.008, r2 = 0.33, P < 0.04, n = 13). Circulating plasma leptin concentrations were not related to leptin mRNA expression, and there was also no difference in the mean fetal plasma concentrations of leptin between the well-fed and control groups in the last 5 d before postmortem (well-fed group, 4.3 ± 0.8 ng/ml; control group, 5.5 ± 0.4 ng/ml).


Figure 6
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  		FIG. 6. The relative expression of leptin mRNA in fetal perirenal fat in control (open histograms) and well-fed (closed histograms) groups at 139–141 d gestation. Asterisks (*) denote a significant effect of increased maternal nutrition (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated for the first time that exposure to an increase in maternal nutrition during late gestation results in an increase in the expression of genes that regulate adipogenesis and lipogenesis and the synthesis of adipokines in fetal adipose tissue. The expression of PPAR{gamma}, a key adipogenic and lipogenic factor in perirenal adipose tissue, was directly related to circulating fetal glucose concentrations in late gestation and the expression of LPL and G3PDH and the insulin-sensitizing adipokine, adiponectin, in perirenal fat were each directly related to expression of PPAR{gamma} mRNA in this adipose depot.

Maternal nutrition, fetal glucose, and PPAR{gamma} expression
Increasing maternal nutrition to approximately 155% of maintenance energy requirements resulted in a significant increase in maternal and fetal glucose concentrations and was associated with a significant increase in the expression of PPAR{gamma} mRNA in fetal perirenal adipose tissue. Importantly, the relative expression of PPAR{gamma} mRNA was directly related to glucose concentrations in the fetal plasma in late gestation, and this relationship persisted after controlling for the effects of plasma insulin. It would therefore appear that the expression of PPAR{gamma} in fetal adipose tissue is regulated by the prevailing concentration of glucose across the physiological range present in this study, i.e. between 1.0 and 2.6 mmol/liter. The demonstration that LPL and G3PDH mRNA levels are each directly related to PPAR{gamma} mRNA expression in fetal perirenal adipose tissue is consistent with studies in the adult rodent in which the activation of PPAR{gamma} promotes lipogenesis within mature adipocytes by increasing the expression of genes, which regulate the incorporation of circulating free fatty acids (LPL) or nutrient-derived factors (G3PDH) into adipose cells (14). Although the expression of both lipogenic factors was also directly related to fetal glucose concentrations, these relationships did not persist when the effects of PPAR{gamma} mRNA level were controlled for in the analysis. This would therefore suggest that the effects of glucose on expression of these lipogenic factors are mediated by the effect of glucose on expression of PPAR{gamma}.

In the adult rodent, an increase in abundance of PPAR{gamma} expression in adipose cells is associated with an increase in lipid locule size and increased fat mass (20). Unlike fat depots in the adult, which are made up almost exclusively of white adipose cells, fetal fat depots consist predominantly of brown adipocytes, which are multilocular and express high amounts of UCP-1 (7, 8, 10, 21, 22). After birth, and the onset of suckling in the lamb, the perirenal adipose tissue takes on the physical and biochemical properties of white adipose tissue during the first month of life (11, 12). The results of this study provide evidence that early exposure to an increase in maternal, and hence fetal, nutrition results in an up-regulation of PPAR{gamma} and an increase in abundance of LPL, adiponectin, and leptin mRNA in fetal perirenal fat.

We have recently found that PPAR{gamma} and adiponectin mRNA levels are higher in perirenal adipose tissue in lambs at 1 month after birth (PPAR{gamma}: 1.15 ± 0.12; adiponectin 2.80 ± 0.45; Muhlhausler, unpublished observations) when compared with the level of expression in fetal adipose tissue present in the current study. This supports the hypothesis that exposure to an elevated nutrient supply before birth may result in a precocial activation of the expression of these genes in the perirenal adipose tissue and an accelerated transition from a predominantly thermogenic to a lipid storage function in this adipose depot.

The lack of a significant increase in G3PDH in the fetal perirenal tissue of well-fed ewes may indicate that the fetal plasma glucose concentrations present in the well-nourished ewes were below the threshold level at which G3PDH expression may be up-regulated. Although plasma glucose concentrations were increased in the fetus of the well-fed ewes, they were lower, however, than the plasma glucose concentrations normally present in the lamb after birth (9). The absence of an increase in G3PDH expression in perirenal adipose tissue of fetuses may explain the lack of an increase in the relative mass of this fetal adipose depot, and it may be that exposure to either higher or more prolonged increases in fetal plasma nutrients are required to stimulate an increase in fetal fat mass (8). It has been demonstrated that lipogenic activity is increased in the fetal perirenal adipocytes of sows in which diabetes, and therefore fetal hyperglycemia, is induced in late pregnancy (23).

We have previously reported that exposure to maternal overnutrition does not result in a significant alteration in the morphometric properties of the perirenal adipose tissue, but we demonstrated that there is a direct relationship between glucose concentrations and the mean size of lipid locules in the perirenal adipose tissue in fetuses of ewes fed at or above maintenance energy requirements in late pregnancy, which provides evidence that glucose can increase lipogenesis in fat depots before birth (8). The results of the present study extend these findings and suggest that glucose increases expression of key lipogenic genes in fetal adipose tissue with potential consequences for adipose tissue accumulation after birth when the availability of substrates is no longer limiting. Interestingly, preliminary studies indicate that the level of G3PDH mRNA in perirenal adipose tissue at 1 month of postnatal age (0.57 ± 0.09; our unpublished observations) is higher than in fetal tissues.

In the present study, leptin mRNA expression in perirenal fat was increased in the fetuses of well-fed ewes, and this increase in leptin mRNA expression was directly related to fetal plasma insulin concentrations. This is consistent with previous results from our laboratory (8) and with the results of studies by Devaskar and colleagues, which have demonstrated that intrafetal infusion of insulin for 24 h results in an increase in leptin mRNA expression in the perirenal adipose tissue of the fetal sheep (24). Although the expression of leptin mRNA was increased in fetuses of well-fed ewes, there was no difference in circulating plasma leptin concentrations and plasma leptin concentrations were not related to leptin mRNA expression in perirenal fat. One possible explanation is that there are mechanisms present within the perirenal adipocyte, which act to limit fetal leptin secretion when nutritional supply is increased. The abundance of the leptin mRNA transcript in adipose tissue in fetal life has been shown to be lower than in postnatal adipose tissue (25), and it has been proposed that this is a consequence of the difference in lipid accumulation in adipose tissue depots before and after birth.

In the present study, we have found that adiponectin was expressed in the perirenal adipose depot before birth and that adiponectin mRNA expression in fetal adipose tissue was also directly related to the expression of PPAR{gamma} mRNA. These observations in the fetus are consistent with the stimulatory effects of PPAR{gamma} activation on adiponectin mRNA expression in white adipocytes in the adult rodent (26). Thus, it appears that PPAR{gamma} may act to up-regulate adiponectin expression before birth. In the adult rodent, adiponectin is secreted exclusively from adipose tissue and acts to increase peripheral insulin sensitivity and promote glucose uptake (27).

It has been demonstrated that overexpression of adiponectin in vitro results in accelerated adipocyte differentiation in preadipocytes and increased accumulation of lipid and higher rates of insulin-stimulated glucose uptake in fully differentiated adipocytes (28). Furthermore, plasma adiponectin concentrations are positively related to adiposity in human neonates (15). There is also substantial evidence from human studies that weight gain and food intake in the early neonatal period are each critical in determining patterns of food intake and fat deposition throughout infancy and later life (29, 30). One possibility is that an increased expression of adiponectin in adipose tissue may result in the increase in fat deposition present in the hyperglycemic fetus of the diabetic mother or in the hyperglycemic lamb of the well-fed ewe after birth. Interestingly, lambs of well-fed ewes develop an increase in subcutaneous, but not perirenal, adiposity at 1 month of age (9). Unlike the perirenal depot, which develops before birth, the subcutaneous depot develops predominantly in postnatal life in the lamb (21). One speculation, therefore, is that there may be an important relationship between patterns of gene expression in perirenal adipose tissue in fetal life and the subsequent development of subcutaneous adiposity in early postnatal life.

Summary
We have demonstrated for the first time that exposure to an increased nutrient supply during late fetal development results in an increased expression of genes that regulate adipogenesis and lipogenesis in fetal perirenal adipose tissue. The results of this study provide evidence that early exposure to an increase in maternal, and hence fetal, nutrition results in an up-regulation of PPAR{gamma} and an increase in abundance of LPL and adiponectin mRNA in fetal perirenal fat. These findings suggest that exposure to increased nutrition before birth results in a precocial increase in lipogenic capacity in adipose tissue (as a consequence of either a increase in adipogenesis or lipogenic gene expression), which may result in premature transition of adipose depots from a predominantly thermogenic to a lipid storage function. The results of the present study also provide evidence that PPAR{gamma} and the insulin-sensitizing hormone, adiponectin, may play an important role in the association between exposure to increased nutrition before birth and the programing of childhood and adult obesity. These findings provide new insights into the mechanisms through which exposure to increased nutrition before birth may result in an increased risk of child and adult obesity.


   Acknowledgments
 
The authors acknowledge Anne Jurisevic and Laura O’Carroll for expert assistance with animal surgery and maintenance. The sequences for the primers for real-time PCR experiments were generously supplied by Dr. Ross Tellam (Commonwealth Scientific and Industrial Research Organisation Livestock Industries, Queensland, Australia).


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

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 26, 2006

Abbreviations: G3PDH, Glycerol-3-phosphate dehydrogenase; LPL, lipoprotein lipase; ME, metabolizable energy; PAT, perirenal adipose depot; PPAR, peroxisome proliferator-activated receptor; qRT-PCR, quantitative RT-PCR; UCP, uncoupling protein.

Received August 14, 2006.

Accepted for publication October 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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