Increased uncoupling protein-2 mRNA abundance and glucocorticoid action in adipose tissue in the sheep fetus during late gestation is depend
1 Centre for Reproduction and Early Life, Institute of Clinical Research, University of Nottingham NG7 2UH, UK
2 Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK
Abstract
The endocrine regulation of uncoupling protein-2 (UCP2), an inner mitochondrial protein, in fetal adipose tissue remains unclear. The present study aimed to determine if fetal plasma cortisol and triiodothyronine (T3) influenced the mRNA abundance of UCP2, glucocorticoid receptor (GR) and 11hydroxysteroid dehydrogenase type 1 (11HSD1) and 2 (11HSD2) in fetal adipose tissue in the sheep during late gestation. Perirenal–abdominal adipose tissue was sampled from ovine fetuses to which either cortisol (2–3 mg kg–1 day–1) or saline was infused for 5 days up to 127–130 days gestation, or near term fetuses (i.e. 142–145 days gestation) that were either adrenalectomised (AX) or remained intact. Fetal plasma cortisol and T3 concentrations were higher in the cortisol infused animals and lower in AX fetuses compared with their corresponding control group, and increased with gestational age. UCP2 and GR mRNA abundance were significantly lower in AX fetuses compared with age-matched controls, and increased with gestational age and by cortisol infusion. Glucocorticoid action in fetal adipose tissue was augmented by AX and suppressed by cortisol infusion, the latter also preventing the gestational increase in 11HSD1 mRNA and decrease in 11HSD2 mRNA. When all treatment groups were combined, both fetal plasma cortisol and T3 concentrations were positively correlated with UCP2, GR and 11HSD2 mRNA abundance, but negatively correlated with 11HSD1 mRNA abundance. In conclusion, plasma cortisol and T3 are both required for the late gestation rise in UCP2 mRNA and differentially regulate glucocorticoid action in fetal adipose tissue in the sheep during late gestation.
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Introduction
Fetal fat possesses the dual characteristics of brown and white adipocytes (Devasker et al. 2002). In species such as humans and sheep that are born with a mature hypothalamic–pituitary–adrenal axis and are precocial thermoregulators, brown adipose tissue abundance is maximal around the time of birth (Casteilla et al. 1989; Clarke et al. 1997a) and is then not normally detectable after the postnatal period (Lean, 1989; Clarke et al. 1997b). This transition includes the acquisition of white adipose tissue characteristics, by proliferation and differentiation of preadipocytes and cell loss via apoptosis of preadipocytes and adipocytes, and possibly by other processes such as adipocyte dedifferentiation (Clarke et al. 1997b; Prins & O'Rahilly, 1997). Uncoupling protein (UCP)-2, a recently discovered inner mitochondrial protein (Fleury et al. 1997), has postulated roles in reactive oxygen species production (Negre-Salvayre et al. 1997; Kizaki et al. 2002) and apoptosis (Voehringer et al. 2000), as well as in energy regulation (Boss et al. 2000; Buemann et al. 2001). The abundance of UCP2 peaks around the time of birth (Gnanalingham et al. 2005b) at least in the ovine lung where its ontogeny is similar to that of the brown adipose tissue-specific UCP1. It has recently been shown that chronic fetal stress induced by umbilical cord occlusion results in a precocious rise in both UCP1 and 2 mRNA suggesting that both of these proteins may be important in promoting metabolic adaptation in the newborn (Gnanalingham et al. 2005a). The increase in fetal UCP1 near to term is dependent on an intact adrenal gland and the prepartum surge in fetal plasma cortisol and triiodothyronine (T3) (Mostyn et al. 2003), but this has yet to be established for UCP2.
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In fetal adipose tissue, glucocorticoid action is determined by its ability to both synthesize and inactivate cortisol, and by the expression of glucocorticoid receptor (GR, type 2) and isoforms of 11hydroxysteroid dehydrogenase (11HSD) (Whorwood et al. 2001). In this regard, 11HSD type 1 (11HSD1) behaves predominantly as an 11-oxoreductase, catalysing the conversion of inactive cortisone to active cortisol, thereby amplifying activation of intracellular GR, while 11HSD type 2 (11HSD2) behaves as an 11-dehydrogenase, catalysing the inactivation of cortisol to inert cortisone, and thereby maintains the specificity of the mineralocorticoid receptor for aldosterone (Bamberger et al. 1996; Stewart & Krozowski, 1999). However, the direct influence of fetal plasma cortisol and possibly T3 on local glucocorticoid action in fetal adipose tissue in preparation for extrauterine life remains to be determined.
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During the prepartum period, cortisol regulates the expression of a wide range of enzymes, receptors, hormones and binding proteins in fetal tissues, including GR and 11HSD isoforms in the fetal liver and placenta (Fowden et al. 1998). In several instances, the maturational effects of cortisol are mediated by the cortisol-dependent rise in fetal plasma T3 (Fowden et al. 1998). In humans, the UCP2 gene has putative glucocorticoid and thyroid hormone response elements (Tu et al. 1999) and is up regulated by T3 in adipose tissue in vitro and in vivo (Barbe et al. 2001). However, the role of cortisol in UCP2 expression and glucocorticoid action in fetal adipose tissue remains unknown. Hence, the aim of this study was to investigate the effects of cortisol and the concomitant rise in T3 on the expression of UCP2, GR and 11HSD isoforms in adipose tissue from the fetal sheep during late gestation, when endogenous cortisol levels rise and after experimental manipulation of fetal plasma cortisol concentration by exogenous infusion and fetal adrenalectomy. This was examined by determining the effect of (1) infusing cortisol into the fetus between 122 and 125 days gestation in order to increase fetal plasma cortisol to values observed near to term and (2) removing both adrenal glands (adrenalectomised, AX) thereby preventing the normal prepartum surges in cortisol and T3. Effects on fetal adipose tissue maturation were then compared with age-matched controls with respect to the abundance of UCP2, GR, 11HSD type 1 and 2 mRNA in perirenal–abdominal adipose tissue (which constitutes up to 80% of fetal adipose tissue).
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Methods
Animals, surgical and experimental procedures
A total of 27 Welsh Mountain sheep fetuses of known gestational age were used in this the study, A summary of the experimental protocol is given in Fig. 1 and full details of the animals, surgical and experimental procedures have been published previously (Mostyn et al. 2003). In brief, under halothane anaesthesia (1.5% in O2–N2O), one of the following procedures were carried out between 115 and 118 days gestation (term 145 days) using the surgical methods previously described (Fowden et al. 1996). Through a midline incision in the ewe the uterus was exteriorized and catheters were inserted into the caudal aorta and vena cava of the fetus via the tarsal vessels (n = 15) or both fetal adrenals were removed (n = 6). Six additional age-matched intact unoperated fetuses acted as controls for the adrenalectomized fetuses.
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AT, adipose tissue; G, days of gestation (term 145 days); AX, adrenalectomised; C/S, Caesarean section.
The study aimed to determine the effect of (1) a precocious rise in fetal cortisol and (2) a prolonged reduction in fetal cortisol. The first objective was achieved by comparing the effect of a 5-day period of cortisol infusion (n = 8, 2–3 mg kg–1 day–1 in 3 ml 0.9% (w/v) saline (EF-Cortelan; Glaxo Ltd, Greenford, Middlesex, UK)) with 5-day saline infusion (n = 7, 3 ml day–1, 0.9% w/v), before tissue collection at 127–130 days gestation. This dose of cortisol was sufficient to achieve plasma cortisol concentrations similar to those observed close to term, i.e. 40–50 ng ml–1 (Fowden et al. 1996). The second objective was achieved by bilateral adrenalectomy which prevented the prepartum rise in fetal cortisol and T3 and then comparing adipose tissue composition from these animals with non-operated age matched controls (n = 6) near to term, i.e. 142–145 days gestation. We have previously shown no difference in adipose tissue composition in neonatal lambs between those which have undergone a sham operation and those not subjected to this procedure (Schermer et al. 1996). Blood samples of 2 ml were taken daily throughout the experimental period from all the catheterized fetuses to determine plasma cortisol and thyroid hormone concentrations and to monitor fetal well-being using blood pH, PO2and glucose levels (ABL5 Radiometer and YSI glucose analyser). The distribution of male and female fetuses between treatment groups was saline 3: 4, cortisol 3: 5, Ax 3: 3 and sham 2: 4 and there was no effect of sex on any of the measurements made. All procedures were carried out under the UK Animals (Scientific Procedures) Act 1986 and approved by the local ethical review board.
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Sample collection
All fetuses were delivered by Caesarean section under sodium pentobarbitone anaesthesia (20 mg kg–1 I.V.) between 127 and 130 days gestation for cortisol- and saline-infused fetuses and between 142 and 145 days for AX and intact control fetuses. Fetal blood samples were taken at the time of delivery either through the indwelling arterial catheter (saline and cortisol infused fetuses) or by venipuncture from the umbilical artery in the cord (adrenalectomised fetuses and age matched controls). After administration of a lethal dose of anaesthetic (200 mg kg–1 sodium pentobarbitone), samples of perirenal adipose tissue were collected and frozen rapidly in liquid nitrogen before storage at –80°C. All blood samples were centrifuged immediately at 4°C and the plasma stored at –20°C before plasma analyses. No adrenal remnants were found in any of the AX fetuses at autopsy.
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Laboratory analyses
Messenger RNA detection. Total RNA was isolated from fetal adipose tissue using Tri-Reagent (Sigma, Poole, UK). In order to maximize sensitivity, a two-tube approach to reverse transcription (RT) was adopted. The conditions used to generate first strand cDNA RT were: 70°C (5 min), 4°C (5 min), 25°C (5 min), 25°C (10 min), 42°C (1 h), 72°C (10 min), 4°C (5 min). The RT reaction (final volume 20 μl) contained: 5 x cDNA (first-strand), buffer (250 mM Tris-HCl, 40 mM MgCl2, 150 mM KCl, 5 mM dithioerythritol pH 8.5), 2 mM dNTPs, 1 x hexanucleotide mix, 10 units RNase inhibitor, 10 units M-MLV reverse transcriptase and 1 μg total RNA. All of these commercially available products were purchased from Roche Diagnostics Ltd (Lewes, UK).
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The expression of UCP2, GR (type 2), and 11HSD type 1 and 2 mRNA were determined by reverse transcriptase–polymerase chain reaction (RT-PCR), as previously described by (Gnanalingham et al. 2005b). The analysis used oligonucleotide cDNA primers to UCP2, GR (type 2), and 11HSD type 1 and 2 genes generating specific intron spanning products (Table 1). Briefly, the PCR programme consisted of an initial denaturation (95°C (15 min)), amplification (stage I, 94°C (30 s); stage II, annealing temperature (30 s); stage III, 72°C (60 s)) and final extension (72°C (7 min); 8°C ‘hold’). The PCR mixture (final volume 20 μl) contained 7 μl diethylpyrocarbonate (DEPC) H2O, 10 μl Thermo-Start PCR Master Mix (50 μl contains 1.25 units Thermo-Start DNA Polymerase, 1 x Thermo-Start reaction buffer, 1.5 mM MgCl2 and 0.2 mM each of dATP, dCTP, dGTP and dTTP; ABgene, Epsom, UK, cat. no. AB-0938-DC-15), 1 μl Forward Primer, 1 μl Reverse Primer and 1 μl RT (cDNA) product. The annealing temperature and cycle numbers of all primers were optimized so as to be in the linear range (see Table 1). Agrose gel electrophoresis (2.0–2.5%) and ethidium bromide staining confirmed the presence of both the product and 18S at the expected sizes. Densitometric analysis was performed on each gel by image detection using a Fujifilm LAS-1000 cooled charge-coupled device camera and UCP2, GR, 11HSD type 1 and 2 and 18S mRNA abundance determined. Consistency of lane loading for each sample was verified and all results expressed as a ratio of a reference sample to r18S abundance. All analyses and gels were conducted in duplicate, with appropriate positive and negative controls, and a range of molecular weight markers. The resultant PCR product was extracted (QIAquick gel extraction kit, Qiagen, West Sussex, UK, cat. no. 28704), sequenced and results cross-referenced against the GenBank website to determine specificity of the target gene. In this present study, the abundance of UCP1 mRNA and protein in perirenal adipose tissue in the fetuses were determined previously, as described by Mostyn et al. (2003).
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Radioimmunoassays. The effect of cortisol infusion and bilateral adrenalectomy on plasma cortisol, T3 and UCP1 in the sheep fetus during late gestation have been previously published (Mostyn et al. 2003), but brief details are included as the same fetuses were used in both the previous and current study and to enable full interpretation of significant correlations outlined below. Cortisol concentrations were measured by radioimmunoassay and validated for use with ovine plasma (Robinson et al. 1983). The minimum detectable quantity of cortisol was 1.5 ng ml–1 and interassay coefficient of variation was 11%. Total plasma T3 concentrations were also measured by radioimmunoassay, using a commercial kit (ICN Biomedicals, Thame, Oxon, UK) validated for ovine plasma (Forhead et al. 2003). The lower unit of detection was 0.1 ng ml–1 and interassay coefficient of variation was 10%.
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Statistical analyses
All data are presented as means ± S.E.M. Significant differences (P < 0.05) between mean values obtained between each treatment group were carried out using Mann–Whitney U tests. Significant correlations between fetal plasma hormone concentrations and molecular parameters measured in the study were analysed by Spearman's rank order test using SPSS v11.0 (SPSS Inc., Chicago, IL, USA). Partial correlation analysis was undertaken when there was more than one variable correlated with the measured molecular parameter, to determine which variable had the greatest effect.
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Results
Effect of cortisol infusion and adrenalectomy on plasma cortisol, T3 and UCP1
Fetal plasma cortisol and T3 concentrations were higher in the cortisol infused animals (cortisol: saline, 14.0 ± 2.3; cortisol, 45.7 ± 5.3 ng ml–1 (P < 0.01); T3: saline, 0.28 ± 0.04; cortisol, 0.57 ± 0.16 ng ml–1 (P < 0.01)) and lower in AX fetuses (cortisol: intact, 49.5 ± 12.3; AX, 7.2 ± 0.6 ng ml–1 (P < 0.01); T3: intact, 0.49 ± 0.07; AX, 0.29 ± 0.04 ng ml–1 (P < 0.05)) compared with their corresponding control group, and increased (P < 0.05) with gestational age (130 days gestation: cortisol: 14.0 ± 2.3; T3: 0.28 ± 0.04; 145 days gestation: cortisol: 49.5 ± 12.3; T3: 0.49 ± 0.07 ng ml–1 (P < 0.05). UCP1 protein abundance was significantly lower in AX fetuses compared with age-matched controls (intact, 38.5 ± 7.3; AX, 12.1 ± 2.7% of reference (P < 0.01)), and was increased by cortisol infusion and with gestational age (saline, 22.1 ± 3.7; cortisol, 52.1 ± 4.8% of reference (P < 0.05)), in contrast to UCP1 mRNA that was not statistically altered by cortisol status (results not shown). Fetal weight was not significantly different between sex or two treatment groups at each gestational age (e.g. saline: males, 3.19 ± 0.28; females, 3.29 ± 0.25; cortisol: males, 3.17 ± 0.27; females, 2.73 ± 0.22 kg). Total perirena–abdominal adipose tissue weights were not recorded.
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Effect of fetal cortisol infusion and adrenalectomy on UCP2, GR, and 11HSD type 1 and 2 mRNA abundance in fetal adipose tissue
There was a gestational increase (P < 0.01) in the abundance of UCP2, GR and 11HSD1 mRNA and a decrease (P = 0.01) in the abundance of 11HSD2 mRNA in fetal adipose tissue (Figs 2 and 3). Fetal cortisol infusion increased (P < 0.05) UCP2 mRNA abundance compared to age-matched controls and to a level similar to that of late gestation controls at 142–145 days. While fetal cortisol infusion increased GR (P < 0.05) and 11HSD2 (P < 0.01) mRNA abundance compared to age-matched controls, but to a level lower (P < 0.01) than late gestation controls. In contrast, fetal cortisol infusion decreased (P < 0.05) 11HSD1 mRNA abundance compared to age-matched controls and to a level higher (P < 0.01) than late gestation controls. Bilateral AX decreased (P < 0.05) the prepartum rise in UCP2 and GR mRNA abundance near term, resulting in comparable abundance to saline infused control animals at 127–130 days gestation (UCP2: saline, 19.5 ± 4.1; AX, 28.1 ± 3.8% 18S/reference (NS); GCR: saline, 32.6 ± 3.8; AX, 44.4 ± 3.0% 18S/reference (NS)). In contrast, bilateral AX augmented the prepartum rise (P < 0.05) in 11HSD1 mRNA and diminished (P < 0.01) in 11HSD2 mRNA abundance near term, resulting in significantly different abundance compared to saline infused control animals at 127–130 days gestation (11HSD1: saline, 11.5 ± 1.8; AX, 34.4 ± 2.7% 18S/reference (P < 0.001); 11HSD2: saline, 65.1 ± 3.7; AX, 18.9 ± 3.2% 18S/reference (P < 0.001)). When all treatment groups were combined, UCP2 mRNA was positively (P < 0.001) correlated to GR mRNA abundance in fetal adipose tissue, and 11HSD1 mRNA was negatively (P < 0.001) correlated to 11HSD2 mRNA abundance (Fig. 4).
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Effect on uncoupling protein-2 (UCP2) mRNA (A) and glucocorticoid receptor (GR) mRNA (B) in fetal perirenal-abdominal adipose tissue collected at 127–130 days gestation (term 145 days) following cortisol (C, 2–3 mg kg–1 day–1 for 5 days) or saline (S) infusion, and at 142–145 days gestation following bilateral adrenalectomy (AX, 115–118 days gestation) or intact (I) controls. Examples of charge-coupled device camera images of gene mRNA expression are given. Values are means with their standard errors (n = 5–6 per group). G = mean gestational age. *P < 0.05, **P < 0.01, mean value significantly different from respective fetal control, and between 127 and 130 and 142–145 days gestational age.
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Effect on 11hydroxysteroid dehydrogenase type 1 (11HSD1) mRNA (A) and 11hydroxysteroid dehydrogenase type 2 (11HSD2) mRNA (B) in fetal perirenal–abdominal adipose tissue collected at 127–130 days gestation (term 145 days) following cortisol (C, 2–3 mg kg–1 day–1 for 5 days) or saline (S) infusion, and at 142–145 days gestation following bilateral adrenalectomy (AX, 115–118 days gestation) or intact (I) controls. Examples of charge-coupled device camera images of gene mRNA expression are given. Values are means with their standard errors (n = 5–6 per group). G = mean gestational age. *P < 0.05, **P < 0.01, mean value significantly different from respective fetal control, and between 127 and 130 and 142–145 days gestational age.
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A–B are for perirenal–abdominal adipose tissue sampled from all fetuses (n = 22) irrespective of treatment or gestational age in the sheep during late gestation. A, relationship between uncoupling protein-2 (UCP2) mRNA and glucocorticoid receptor (GR) mRNA abundance (r2 = 0.43, P < 0.001, where y = 0.53x + 6.65). B, relationship between 11hydroxysteroid dehydrogenase type 1 (11HSD1) mRNA and 11hydroxysteroid dehydrogenase type 2 (11HSD2) mRNA abundance (r2 = 0.84, P < 0.001, where y = –0.37x + 38.70).
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Since UCP2 mRNA was elevated when fetal plasma cortisol and T3 levels were raised and was low when cortisol and T3 were low, the relationship between UCP2 mRNA abundance in fetal adipose tissue and the plasma concentrations of these hormones were examined in more detail. When individual results from all fetuses were combined regardless of gestational age or treatment, there was a significant positive correlation between UCP2 mRNA abundance in fetal adipose tissue and both the plasma concentrations of cortisol and T3 (Figs 5 and 6), which were positively correlated to each other (r2 = 0.595, P = 0.001) (Mostyn et al. 2003). However, partial correlation analysis revealed that neither fetal plasma cortisol nor T3 concentrations were significant independent determinants of UCP2 mRNA expression, suggesting that concomitant increments in both cortisol and T3 are required to up regulate expression of this gene in fetal adipose tissue.
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A–D are for perirenal–abdominal adipose tissue sampled from all fetuses (n = 22) irrespective of treatment or gestational age in the sheep during late gestation. A, relationship between uncoupling protein-2 (UCP2) mRNA and fetal plasma cortisol concentration (r2 = 0.44, P = 0.001, where y = 0.49x + 20.08). B, relationship between UCP2 mRNA and fetal plasma triiodothyronine (T3) concentration (r2 = 0.42, P = 0.001, where y = 28.73x + 19.01). C, relationship between glucocorticoid receptor (GR) mRNA and fetal plasma cortisol concentration (r2 = 0.34, P = 0.006, where y = 0.54x +36.04). D, relationship between GR mRNA and fetal plasma triiodothyronine (T3) concentration (r2 = 0.20, P = 0.024, where y = 20.03x + 39.65).
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A–D are for perirenal-abdominal adipose tissue sampled from all fetuses (n = 22) irrespective of treatment or gestational age in the sheep during late gestation. A, relationship between 11hydroxysteroid dehydrogenase type 1 (11HSD1) mRNA and fetal plasma cortisol concentration (r2 = 0.22, P = 0.013, where y = –0.31x + 26.09). B, relationship between 11HSD1 mRNA and fetal plasma triiodothyronine (T3) concentration (r2 = 0.28, P = 0.021, where y = –20.83x + 27.78). C, relationship between 11hydroxysteroid dehydrogenase type 2 (11HSD2) mRNA and fetal plasma cortisol concentration (r2 = 0.33, P = 0.003, where y = 0.96x +31.41). D, relationship between 11HSD2 mRNA and fetal plasma triiodothyronine (T3) concentration (r2 = 0.38, P = 0.020, where y = 60.96x + 27.48).
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A number of other statistically significant correlations were also noted (Figs 5 and 6). GR mRNA abundance was positively correlated to fetal plasma cortisol concentration and to a lesser extent to T3 concentration. Fetal plasma cortisol and T3 concentrations were negatively correlated to 11HSD1 mRNA abundance, but positively correlated to 11HSD2 mRNA abundance. UCP1 mRNA and protein were negatively correlated to 11HSD1 mRNA abundance, but positively correlated to 11HSD2 mRNA abundance (Table 2). UCP1 protein was also positively correlated to GR mRNA. Partial correlation analysis revealed that neither fetal plasma cortisol nor T3 concentrations were significant independent determinants of GR, or 11HSD type 1 or 2 mRNA expression, suggesting that concomitant increments in both cortisol and T3 are required to regulate the expression of these genes in fetal adipose tissue, similar to UCP2 expression.
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Discussion
This present study has shown for the first time that cortisol and T3 potentially regulate UCP2 mRNA expression in adipose tissue in the fetal sheep during late gestation, and that both hormones are required for this regulation. We have also shown that cortisol and T3 affect glucocorticoid action in fetal adipose tissue by specifically influencing the expression of GR and 11HSD isoforms. The abundance of UCP2 and GR mRNA increased with gestational age in parallel with the rise in plasma cortisol and T3 towards term. When this prepartum increase in cortisol and T3 was prevented by fetal adrenalectomy, the rise in UCP2 and GR mRNA was abolished, resulting in comparable abundance to control animals at 127–130 days gestation, as was the case for UCP1 protein (Mostyn et al. 2003). Conversely, raising cortisol and T3 levels at a time when concentrations are normally low, increased UCP2 mRNA abundance to values close to those seen in older animals nearer to term, as well as GR mRNA abundance, but to values lower than control animals nearer to term. Regulation of fetal UCP2 mRNA by cortisol is in accord with the effects of umbilical cord occlusion which results in a precocious rise in both UCP1 and 2 (Gnanalingham et al. 2005a), although under these adverse hypoxic conditions it is cortisol rather than T3 which regulates UCP abundance. Taken together our findings indicate the very different developmental regulation of UCP2 in large compared with small mammals (Gnanalingham et al. 2005b), which is in accord with UCP1 (Symonds et al. 2003). It is not therefore unexpected that deletion of UCP2 has no immediate effect on postnatal survival in small mammals (Rousset et al. 2003).
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Potential glucocorticoid and thyroid response elements have been identified in the promoter region of human UCP2 (Tu et al. 1999), suggesting that the developmental expression of UCP2 mRNA in fetal adipose tissue could be directly or indirectly regulated by glucocorticoids and thyroid hormones, as is the case for brown adipose tissue-specific UCP1 (Mostyn et al. 2003). Indeed, when the prepartum rise in cortisol and T3 was prevented by adrenalectomy, UCP2 mRNA levels were abolished, as was the case with UCP1 protein (Mostyn et al. 2003). However, we were not able to confirm translated UCP2 protein levels in fetal adipose tissue, as has been previously published for UCP1 in this study (Mostyn et al. 2003), since the antibody raised against UCP2 cross-reacts with UCP1 (Pecqueur et al. 2001); thus it is not possible to determine the abundance of UCP2 in mitochondria that possessed UCP1. In addition, it is known that the UCP2 gene is down-regulated at the translational level by an upstream open reading frame located in exon 2 of the gene, so it may be possible that UCP2 mRNA levels may not accurately represent that of the protein in fetal adipose tissue (Pecqueur et al. 2001). However, in the postnatal lung where we have been able to make measurements of both mRNA and protein we have found these to be closely correlated (Gnanalingham et al. 2005b) suggesting that it is not only UCP2 function but also the translational regulation that differs between small and large mammals.
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The current study has also demonstrated that the fetal cortisol status is important in determining glucocorticoid action in adipose tissue in the fetal sheep during late gestation, with cortisol infusion and bilateral adrenalectomy having opposing effects. While fetal cortisol infusion decreased local glucocorticoid action, by increasing 11HSD2 and decreasing 11HSD1 mRNA, this pattern was reversed by fetal bilateral adrenalectomy. These 11HSD isoform mRNA changes are likely to be accompanied by parallel changes in enzyme activity (Whorwood et al. 2001). In contrast to the comparable UCP2 and GR mRNA abundance in fetal adipose tissue following adrenalectomy at 142–145 days gestation and saline-infused controls at 127–130 days of gestation, the mRNA abundance of the 11HSD isoforms following adrenalectomy were markedly different from saline-infused controls, and did not prevent gestational changes in their abundance nearer to term. Collectively, these observations suggest potentially different control mechanisms in the regulation of the 11HSD isoforms in contrast to that for UCP2 and GR expression in fetal adipose tissue. The precise control mechanisms remain to be fully elucidated although under conditions of fetal stress cortisol appears to the primary regulator of 11HSD2 (Gnanalingham et al. 2005a) and a switch from 11HSD type 2 to type 1 dominance may be important in causing a switch from adipocyte proliferation to differentiation (Stewart et al. 2001).
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In contrast to our findings in fetal adipose tissue, cortisol infusion has been shown to enhance glucocorticoid action in the fetal liver by increasing GR and 11HSD1 expression in the sheep during late gestation (Gupta et al. 2003), while adrenalectomy reduced the enhanced 11HSD1 activity in omental fat of obese Zucker rats (Livingstone et al. 2000) and did not affect 11HSD2 expression in the kidney in male rats (Zallocchi et al. 2004). Collectively, these findings suggest that local glucocorticoid action within tissues is differentially regulated by cortisol status, as well as highlighting that the 11HSD isoforms are differentially expressed in fetal ovine tissues, such as the liver and kidney (Langlois & Matthews, 1995). Interestingly, 11HSD type 1 and 2 mRNA were also differentially regulated by fetal plasma cortisol and T3, having a positive relationship with 11HSD type 2 mRNA and a negative relationship with 11HSD type 1 mRNA, as also found in the subcutaneous adipose tissue of postmenopausal women (Engeli et al. 2004). This differential regulation of the 11HSD isoforms by fetal plasma cortisol and T3 in fetal adipose tissue may be important in promoting adipose tissue deposition during late gestation, which primarily occurs over the final third of gestation (Clarke et al. 1997a). While the relationship between cortisol, T3 and 11HSD isoforms in fetal adipose tissue have not been previously investigated, cortisol infusion increased 11HSD1 expression in the fetal liver in the late gestation sheep, suggesting increased hepatic responsiveness to glucocorticoids in late gestation (Gupta et al. 2003). In addition, cortisol increased 11HSD1 gene expression in isolated human adipocytes in vitro, in contrast to T3, which had no influence (Engeli et al. 2004). The 11HSD isoforms also had similar opposing relationships with UCP1 mRNA and its translated protein, suggesting that these enzymes may also be important in the regulation of UCP1 during late gestation in the sheep fetus. Similar mechanisms may also underlie the differential regulation of UCP1 mRNA and protein with the manipulation of fetal plasma cortisol and T3 concentrations (Mostyn et al. 2003). These relationships may be important in preparing the brown adipose tissue for effective non-shivering thermogenesis following birth and cold exposure (Mostyn et al. 2003).
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In conclusion, we have shown for the first time that plasma cortisol and T3 potentially regulate the late gestation rise in UCP2 mRNA in fetal adipose tissue, and that local glucocorticoid action is differentially affected by cortisol status. These changes may be important in ensuring optimal development fetal adipose tissue development in preparation for life after birth.
References
Bamberger CM, Schulte HM & Chrousos GP (1996). Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids. Endocrinol Rev 17, 245–261.
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Barbe P, Larrouy D, Boulanger C, Chevillotte E, Viguerie N, Thalamas C, Trastoy MO, Roques M, Vidal H & Langin D (2001). Triiodothyronine-mediated up-regulation of UCP2 and UCP3 mRNA expression in human skeletal muscle without coordinated induction of mitochondrial respiratory chain genes. FASEB J 15, 13–15.
Boss O, Hagen T & Lowell BB (2000). Uncoupling proteins 2 and 3, potential regulators of mitochondrial energy metabolism. Diabetes 49, 143–156.
, 百拇医药
Buemann B, Schierning B, Toubro S, Bibby BM, Srensen T, Dalgaard L, Pedersen O & Astrup A (2001). The association between the val/ala-55 polymorphism of the uncoupling protein 2 gene and exercise efficiency. Int J Obesity Related Metabolic Disorders 25, 467–471.
Casteilla L, Champigny O, Bouillaud F, Robelin J & Ricquier D (1989). Sequential changes in the expression of mitochondrial protein mRNA during the development of brown adipose tissue in bovine and ovine species. Biochem J 257, 665–671.
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Clarke L, Bryant MJ, Lomax MA & Symonds ME (1997a). Maternal manipulation of brown adipose tissue and liver development in the ovine fetus during late gestation. Br J Nutrition 77, 871–883.
Clarke L, Buss DS, Juniper DS, Lomax MA & Symonds ME (1997b). Adipose tissue development during early postnatal life in ewe-reared lambs. Exp Physiol 82, 1015–1017.
Devasker SU, Anthony RV & Hay WW (2002). Ontogeny and insulin regulation of fetal ovine white adipose tissue leptin expression. Am J Physiol 282, R431–R438.
, http://www.100md.com
Engeli S, Bohnke J, Feldpausch M, Gorzelniak K, Heintze U, Janke J, Luft FC & Sharma AM (2004). Regulation of 11HSD genes in human adipose tissue: influence of central obesity and weight loss. Obesity Res 12, 9–17.
Fleury C, Neverova M, Collins S, Pecquer C, Raimbault S, Champigny O, Meyrueis C, Bouillaud F, Seldin MF, Surwit R, Ricquier D & Warden C (1997). Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nature Genet 15, 269–272.
, http://www.100md.com
Forhead AJ, Poore KR, Mapstone J & Fowden AL (2003). Developmental regulation of hepatic and renal gluconeogenic enzymes by thyroid hormones in fetal sheep during late gestation. J Physiol 548, 941–947.
Fowden AL, Li J & Forhead AJ (1998). Glucocorticoids and the preparation for life after birth: are there long-term consequences of the life insurance Proc Nutrition Soc 57, 113–122.
Fowden AL, Szemere J, Hughes P, Gilmour RS & Forhead AJ (1996). The effects of cortisol on the growth rate of the sheep fetus during late gestation. J Endocrinol 151, 97–105.
, 百拇医药
Gnanalingham MG, Giussani DA, Stephenson T, Symonds ME & Gardner DS (2005a). Chronic umbilical cord compression results in premature maturation of lung and brown adipose tissue in the late gestation ovine fetus. Am J Physiol Endocrinol Metab (in press).
Gnanalingham MG, Mostyn A, Dandrea J, Yakubu DP, Symonds ME & Stephenson T (2005b). Ontogeny and nutritional programming of uncoupling protein-2 (UCP2) and glucocorticoid receptor (GR) mRNA in the ovine lung. J Physiol 565, 159–169.
, http://www.100md.com
Gupta S, Alfaidy N, Holloway A, Whittle W, Lye S, Gibb W & Challis JR (2003). Effects of cortisol and oestradiol on hepatic 11hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor proteins in late-gestation sheep fetus. J Endocrinol 176, 175–184.
Kizaki T, Suzuki K, Hitomi Y, Taniguchi N, Saitoh D, Watanabe K, Onoe K, Day NK, Good RA & Ohno H (2002). Uncoupling protein 2 plays an important role in nitric oxide production of lipopolysaccharide-stimulated macrophages. Proc Natl Acad Sci, USA 99, 9392–9397.
, 百拇医药
Langlois DA, Matthews SG, Yu M & Yang K (1995). Differential expression of 11b-hydroxysteroid dehydrogenase 1 and 2 in the developing ovine fetal liver and kidney. J Endocrinol 147, 405–411.
Lean MEJ (1989). Brown adipose tissue in humans. Proc Nutrition Soc 48, 243–256.
Livingstone D, Kenyon C & Walker B (2000). Mechanisms of dysregulation of 11 beta-hydroxysteroid dehydrogenase type 1 in obese Zucker rats. J Endocrinol 167, 533–539.
, 百拇医药
Mostyn A, Pearce S, Budge H, Elmes M, Forehead AJ, Fowden AL, Symonds ME & Stephenson T (2003). Influence of cortisol on adipose tissue development in the fetal sheep during late gestation. J Endocrinol 176, 23–30.
Negre-Salvayre A, Hirtz C, Carrera G, Cazenave R, Troly M, Salvayre R, Penicaud L & Casteilla L (1997). A role for uncoupling protein-2 as a regulator of mitochondrial hydrogen peroxide generation. FASEB J 11, 809–815.
, 百拇医药
Pecqueur C, Alves-Guerra M-C, Gelly C, Levi-Meyrueis C, Couplan E, Collins S, Ricquier D, Bouillaud F & Miroux B (2001). Uncoupling protein-2: in vivo distribution, induction upon oxidative stress and evidence for translational regulation. J Biol Chem 276, 8705–8712.
Prins JB & O'Rahilly S (1997). Regulation of adipose cell number in man. Clin Sci 92, 3–11.
Robinson PM, Comline RS, Fowden AL & Silver M (1983). Adrenal cortex of fetal lamb: changes after hypophysectomy and effects of synacthen on cytoarchitecture and secretory activity. Q J Exp Physiol 68, 15–27.
, 百拇医药
Rousset S, Alves-Guerra M-C, Ouadghiri-Bencherif S, Kozak LP, Miroux B, Richard D, Bouillaud F, Ricquier D & Cassard-Doulcier A-M (2003). UCP2 is expressed in the female mice reproductive tract whereas UCP1 is not. J Biol Chem 278, 45843–45847.
Schermer SJ, Bird JA, Lomax MA, Shepherd DAL & Symonds ME (1996). Effect of fetal thyroidectomy on brown adipose tissue and thermoregulation in newborn lambs. Reprod Fertil Dev 8, 995–1002.
, http://www.100md.com
Stewart PM & Krozowski ZS (1999). 11hydroxysteroid dehydrogenase. Vitamins Hormones 57, 249–324.
Stewart PM, Toogood AA & Tomlinson JW (2001). Growth hormone, insulin-like growth factor-I and the cortisol-cortisone shuttle. Hormone Res 56, S1–S6.
Symonds ME, Mostyn A, Pearce S, Budge H & Stephenson T (2003). Endocrine and nutritional regulation of fetal adipose tissue development. J Endocrinol 179, 293–299.
, 百拇医药
Tu N, Chen H, Winnikes U, Reinert I, Marmann G, Pirke KM & Lentes KU (1999). Molecular cloning and functional characterization of the promoter region of the human uncoupling protein-2 gene. Biochem Biophys Res Comms 265, 326–334.
Voehringer DW, Hirschberg DL, Xiao J, Lu Q, Roederer M, Lock CB, Herzenberg LA, Steinman L & Herzenberg LA (2000). Gene microarray identification of redox and mitochondrial elements that control resistance or sensitivity to apoptosis. Proc Natl Acad Sci, USA 97, 2680–2685.
, 百拇医药
Whorwood CB, Firth KM, Budge H & Symonds ME (2001). Maternal undernutrition during early- to mid-gestation programmes tissue-specific alterations in the expression of the glucocorticoid receptor, 11hydroxysteroid dehydrogenase isoforms and type 1 angiotensin II receptor in neonatal sheep. Endocrinology 142, 2554–2864.
Zallocchi ML, Matkovic L, Calvo JC & Damasco MC (2004). Adrenal gland involvement in the regulation of renal 11Hydroxysteroid dehydrogenase 2. J Cellular Biochem 92, 591–602., 百拇医药(M. G Gnanalingham, A Most)
2 Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK
Abstract
The endocrine regulation of uncoupling protein-2 (UCP2), an inner mitochondrial protein, in fetal adipose tissue remains unclear. The present study aimed to determine if fetal plasma cortisol and triiodothyronine (T3) influenced the mRNA abundance of UCP2, glucocorticoid receptor (GR) and 11hydroxysteroid dehydrogenase type 1 (11HSD1) and 2 (11HSD2) in fetal adipose tissue in the sheep during late gestation. Perirenal–abdominal adipose tissue was sampled from ovine fetuses to which either cortisol (2–3 mg kg–1 day–1) or saline was infused for 5 days up to 127–130 days gestation, or near term fetuses (i.e. 142–145 days gestation) that were either adrenalectomised (AX) or remained intact. Fetal plasma cortisol and T3 concentrations were higher in the cortisol infused animals and lower in AX fetuses compared with their corresponding control group, and increased with gestational age. UCP2 and GR mRNA abundance were significantly lower in AX fetuses compared with age-matched controls, and increased with gestational age and by cortisol infusion. Glucocorticoid action in fetal adipose tissue was augmented by AX and suppressed by cortisol infusion, the latter also preventing the gestational increase in 11HSD1 mRNA and decrease in 11HSD2 mRNA. When all treatment groups were combined, both fetal plasma cortisol and T3 concentrations were positively correlated with UCP2, GR and 11HSD2 mRNA abundance, but negatively correlated with 11HSD1 mRNA abundance. In conclusion, plasma cortisol and T3 are both required for the late gestation rise in UCP2 mRNA and differentially regulate glucocorticoid action in fetal adipose tissue in the sheep during late gestation.
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Introduction
Fetal fat possesses the dual characteristics of brown and white adipocytes (Devasker et al. 2002). In species such as humans and sheep that are born with a mature hypothalamic–pituitary–adrenal axis and are precocial thermoregulators, brown adipose tissue abundance is maximal around the time of birth (Casteilla et al. 1989; Clarke et al. 1997a) and is then not normally detectable after the postnatal period (Lean, 1989; Clarke et al. 1997b). This transition includes the acquisition of white adipose tissue characteristics, by proliferation and differentiation of preadipocytes and cell loss via apoptosis of preadipocytes and adipocytes, and possibly by other processes such as adipocyte dedifferentiation (Clarke et al. 1997b; Prins & O'Rahilly, 1997). Uncoupling protein (UCP)-2, a recently discovered inner mitochondrial protein (Fleury et al. 1997), has postulated roles in reactive oxygen species production (Negre-Salvayre et al. 1997; Kizaki et al. 2002) and apoptosis (Voehringer et al. 2000), as well as in energy regulation (Boss et al. 2000; Buemann et al. 2001). The abundance of UCP2 peaks around the time of birth (Gnanalingham et al. 2005b) at least in the ovine lung where its ontogeny is similar to that of the brown adipose tissue-specific UCP1. It has recently been shown that chronic fetal stress induced by umbilical cord occlusion results in a precocious rise in both UCP1 and 2 mRNA suggesting that both of these proteins may be important in promoting metabolic adaptation in the newborn (Gnanalingham et al. 2005a). The increase in fetal UCP1 near to term is dependent on an intact adrenal gland and the prepartum surge in fetal plasma cortisol and triiodothyronine (T3) (Mostyn et al. 2003), but this has yet to be established for UCP2.
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In fetal adipose tissue, glucocorticoid action is determined by its ability to both synthesize and inactivate cortisol, and by the expression of glucocorticoid receptor (GR, type 2) and isoforms of 11hydroxysteroid dehydrogenase (11HSD) (Whorwood et al. 2001). In this regard, 11HSD type 1 (11HSD1) behaves predominantly as an 11-oxoreductase, catalysing the conversion of inactive cortisone to active cortisol, thereby amplifying activation of intracellular GR, while 11HSD type 2 (11HSD2) behaves as an 11-dehydrogenase, catalysing the inactivation of cortisol to inert cortisone, and thereby maintains the specificity of the mineralocorticoid receptor for aldosterone (Bamberger et al. 1996; Stewart & Krozowski, 1999). However, the direct influence of fetal plasma cortisol and possibly T3 on local glucocorticoid action in fetal adipose tissue in preparation for extrauterine life remains to be determined.
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During the prepartum period, cortisol regulates the expression of a wide range of enzymes, receptors, hormones and binding proteins in fetal tissues, including GR and 11HSD isoforms in the fetal liver and placenta (Fowden et al. 1998). In several instances, the maturational effects of cortisol are mediated by the cortisol-dependent rise in fetal plasma T3 (Fowden et al. 1998). In humans, the UCP2 gene has putative glucocorticoid and thyroid hormone response elements (Tu et al. 1999) and is up regulated by T3 in adipose tissue in vitro and in vivo (Barbe et al. 2001). However, the role of cortisol in UCP2 expression and glucocorticoid action in fetal adipose tissue remains unknown. Hence, the aim of this study was to investigate the effects of cortisol and the concomitant rise in T3 on the expression of UCP2, GR and 11HSD isoforms in adipose tissue from the fetal sheep during late gestation, when endogenous cortisol levels rise and after experimental manipulation of fetal plasma cortisol concentration by exogenous infusion and fetal adrenalectomy. This was examined by determining the effect of (1) infusing cortisol into the fetus between 122 and 125 days gestation in order to increase fetal plasma cortisol to values observed near to term and (2) removing both adrenal glands (adrenalectomised, AX) thereby preventing the normal prepartum surges in cortisol and T3. Effects on fetal adipose tissue maturation were then compared with age-matched controls with respect to the abundance of UCP2, GR, 11HSD type 1 and 2 mRNA in perirenal–abdominal adipose tissue (which constitutes up to 80% of fetal adipose tissue).
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Methods
Animals, surgical and experimental procedures
A total of 27 Welsh Mountain sheep fetuses of known gestational age were used in this the study, A summary of the experimental protocol is given in Fig. 1 and full details of the animals, surgical and experimental procedures have been published previously (Mostyn et al. 2003). In brief, under halothane anaesthesia (1.5% in O2–N2O), one of the following procedures were carried out between 115 and 118 days gestation (term 145 days) using the surgical methods previously described (Fowden et al. 1996). Through a midline incision in the ewe the uterus was exteriorized and catheters were inserted into the caudal aorta and vena cava of the fetus via the tarsal vessels (n = 15) or both fetal adrenals were removed (n = 6). Six additional age-matched intact unoperated fetuses acted as controls for the adrenalectomized fetuses.
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AT, adipose tissue; G, days of gestation (term 145 days); AX, adrenalectomised; C/S, Caesarean section.
The study aimed to determine the effect of (1) a precocious rise in fetal cortisol and (2) a prolonged reduction in fetal cortisol. The first objective was achieved by comparing the effect of a 5-day period of cortisol infusion (n = 8, 2–3 mg kg–1 day–1 in 3 ml 0.9% (w/v) saline (EF-Cortelan; Glaxo Ltd, Greenford, Middlesex, UK)) with 5-day saline infusion (n = 7, 3 ml day–1, 0.9% w/v), before tissue collection at 127–130 days gestation. This dose of cortisol was sufficient to achieve plasma cortisol concentrations similar to those observed close to term, i.e. 40–50 ng ml–1 (Fowden et al. 1996). The second objective was achieved by bilateral adrenalectomy which prevented the prepartum rise in fetal cortisol and T3 and then comparing adipose tissue composition from these animals with non-operated age matched controls (n = 6) near to term, i.e. 142–145 days gestation. We have previously shown no difference in adipose tissue composition in neonatal lambs between those which have undergone a sham operation and those not subjected to this procedure (Schermer et al. 1996). Blood samples of 2 ml were taken daily throughout the experimental period from all the catheterized fetuses to determine plasma cortisol and thyroid hormone concentrations and to monitor fetal well-being using blood pH, PO2and glucose levels (ABL5 Radiometer and YSI glucose analyser). The distribution of male and female fetuses between treatment groups was saline 3: 4, cortisol 3: 5, Ax 3: 3 and sham 2: 4 and there was no effect of sex on any of the measurements made. All procedures were carried out under the UK Animals (Scientific Procedures) Act 1986 and approved by the local ethical review board.
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Sample collection
All fetuses were delivered by Caesarean section under sodium pentobarbitone anaesthesia (20 mg kg–1 I.V.) between 127 and 130 days gestation for cortisol- and saline-infused fetuses and between 142 and 145 days for AX and intact control fetuses. Fetal blood samples were taken at the time of delivery either through the indwelling arterial catheter (saline and cortisol infused fetuses) or by venipuncture from the umbilical artery in the cord (adrenalectomised fetuses and age matched controls). After administration of a lethal dose of anaesthetic (200 mg kg–1 sodium pentobarbitone), samples of perirenal adipose tissue were collected and frozen rapidly in liquid nitrogen before storage at –80°C. All blood samples were centrifuged immediately at 4°C and the plasma stored at –20°C before plasma analyses. No adrenal remnants were found in any of the AX fetuses at autopsy.
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Laboratory analyses
Messenger RNA detection. Total RNA was isolated from fetal adipose tissue using Tri-Reagent (Sigma, Poole, UK). In order to maximize sensitivity, a two-tube approach to reverse transcription (RT) was adopted. The conditions used to generate first strand cDNA RT were: 70°C (5 min), 4°C (5 min), 25°C (5 min), 25°C (10 min), 42°C (1 h), 72°C (10 min), 4°C (5 min). The RT reaction (final volume 20 μl) contained: 5 x cDNA (first-strand), buffer (250 mM Tris-HCl, 40 mM MgCl2, 150 mM KCl, 5 mM dithioerythritol pH 8.5), 2 mM dNTPs, 1 x hexanucleotide mix, 10 units RNase inhibitor, 10 units M-MLV reverse transcriptase and 1 μg total RNA. All of these commercially available products were purchased from Roche Diagnostics Ltd (Lewes, UK).
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The expression of UCP2, GR (type 2), and 11HSD type 1 and 2 mRNA were determined by reverse transcriptase–polymerase chain reaction (RT-PCR), as previously described by (Gnanalingham et al. 2005b). The analysis used oligonucleotide cDNA primers to UCP2, GR (type 2), and 11HSD type 1 and 2 genes generating specific intron spanning products (Table 1). Briefly, the PCR programme consisted of an initial denaturation (95°C (15 min)), amplification (stage I, 94°C (30 s); stage II, annealing temperature (30 s); stage III, 72°C (60 s)) and final extension (72°C (7 min); 8°C ‘hold’). The PCR mixture (final volume 20 μl) contained 7 μl diethylpyrocarbonate (DEPC) H2O, 10 μl Thermo-Start PCR Master Mix (50 μl contains 1.25 units Thermo-Start DNA Polymerase, 1 x Thermo-Start reaction buffer, 1.5 mM MgCl2 and 0.2 mM each of dATP, dCTP, dGTP and dTTP; ABgene, Epsom, UK, cat. no. AB-0938-DC-15), 1 μl Forward Primer, 1 μl Reverse Primer and 1 μl RT (cDNA) product. The annealing temperature and cycle numbers of all primers were optimized so as to be in the linear range (see Table 1). Agrose gel electrophoresis (2.0–2.5%) and ethidium bromide staining confirmed the presence of both the product and 18S at the expected sizes. Densitometric analysis was performed on each gel by image detection using a Fujifilm LAS-1000 cooled charge-coupled device camera and UCP2, GR, 11HSD type 1 and 2 and 18S mRNA abundance determined. Consistency of lane loading for each sample was verified and all results expressed as a ratio of a reference sample to r18S abundance. All analyses and gels were conducted in duplicate, with appropriate positive and negative controls, and a range of molecular weight markers. The resultant PCR product was extracted (QIAquick gel extraction kit, Qiagen, West Sussex, UK, cat. no. 28704), sequenced and results cross-referenced against the GenBank website to determine specificity of the target gene. In this present study, the abundance of UCP1 mRNA and protein in perirenal adipose tissue in the fetuses were determined previously, as described by Mostyn et al. (2003).
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Radioimmunoassays. The effect of cortisol infusion and bilateral adrenalectomy on plasma cortisol, T3 and UCP1 in the sheep fetus during late gestation have been previously published (Mostyn et al. 2003), but brief details are included as the same fetuses were used in both the previous and current study and to enable full interpretation of significant correlations outlined below. Cortisol concentrations were measured by radioimmunoassay and validated for use with ovine plasma (Robinson et al. 1983). The minimum detectable quantity of cortisol was 1.5 ng ml–1 and interassay coefficient of variation was 11%. Total plasma T3 concentrations were also measured by radioimmunoassay, using a commercial kit (ICN Biomedicals, Thame, Oxon, UK) validated for ovine plasma (Forhead et al. 2003). The lower unit of detection was 0.1 ng ml–1 and interassay coefficient of variation was 10%.
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Statistical analyses
All data are presented as means ± S.E.M. Significant differences (P < 0.05) between mean values obtained between each treatment group were carried out using Mann–Whitney U tests. Significant correlations between fetal plasma hormone concentrations and molecular parameters measured in the study were analysed by Spearman's rank order test using SPSS v11.0 (SPSS Inc., Chicago, IL, USA). Partial correlation analysis was undertaken when there was more than one variable correlated with the measured molecular parameter, to determine which variable had the greatest effect.
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Results
Effect of cortisol infusion and adrenalectomy on plasma cortisol, T3 and UCP1
Fetal plasma cortisol and T3 concentrations were higher in the cortisol infused animals (cortisol: saline, 14.0 ± 2.3; cortisol, 45.7 ± 5.3 ng ml–1 (P < 0.01); T3: saline, 0.28 ± 0.04; cortisol, 0.57 ± 0.16 ng ml–1 (P < 0.01)) and lower in AX fetuses (cortisol: intact, 49.5 ± 12.3; AX, 7.2 ± 0.6 ng ml–1 (P < 0.01); T3: intact, 0.49 ± 0.07; AX, 0.29 ± 0.04 ng ml–1 (P < 0.05)) compared with their corresponding control group, and increased (P < 0.05) with gestational age (130 days gestation: cortisol: 14.0 ± 2.3; T3: 0.28 ± 0.04; 145 days gestation: cortisol: 49.5 ± 12.3; T3: 0.49 ± 0.07 ng ml–1 (P < 0.05). UCP1 protein abundance was significantly lower in AX fetuses compared with age-matched controls (intact, 38.5 ± 7.3; AX, 12.1 ± 2.7% of reference (P < 0.01)), and was increased by cortisol infusion and with gestational age (saline, 22.1 ± 3.7; cortisol, 52.1 ± 4.8% of reference (P < 0.05)), in contrast to UCP1 mRNA that was not statistically altered by cortisol status (results not shown). Fetal weight was not significantly different between sex or two treatment groups at each gestational age (e.g. saline: males, 3.19 ± 0.28; females, 3.29 ± 0.25; cortisol: males, 3.17 ± 0.27; females, 2.73 ± 0.22 kg). Total perirena–abdominal adipose tissue weights were not recorded.
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Effect of fetal cortisol infusion and adrenalectomy on UCP2, GR, and 11HSD type 1 and 2 mRNA abundance in fetal adipose tissue
There was a gestational increase (P < 0.01) in the abundance of UCP2, GR and 11HSD1 mRNA and a decrease (P = 0.01) in the abundance of 11HSD2 mRNA in fetal adipose tissue (Figs 2 and 3). Fetal cortisol infusion increased (P < 0.05) UCP2 mRNA abundance compared to age-matched controls and to a level similar to that of late gestation controls at 142–145 days. While fetal cortisol infusion increased GR (P < 0.05) and 11HSD2 (P < 0.01) mRNA abundance compared to age-matched controls, but to a level lower (P < 0.01) than late gestation controls. In contrast, fetal cortisol infusion decreased (P < 0.05) 11HSD1 mRNA abundance compared to age-matched controls and to a level higher (P < 0.01) than late gestation controls. Bilateral AX decreased (P < 0.05) the prepartum rise in UCP2 and GR mRNA abundance near term, resulting in comparable abundance to saline infused control animals at 127–130 days gestation (UCP2: saline, 19.5 ± 4.1; AX, 28.1 ± 3.8% 18S/reference (NS); GCR: saline, 32.6 ± 3.8; AX, 44.4 ± 3.0% 18S/reference (NS)). In contrast, bilateral AX augmented the prepartum rise (P < 0.05) in 11HSD1 mRNA and diminished (P < 0.01) in 11HSD2 mRNA abundance near term, resulting in significantly different abundance compared to saline infused control animals at 127–130 days gestation (11HSD1: saline, 11.5 ± 1.8; AX, 34.4 ± 2.7% 18S/reference (P < 0.001); 11HSD2: saline, 65.1 ± 3.7; AX, 18.9 ± 3.2% 18S/reference (P < 0.001)). When all treatment groups were combined, UCP2 mRNA was positively (P < 0.001) correlated to GR mRNA abundance in fetal adipose tissue, and 11HSD1 mRNA was negatively (P < 0.001) correlated to 11HSD2 mRNA abundance (Fig. 4).
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Effect on uncoupling protein-2 (UCP2) mRNA (A) and glucocorticoid receptor (GR) mRNA (B) in fetal perirenal-abdominal adipose tissue collected at 127–130 days gestation (term 145 days) following cortisol (C, 2–3 mg kg–1 day–1 for 5 days) or saline (S) infusion, and at 142–145 days gestation following bilateral adrenalectomy (AX, 115–118 days gestation) or intact (I) controls. Examples of charge-coupled device camera images of gene mRNA expression are given. Values are means with their standard errors (n = 5–6 per group). G = mean gestational age. *P < 0.05, **P < 0.01, mean value significantly different from respective fetal control, and between 127 and 130 and 142–145 days gestational age.
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Effect on 11hydroxysteroid dehydrogenase type 1 (11HSD1) mRNA (A) and 11hydroxysteroid dehydrogenase type 2 (11HSD2) mRNA (B) in fetal perirenal–abdominal adipose tissue collected at 127–130 days gestation (term 145 days) following cortisol (C, 2–3 mg kg–1 day–1 for 5 days) or saline (S) infusion, and at 142–145 days gestation following bilateral adrenalectomy (AX, 115–118 days gestation) or intact (I) controls. Examples of charge-coupled device camera images of gene mRNA expression are given. Values are means with their standard errors (n = 5–6 per group). G = mean gestational age. *P < 0.05, **P < 0.01, mean value significantly different from respective fetal control, and between 127 and 130 and 142–145 days gestational age.
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A–B are for perirenal–abdominal adipose tissue sampled from all fetuses (n = 22) irrespective of treatment or gestational age in the sheep during late gestation. A, relationship between uncoupling protein-2 (UCP2) mRNA and glucocorticoid receptor (GR) mRNA abundance (r2 = 0.43, P < 0.001, where y = 0.53x + 6.65). B, relationship between 11hydroxysteroid dehydrogenase type 1 (11HSD1) mRNA and 11hydroxysteroid dehydrogenase type 2 (11HSD2) mRNA abundance (r2 = 0.84, P < 0.001, where y = –0.37x + 38.70).
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Since UCP2 mRNA was elevated when fetal plasma cortisol and T3 levels were raised and was low when cortisol and T3 were low, the relationship between UCP2 mRNA abundance in fetal adipose tissue and the plasma concentrations of these hormones were examined in more detail. When individual results from all fetuses were combined regardless of gestational age or treatment, there was a significant positive correlation between UCP2 mRNA abundance in fetal adipose tissue and both the plasma concentrations of cortisol and T3 (Figs 5 and 6), which were positively correlated to each other (r2 = 0.595, P = 0.001) (Mostyn et al. 2003). However, partial correlation analysis revealed that neither fetal plasma cortisol nor T3 concentrations were significant independent determinants of UCP2 mRNA expression, suggesting that concomitant increments in both cortisol and T3 are required to up regulate expression of this gene in fetal adipose tissue.
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A–D are for perirenal–abdominal adipose tissue sampled from all fetuses (n = 22) irrespective of treatment or gestational age in the sheep during late gestation. A, relationship between uncoupling protein-2 (UCP2) mRNA and fetal plasma cortisol concentration (r2 = 0.44, P = 0.001, where y = 0.49x + 20.08). B, relationship between UCP2 mRNA and fetal plasma triiodothyronine (T3) concentration (r2 = 0.42, P = 0.001, where y = 28.73x + 19.01). C, relationship between glucocorticoid receptor (GR) mRNA and fetal plasma cortisol concentration (r2 = 0.34, P = 0.006, where y = 0.54x +36.04). D, relationship between GR mRNA and fetal plasma triiodothyronine (T3) concentration (r2 = 0.20, P = 0.024, where y = 20.03x + 39.65).
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A–D are for perirenal-abdominal adipose tissue sampled from all fetuses (n = 22) irrespective of treatment or gestational age in the sheep during late gestation. A, relationship between 11hydroxysteroid dehydrogenase type 1 (11HSD1) mRNA and fetal plasma cortisol concentration (r2 = 0.22, P = 0.013, where y = –0.31x + 26.09). B, relationship between 11HSD1 mRNA and fetal plasma triiodothyronine (T3) concentration (r2 = 0.28, P = 0.021, where y = –20.83x + 27.78). C, relationship between 11hydroxysteroid dehydrogenase type 2 (11HSD2) mRNA and fetal plasma cortisol concentration (r2 = 0.33, P = 0.003, where y = 0.96x +31.41). D, relationship between 11HSD2 mRNA and fetal plasma triiodothyronine (T3) concentration (r2 = 0.38, P = 0.020, where y = 60.96x + 27.48).
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A number of other statistically significant correlations were also noted (Figs 5 and 6). GR mRNA abundance was positively correlated to fetal plasma cortisol concentration and to a lesser extent to T3 concentration. Fetal plasma cortisol and T3 concentrations were negatively correlated to 11HSD1 mRNA abundance, but positively correlated to 11HSD2 mRNA abundance. UCP1 mRNA and protein were negatively correlated to 11HSD1 mRNA abundance, but positively correlated to 11HSD2 mRNA abundance (Table 2). UCP1 protein was also positively correlated to GR mRNA. Partial correlation analysis revealed that neither fetal plasma cortisol nor T3 concentrations were significant independent determinants of GR, or 11HSD type 1 or 2 mRNA expression, suggesting that concomitant increments in both cortisol and T3 are required to regulate the expression of these genes in fetal adipose tissue, similar to UCP2 expression.
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Discussion
This present study has shown for the first time that cortisol and T3 potentially regulate UCP2 mRNA expression in adipose tissue in the fetal sheep during late gestation, and that both hormones are required for this regulation. We have also shown that cortisol and T3 affect glucocorticoid action in fetal adipose tissue by specifically influencing the expression of GR and 11HSD isoforms. The abundance of UCP2 and GR mRNA increased with gestational age in parallel with the rise in plasma cortisol and T3 towards term. When this prepartum increase in cortisol and T3 was prevented by fetal adrenalectomy, the rise in UCP2 and GR mRNA was abolished, resulting in comparable abundance to control animals at 127–130 days gestation, as was the case for UCP1 protein (Mostyn et al. 2003). Conversely, raising cortisol and T3 levels at a time when concentrations are normally low, increased UCP2 mRNA abundance to values close to those seen in older animals nearer to term, as well as GR mRNA abundance, but to values lower than control animals nearer to term. Regulation of fetal UCP2 mRNA by cortisol is in accord with the effects of umbilical cord occlusion which results in a precocious rise in both UCP1 and 2 (Gnanalingham et al. 2005a), although under these adverse hypoxic conditions it is cortisol rather than T3 which regulates UCP abundance. Taken together our findings indicate the very different developmental regulation of UCP2 in large compared with small mammals (Gnanalingham et al. 2005b), which is in accord with UCP1 (Symonds et al. 2003). It is not therefore unexpected that deletion of UCP2 has no immediate effect on postnatal survival in small mammals (Rousset et al. 2003).
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Potential glucocorticoid and thyroid response elements have been identified in the promoter region of human UCP2 (Tu et al. 1999), suggesting that the developmental expression of UCP2 mRNA in fetal adipose tissue could be directly or indirectly regulated by glucocorticoids and thyroid hormones, as is the case for brown adipose tissue-specific UCP1 (Mostyn et al. 2003). Indeed, when the prepartum rise in cortisol and T3 was prevented by adrenalectomy, UCP2 mRNA levels were abolished, as was the case with UCP1 protein (Mostyn et al. 2003). However, we were not able to confirm translated UCP2 protein levels in fetal adipose tissue, as has been previously published for UCP1 in this study (Mostyn et al. 2003), since the antibody raised against UCP2 cross-reacts with UCP1 (Pecqueur et al. 2001); thus it is not possible to determine the abundance of UCP2 in mitochondria that possessed UCP1. In addition, it is known that the UCP2 gene is down-regulated at the translational level by an upstream open reading frame located in exon 2 of the gene, so it may be possible that UCP2 mRNA levels may not accurately represent that of the protein in fetal adipose tissue (Pecqueur et al. 2001). However, in the postnatal lung where we have been able to make measurements of both mRNA and protein we have found these to be closely correlated (Gnanalingham et al. 2005b) suggesting that it is not only UCP2 function but also the translational regulation that differs between small and large mammals.
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The current study has also demonstrated that the fetal cortisol status is important in determining glucocorticoid action in adipose tissue in the fetal sheep during late gestation, with cortisol infusion and bilateral adrenalectomy having opposing effects. While fetal cortisol infusion decreased local glucocorticoid action, by increasing 11HSD2 and decreasing 11HSD1 mRNA, this pattern was reversed by fetal bilateral adrenalectomy. These 11HSD isoform mRNA changes are likely to be accompanied by parallel changes in enzyme activity (Whorwood et al. 2001). In contrast to the comparable UCP2 and GR mRNA abundance in fetal adipose tissue following adrenalectomy at 142–145 days gestation and saline-infused controls at 127–130 days of gestation, the mRNA abundance of the 11HSD isoforms following adrenalectomy were markedly different from saline-infused controls, and did not prevent gestational changes in their abundance nearer to term. Collectively, these observations suggest potentially different control mechanisms in the regulation of the 11HSD isoforms in contrast to that for UCP2 and GR expression in fetal adipose tissue. The precise control mechanisms remain to be fully elucidated although under conditions of fetal stress cortisol appears to the primary regulator of 11HSD2 (Gnanalingham et al. 2005a) and a switch from 11HSD type 2 to type 1 dominance may be important in causing a switch from adipocyte proliferation to differentiation (Stewart et al. 2001).
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In contrast to our findings in fetal adipose tissue, cortisol infusion has been shown to enhance glucocorticoid action in the fetal liver by increasing GR and 11HSD1 expression in the sheep during late gestation (Gupta et al. 2003), while adrenalectomy reduced the enhanced 11HSD1 activity in omental fat of obese Zucker rats (Livingstone et al. 2000) and did not affect 11HSD2 expression in the kidney in male rats (Zallocchi et al. 2004). Collectively, these findings suggest that local glucocorticoid action within tissues is differentially regulated by cortisol status, as well as highlighting that the 11HSD isoforms are differentially expressed in fetal ovine tissues, such as the liver and kidney (Langlois & Matthews, 1995). Interestingly, 11HSD type 1 and 2 mRNA were also differentially regulated by fetal plasma cortisol and T3, having a positive relationship with 11HSD type 2 mRNA and a negative relationship with 11HSD type 1 mRNA, as also found in the subcutaneous adipose tissue of postmenopausal women (Engeli et al. 2004). This differential regulation of the 11HSD isoforms by fetal plasma cortisol and T3 in fetal adipose tissue may be important in promoting adipose tissue deposition during late gestation, which primarily occurs over the final third of gestation (Clarke et al. 1997a). While the relationship between cortisol, T3 and 11HSD isoforms in fetal adipose tissue have not been previously investigated, cortisol infusion increased 11HSD1 expression in the fetal liver in the late gestation sheep, suggesting increased hepatic responsiveness to glucocorticoids in late gestation (Gupta et al. 2003). In addition, cortisol increased 11HSD1 gene expression in isolated human adipocytes in vitro, in contrast to T3, which had no influence (Engeli et al. 2004). The 11HSD isoforms also had similar opposing relationships with UCP1 mRNA and its translated protein, suggesting that these enzymes may also be important in the regulation of UCP1 during late gestation in the sheep fetus. Similar mechanisms may also underlie the differential regulation of UCP1 mRNA and protein with the manipulation of fetal plasma cortisol and T3 concentrations (Mostyn et al. 2003). These relationships may be important in preparing the brown adipose tissue for effective non-shivering thermogenesis following birth and cold exposure (Mostyn et al. 2003).
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In conclusion, we have shown for the first time that plasma cortisol and T3 potentially regulate the late gestation rise in UCP2 mRNA in fetal adipose tissue, and that local glucocorticoid action is differentially affected by cortisol status. These changes may be important in ensuring optimal development fetal adipose tissue development in preparation for life after birth.
References
Bamberger CM, Schulte HM & Chrousos GP (1996). Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids. Endocrinol Rev 17, 245–261.
, http://www.100md.com
Barbe P, Larrouy D, Boulanger C, Chevillotte E, Viguerie N, Thalamas C, Trastoy MO, Roques M, Vidal H & Langin D (2001). Triiodothyronine-mediated up-regulation of UCP2 and UCP3 mRNA expression in human skeletal muscle without coordinated induction of mitochondrial respiratory chain genes. FASEB J 15, 13–15.
Boss O, Hagen T & Lowell BB (2000). Uncoupling proteins 2 and 3, potential regulators of mitochondrial energy metabolism. Diabetes 49, 143–156.
, 百拇医药
Buemann B, Schierning B, Toubro S, Bibby BM, Srensen T, Dalgaard L, Pedersen O & Astrup A (2001). The association between the val/ala-55 polymorphism of the uncoupling protein 2 gene and exercise efficiency. Int J Obesity Related Metabolic Disorders 25, 467–471.
Casteilla L, Champigny O, Bouillaud F, Robelin J & Ricquier D (1989). Sequential changes in the expression of mitochondrial protein mRNA during the development of brown adipose tissue in bovine and ovine species. Biochem J 257, 665–671.
, 百拇医药
Clarke L, Bryant MJ, Lomax MA & Symonds ME (1997a). Maternal manipulation of brown adipose tissue and liver development in the ovine fetus during late gestation. Br J Nutrition 77, 871–883.
Clarke L, Buss DS, Juniper DS, Lomax MA & Symonds ME (1997b). Adipose tissue development during early postnatal life in ewe-reared lambs. Exp Physiol 82, 1015–1017.
Devasker SU, Anthony RV & Hay WW (2002). Ontogeny and insulin regulation of fetal ovine white adipose tissue leptin expression. Am J Physiol 282, R431–R438.
, http://www.100md.com
Engeli S, Bohnke J, Feldpausch M, Gorzelniak K, Heintze U, Janke J, Luft FC & Sharma AM (2004). Regulation of 11HSD genes in human adipose tissue: influence of central obesity and weight loss. Obesity Res 12, 9–17.
Fleury C, Neverova M, Collins S, Pecquer C, Raimbault S, Champigny O, Meyrueis C, Bouillaud F, Seldin MF, Surwit R, Ricquier D & Warden C (1997). Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nature Genet 15, 269–272.
, http://www.100md.com
Forhead AJ, Poore KR, Mapstone J & Fowden AL (2003). Developmental regulation of hepatic and renal gluconeogenic enzymes by thyroid hormones in fetal sheep during late gestation. J Physiol 548, 941–947.
Fowden AL, Li J & Forhead AJ (1998). Glucocorticoids and the preparation for life after birth: are there long-term consequences of the life insurance Proc Nutrition Soc 57, 113–122.
Fowden AL, Szemere J, Hughes P, Gilmour RS & Forhead AJ (1996). The effects of cortisol on the growth rate of the sheep fetus during late gestation. J Endocrinol 151, 97–105.
, 百拇医药
Gnanalingham MG, Giussani DA, Stephenson T, Symonds ME & Gardner DS (2005a). Chronic umbilical cord compression results in premature maturation of lung and brown adipose tissue in the late gestation ovine fetus. Am J Physiol Endocrinol Metab (in press).
Gnanalingham MG, Mostyn A, Dandrea J, Yakubu DP, Symonds ME & Stephenson T (2005b). Ontogeny and nutritional programming of uncoupling protein-2 (UCP2) and glucocorticoid receptor (GR) mRNA in the ovine lung. J Physiol 565, 159–169.
, http://www.100md.com
Gupta S, Alfaidy N, Holloway A, Whittle W, Lye S, Gibb W & Challis JR (2003). Effects of cortisol and oestradiol on hepatic 11hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor proteins in late-gestation sheep fetus. J Endocrinol 176, 175–184.
Kizaki T, Suzuki K, Hitomi Y, Taniguchi N, Saitoh D, Watanabe K, Onoe K, Day NK, Good RA & Ohno H (2002). Uncoupling protein 2 plays an important role in nitric oxide production of lipopolysaccharide-stimulated macrophages. Proc Natl Acad Sci, USA 99, 9392–9397.
, 百拇医药
Langlois DA, Matthews SG, Yu M & Yang K (1995). Differential expression of 11b-hydroxysteroid dehydrogenase 1 and 2 in the developing ovine fetal liver and kidney. J Endocrinol 147, 405–411.
Lean MEJ (1989). Brown adipose tissue in humans. Proc Nutrition Soc 48, 243–256.
Livingstone D, Kenyon C & Walker B (2000). Mechanisms of dysregulation of 11 beta-hydroxysteroid dehydrogenase type 1 in obese Zucker rats. J Endocrinol 167, 533–539.
, 百拇医药
Mostyn A, Pearce S, Budge H, Elmes M, Forehead AJ, Fowden AL, Symonds ME & Stephenson T (2003). Influence of cortisol on adipose tissue development in the fetal sheep during late gestation. J Endocrinol 176, 23–30.
Negre-Salvayre A, Hirtz C, Carrera G, Cazenave R, Troly M, Salvayre R, Penicaud L & Casteilla L (1997). A role for uncoupling protein-2 as a regulator of mitochondrial hydrogen peroxide generation. FASEB J 11, 809–815.
, 百拇医药
Pecqueur C, Alves-Guerra M-C, Gelly C, Levi-Meyrueis C, Couplan E, Collins S, Ricquier D, Bouillaud F & Miroux B (2001). Uncoupling protein-2: in vivo distribution, induction upon oxidative stress and evidence for translational regulation. J Biol Chem 276, 8705–8712.
Prins JB & O'Rahilly S (1997). Regulation of adipose cell number in man. Clin Sci 92, 3–11.
Robinson PM, Comline RS, Fowden AL & Silver M (1983). Adrenal cortex of fetal lamb: changes after hypophysectomy and effects of synacthen on cytoarchitecture and secretory activity. Q J Exp Physiol 68, 15–27.
, 百拇医药
Rousset S, Alves-Guerra M-C, Ouadghiri-Bencherif S, Kozak LP, Miroux B, Richard D, Bouillaud F, Ricquier D & Cassard-Doulcier A-M (2003). UCP2 is expressed in the female mice reproductive tract whereas UCP1 is not. J Biol Chem 278, 45843–45847.
Schermer SJ, Bird JA, Lomax MA, Shepherd DAL & Symonds ME (1996). Effect of fetal thyroidectomy on brown adipose tissue and thermoregulation in newborn lambs. Reprod Fertil Dev 8, 995–1002.
, http://www.100md.com
Stewart PM & Krozowski ZS (1999). 11hydroxysteroid dehydrogenase. Vitamins Hormones 57, 249–324.
Stewart PM, Toogood AA & Tomlinson JW (2001). Growth hormone, insulin-like growth factor-I and the cortisol-cortisone shuttle. Hormone Res 56, S1–S6.
Symonds ME, Mostyn A, Pearce S, Budge H & Stephenson T (2003). Endocrine and nutritional regulation of fetal adipose tissue development. J Endocrinol 179, 293–299.
, 百拇医药
Tu N, Chen H, Winnikes U, Reinert I, Marmann G, Pirke KM & Lentes KU (1999). Molecular cloning and functional characterization of the promoter region of the human uncoupling protein-2 gene. Biochem Biophys Res Comms 265, 326–334.
Voehringer DW, Hirschberg DL, Xiao J, Lu Q, Roederer M, Lock CB, Herzenberg LA, Steinman L & Herzenberg LA (2000). Gene microarray identification of redox and mitochondrial elements that control resistance or sensitivity to apoptosis. Proc Natl Acad Sci, USA 97, 2680–2685.
, 百拇医药
Whorwood CB, Firth KM, Budge H & Symonds ME (2001). Maternal undernutrition during early- to mid-gestation programmes tissue-specific alterations in the expression of the glucocorticoid receptor, 11hydroxysteroid dehydrogenase isoforms and type 1 angiotensin II receptor in neonatal sheep. Endocrinology 142, 2554–2864.
Zallocchi ML, Matkovic L, Calvo JC & Damasco MC (2004). Adrenal gland involvement in the regulation of renal 11Hydroxysteroid dehydrogenase 2. J Cellular Biochem 92, 591–602., 百拇医药(M. G Gnanalingham, A Most)