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Endocrinology Vol. 148, No. 1 461-468
Copyright © 2007 by The Endocrine Society

Ontogenic Loss of Brown Adipose Tissue Sensitivity to ß-Adrenergic Stimulation in the Ovine

Michael A. Lomax, Fouzia Sadiq, Georgios Karamanlidis, Angeliki Karamitri, Paul Trayhurn and David G. Hazlerigg

Divisions of Biomedical Science (M.A.L., G.K., A.K.) and Molecular Biology (F.S.), Imperial College, Wye Campus, Ashford, Kent TN25 5AH, United Kingdom; Liverpool Centre for Nutritional Genomics (P.T.), Obesity Biology Unit, School of Clinical Sciences, University of Liverpool, University Clinical Departments, Liverpool L69 3GA, United Kingdom; and School of Biological Sciences (D.G.H.), University of Aberdeen, Aberdeen AB24 5UH, United Kingdom

Address all correspondence and requests for reprints to: M. A. Lomax, Division of Biomedical Science, Imperial College, Ashford, Kent TN25 5AH, United Kingdom. E-mail: m.lomax{at}imperial.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In ruminants and other large animals, expression of uncoupling protein-1 (UCP1) in brown adipose tissue (BAT) is confined to the perinatal period when it plays a key role in nonshivering thermogenesis. This study determined whether loss of expression of the BAT phenotype was due to reduced response to a ß-agonist, isoprenaline, and expression of the peroxisome proliferator-activated receptor (PPAR) family [PPAR{alpha}, PPAR{gamma}, PPAR coactivator 1{alpha} (PGC-1{alpha})], which regulates UCP1 gene expression. Perirenal adipose tissue (PAT) was sampled from ovine fetuses, newborn lambs, and lambs on d 1, 5, 7, and 21 of life. UCP1 mRNA and protein in PAT increased from d 123 of fetal life to reach a maximum at birth followed by a rapid decrease over the first 5 d of life. Expression of the coactivator, PGC-1{alpha} and PPAR {alpha}, peaked between fetal day 123 and birth, and then declined to undetectable levels in the first days of life. In vivo administration of isoprenaline was able to induce expression of UCP1, PGC-1{alpha}, and PPAR{alpha} in BAT up to 5 d of age but thereafter was ineffective. In vitro addition of ß-receptor, PPAR{alpha}, and PPAR{gamma} agonists were unable to overcome the suppression of UCP1, PPAR{alpha}, and PPAR{gamma} expression observed in differentiated adipocytes prepared from 30-d-old compared with 1-d-old lambs. These data are consistent with a model in which postnatal loss of UCP1 expression and ß-adrenergic induction of the brown adipocyte phenotype is due to loss of expression of PGC-1{alpha} and PPAR{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BROWN ADIPOSE TISSUE (BAT) plays a vital role in neonatal mammals to prevent hypothermia (1, 2). In large mammals (primates, dogs, rabbits, and ruminants), BAT development during fetal life reaches a maximum thermogenic activity at birth and then rapidly transforms to white adipose tissue (WAT) within the first weeks of life (1, 3, 4, 5, 6). At a molecular level, this change can be followed by the increased expression during fetal life of the BAT marker gene uncoupling protein 1 (UCP1), which confers the thermogenic function of this tissue (7). Our studies in the ovine have demonstrated that expression of UCP1 in perirenal adipose tissue (PAT) is totally suppressed within 5 d after birth despite being born into an environmental temperature below the thermoneutral zone (8). Attempts to reinduce UCP1 expression in the ovine by administration of a ß3-adrenoreceptor agonist during the first month of life have been met with limited success (9, 10), agreeing with the morphological and thermogenic activity evidence that ovine BAT is irreversibly transformed to WAT during the first weeks of life (2, 11). Furthermore, it has been shown that perirenal preadipocytes taken from 1-month-old lambs cannot be induced to differentiate into brown adipocytes, although this is possible within a few days of birth (12). Combined, this evidence strongly suggests that in ruminants the postnatal transformation of BAT to WAT is ontogenically programmed. In humans, BAT is also restricted to neonates and found in adults only under pathological conditions (e.g. pheochromocytoma), but the extent to which this is similarly ontogenically programmed is a matter of debate (1). Recent studies in human tissues have observed islets of UCP1-expressing brown adipocytes within WAT depots (13, 14).

In contrast, many species such as rodents and dogs retain the ability to recruit BAT into adult life in response to cold environments via ß-adrenergic stimulated pathways (1). The molecular mechanisms responsible for this BAT recruitment have been studied in detail in rodents and have demonstrated that ß-adrenergic agonists activate the protein kinase A pathway, which induces the expression of UCP1 via the peroxisome proliferator-activated receptor (PPAR) and cAMP response element binding protein (CREB) families of transcriptional factors (15). It has also been proposed that the protein kinase A pathway activates the PPAR coactivator 1{alpha} (PGC-1{alpha}), which acts as a master regulator of BAT differentiation (16).

Based on the data, two opposing hypotheses may be advanced to account for the developmental suppression of UCP1 expression in the ovine: either this is due to a loss of expression of transcriptional activators/coactivators (PGC-1{alpha}, PPAR{alpha}, and PPAR{gamma}) or to the appearance of a yet unidentified corepressor. Here we present an in vivo analysis of UCP1, PGC-1{alpha}, and PPAR{alpha} expression that is consistent with the former model and show by cell culture that this effect cannot be overcome by pharmacological stimulation of the PPAR axis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and diets
Multiparous ewes (Scottish blackface x Bluefaced Leicester females crossed with a Suffolk male) were naturally inseminated. Twenty-four ewes bearing twin fetuses were selected by ultrasound scanning on d 90 of predicted fetal age and on d 100, ewes were housed in natural lighting and at ambient temperature (1–8 C) and fed hay plus concentrates to meet the energy requirements of maintenance and pregnancy. Lambs were born naturally and, apart from those sampled within 3 h of birth, allowed to suckle freely except when sampling.

Ontogenic changes in responses to isoprenaline administration
On d 123, 135, and 145 of predicted fetal age, three ewes and six fetuses were humanely slaughtered using an overdose of Pentojet (pentobarbitane sodium, 150 mg/kg). Blood samples were obtained from fetuses for plasma preparation, and PAT was removed quickly, snap frozen in liquid nitrogen, and stored at –80 C. Within 30 min of natural birth (d 0), four lambs were humanely slaughtered, blood sampled, and PAT removed and stored. On d 1, 5, 7, and 21, lambs were treated with either isoprenaline (four lambs) or saline (four lambs) over 13 h before humane slaughter and tissue sampling. Three sc injections of isoprenaline (0.0625 mg/kg in saline sterilized through a 0.2-µm filter) or saline (control) at intervals of 0, 4, and 8 h were administered. For the studies on d 5, 7, and 21, blood samples were taken over the last 5 h of isoprenaline administration; on the day before isoprenaline administration, polyvinyl catheters were inserted into the jugular vein of each lamb, and 1-ml blood samples were taken from lambs (0 h) before receiving the last of the three injections and at 0.5, 1, 1.5, 2, 3, 4, and 5 h. Plasma was prepared from blood samples and were stored at –20 C.

Cell culture
Stroma vascular cells were isolated from ovine adipose tissue taken on d 1 or 30 of life as described previously (12). Cells were then grown in growth medium (M199) supplemented with 20% of fetal calf serum. For differentiation, confluent cells were switched to DMEM/F12 without serum, and 24 h later, cells were also supplemented with adipogenic differentiation media containing T3 (2 nM), insulin (30 nM), and dexamethasone (10 nM) for 10 d. On d 10, the medium was supplemented with dimethylsulfoxide (control), the ß-adrenergic agonist isoprenaline (2 µM), the PPAR{alpha} agonist Wy14643 (5 µM), or the PPAR{gamma} rosiglitazone (100 nM) for 24 h, and then cells were harvested for RNA extraction as described below.

Analytical procedures
Plasma glucose and 3-hydroxybutyrate were assayed using kits from Sigma Diagnostics (St. Louis, MO). For RNA isolation, total RNA was extracted from ground ovine perirenal tissue (~50 µg) or cells using TRI reagent. Tissue samples were homogenized in 0.5 ml TRI reagent and incubated for 10 min at room temperature. Then 200 µl chloroform was added. After vigorous shaking and incubation at room temperature for 10 min, the homogenates were centrifuged at 12,000 x g for 15 min at 4 C to separate the phases. The top aqueous phase containing the RNA was transferred to a clean tube to which an equal volume of 100% isopropyl alcohol was added. Centrifugation at 12,000 x g for 10 min at 4 C yielded an RNA pellet that was washed in 80% ethanol followed by centrifugation at 12,000 x g for 5 min at 4 C. The final resulting RNA pellet was air dried at room temperature and resuspended in 50 µl RNase-free water.

Western blotting
Proteins from nuclear extracts or the cytoplasmic fraction were prepared as described previously (17), with 20 µg separated on an SDS-8% polyacrylamide gel and then transferred onto polyvinylidene fluoride membranes (Amersham Biosciences, Arlington Heights, IL). The membranes were incubated with antibodies against UCP1 (Calbiochem, La Jolla, CA) or PGC-1{alpha} (Calbiochem) according to the manufacturer’s protocol. Bands were visualized using the enhanced chemiluminescence reagent after application of horseradish-peroxidase-conjugated antibody (Cell Signaling Technology, Beverly, MA) according to the manufacturer’s protocol.

Northern blotting
Ten micrograms of total RNA were run on 1% agarose formaldehyde denaturing gel, transferred to Genescreen membrane (NEN Life Science Products, Norwalk, CT) and covalently fixed to the membrane by UV irradiation. {alpha}-32P-labeled probes were synthesized by random priming (QIAGEN, Crawley, UK) of cDNAs for the ovine genes UCP1 (AY371696), PGC-1{alpha} (AY345941), PPAR{alpha} (AY369138), PPAR{gamma} (AY367558), and glyceraldehyde-3-phosphate dehydrogenase (U94889). Serial hybridizations with different probes were performed. Membranes were subjected to autoradiography, and specific hybridization signals were quantified by measuring radioactivity by laser densitometry and expressed relative to the corresponding glyceraldehyde-3-phosphate dehydrogenase mRNA value to normalize for equal loading and transfer of RNA.

In situ hybridization and immunohistochemistry
UCP1 mRNA was detected in sections of adipose tissue using a digoxigenin end-labeled oligonucleotide probe, and UCP1 protein was detected using the techniques described by Finn et al. (8). Results are expressed as the percentage of perirenal adipocytes staining positive for either UCP1 mRNA or protein in four representative sections for each animal.

Quantitative RT-PCR
One microgram RNA served as a template for first-strand synthesis using poly (dT) primers and Omniscript reverse transcriptase (QIAGEN). Quantitative RT-PCR was performed using Sybr green (QIAGEN) according to the manufacturer’s instructions in Rotor Gene 3000 (Corbett Life Sciences, Sydney, Australia). Primer sets used were as follows: UCP1 forward, 5'-GCTAGTTTAGGAAGCAAG TC-3', and reverse, 5'-GCCCCGTCAAGCCTTCTGTTGTTG-3'; PGC-1{alpha} forward, 5'-GCGCCGTGTGATTTACGTT-3', and reverse, 5'-AAAACTTCAAAGCGGTCTCTCAA-3'; PPAR{alpha} forward, 5'-GACGAATGCCAAGATCTGAAAAAG-3', and reverse 5'-GAAGGGCGGATTGTTGTTGGTCT-3'; PPAR{gamma} forward, 5'-CCGCATCTTCCAGGGGTGTC-3', and reverse, 5'-CAAGGAGGCCAGCATCGTGTAAAT-3'; and 18S rRNA forward, 5'-GTAACCCGTTGAACCCCATT-3', and reverse, 5'-CCATCCAATCGGTAGTAGCG-3'. Each reaction yielded amplicons between 80 and 200 bp. PCR conditions were as follows: 20 sec at 95 C, 20 sec at 56 C, and 20 sec at 72 C for 35 cycles. After amplification, a melting curve (0.01 C/sec) was used to confirm product purity. Results are expressed relative to 18S rRNA.

Statistical analysis
Results are means ± SEM. Statistical significance was tested using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of development age on PAT gene expression and PGC-1{alpha} protein translocation to the nucleus
We first investigated the exact timing of the developmental suppression of genes involved in BAT differentiation by conducting an ontogenic gene profile during fetal and early neonatal development. As expected, expression of the key BAT marker gene UCP1 in PAT significantly (P < 0.01) increased 3-fold during the last 3 wk of fetal life, sharply peaking at 3 h of birth (Fig. 1AGo). Exposure to the cold extrauterine environment at birth will induce adrenergic stimulation that stimulates perirenal UCP1 expression (18, 19). Within 24 h after birth, UCP1 mRNA levels had decreased and by d 7 of life had nearly returned to levels observed at d 123 of fetal life. Measurement of UCP1 protein levels by Western blotting in the same samples revealed a similar pattern, although the peak in expression was delayed by 1 d, occurring on d 1 of postnatal life (Fig. 1BGo). Despite the postnatal fall in UCP1 expression, there were still significant amounts of UCP1 mRNA and protein present in PAT at d 21 (Fig. 1Go, A and B) These results clearly suggest that the ovine may be similar to humans in that UCP1-expressing cells can be found in WAT (14).


Figure 1
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  		FIG. 1. Effect of developmental age on gene expression and UCP1 protein content or PAT in fetal and neonatal lambs. A, UCP1 mRNA; B, UCP1 protein; C, PPAR{alpha} mRNA; D, PPAR{gamma} mRNA; E, PGC-1{alpha} mRNA. Results are shown as mean values for four animals ± SEM.

 
Expression of PPAR{alpha} followed a different pattern from UCP1; mRNA levels were high in fetal PAT over the last 3 wk of gestation and at birth, but 5 d after birth, PPAR{alpha} mRNA had precipitously declined (P < 0.001) to below detection limits, and no further change was observed through d 21 of life (Fig. 1CGo). PPAR{gamma} mRNA was expressed in PAT in both fetal and neonatal life (Fig. 1DGo). PGC-1{alpha} mRNA levels were high in fetal PAT over the last 3 wk of gestation and at birth, but within 24 h after birth, PGC-1{alpha} mRNA had precipitously declined (P < 0.001) to below detection limits, and no further change was observed through d 21 of life (Fig. 1EGo). Western blotting for PGC-1{alpha} protein in PAT demonstrated that this was mainly distributed in the cytoplasm during fetal life, but 1 d after birth, there was a rise in PGC-1{alpha} detected in nuclear protein extracts (Fig. 2Go) followed by a return to principally cytoplasmic distribution by d 5 and then loss of protein expression during the remaining neonatal period.


Figure 2
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  		FIG. 2. Effect of developmental age on nuclear and cytoplasmic PGC-1{alpha} protein content in PAT from fetal and neonatal lambs.

 
Effect of the ß-agonist isoprenaline on PAT differentiation, gene expression, and plasma metabolite concentrations in vivo
We have previously developed in situ hybridization and immunohistochemistry protocols for establishing the degree of brown adipocyte differentiation in vivo in ovine PAT based on the percentage of cells staining positive for either UCP1 mRNA or protein (8). Employing these systems, we confirmed our earlier findings that on d 1 of life, about 30% of PAT cells contained UCP1 mRNA, but by d 5 of life, UCP1 mRNA had disappeared from all PAT cells (Fig. 3AGo). At birth, all PAT cells can be classed as BAT because they all express UCP1 protein, but this rapidly declined to zero over the first 21 d of life as BAT is replaced by WAT. These results differ from the real-time PCR and Western blotting tissue measurements (Fig. 1Go, A and B) where UCP1 mRNA and protein could still be detected in PAT from 21-d-old lambs. This is likely to be due to the much greater sensitivity of these techniques compared with the in situ hybridization and immunohistochemistry techniques, respectively. The delayed loss of UCP1 protein compared with mRNA in both the tissue and cell analyses is explained by the long half-life of UCP1 (8).


Figure 3
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  		FIG. 3. Effect of isoprenaline or saline injection on the proportion of perirenal adipocytes staining positive on d 1, 5, 7, and 21 of life in neonatal lambs estimated by in situ hybridization and immunohistochemistry. A, UCP1 mRNA; B, immunoreactive UCP1. Results are shown as mean values ± SEM for four animals in each group from representative sections of adipose tissue.

 
Using this system, we next investigated whether the loss of UCP1-positive staining during ovine development could be reversed by prior administration of isoprenaline for 12 h. On d 1 of life, isoprenaline administration had no effect on the number of PAT cells expressing UCP1 mRNA and protein but on d 5 significantly (P < 0.001) reversed the loss of cells containing both UCP1 mRNA and protein (Fig. 3Go, A and B). By d 7 and 21 of life, isoprenaline was unable to overcome the loss in PAT cellular UCP1 mRNA or protein expression (Fig. 3Go, A and B). Therefore, the loss of BAT is reversible by administration of a mixed ß-agonist for up to 5 d after birth but becomes irreversible by d 7 of life. We then measured the expression of key genes involved in BAT differentiation using real-time PCR. Administration of isoprenaline did not alter PPAR{alpha} mRNA expression in PAT on d 1 of life but reversed the abrupt loss of expression on d 5 (P < 0.001, Fig. 4AGo). By contrast, d 7 PPAR{alpha} expression was completely suppressed in both saline- and isoprenaline-treated lambs (Fig. 4AGo). There was no effect of isoprenaline on expression of PPAR{gamma} at any of the development ages (Fig. 4BGo). On d 1 and 5 of neonatal life, PGC-1{alpha} mRNA was induced (P < 0.001) by isoprenaline administration from undetectable levels observed in the saline treatment, but by d 7, PGC-1{alpha} mRNA expression was totally suppressed in both saline- and isoprenaline-treated lambs (Fig. 4CGo). Therefore, the suppression of UCP1 expression after d 5 of life was accompanied by complete suppression of PPAR{alpha} and PGC-1{alpha} expression. This occurred despite increases in plasma glucose (Fig. 5AGo) and 3-hydroxybutyrate (Fig. 5BGo) over the first hours after the third isoprenaline administration.


Figure 4
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  		FIG. 4. Effect of isoprenaline or saline injection on gene expression in PAT on d 1, 5, 7, and 21 of life in neonatal lambs. A, PPAR{alpha} mRNA; B, PPAR{gamma} mRNA; C, PGC-1{alpha} mRNA. Results are shown as mean values ± SEM for four animals in each group.

 

Figure 5
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  		FIG. 5. Effect of isoprenaline or saline injection on plasma glucose (A) and 3-hydroxybutyrate (B) concentrations on d 7 and 21 of life in neonatal lambs. Results are shown as mean values ± SEM for four animals in each group.

 
Effect of developmental age on differentiation of adipocyte cultures in response to isoprenaline and PPAR agonists
We then determined whether preadipocytes prepared from 1-d-old and 1-month-old lambs could be differentiated into UCP1-expressing brown adipocytes. Stroma vascular preadipocytes were isolated from the ovine PAT depot on d 1 and 30 of life. Preadipocytes were cultured for 10 d in adipogenic differentiation media containing a cocktail of hormones and oil red staining used to establish the presence of lipid droplets and confirm that differentiation took place. The inclusion of T3, insulin, and dexamethasone was necessary for preadipocytes to differentiate into lipid droplet-containing cells. Preadipocytes prepared from 1-d-old lambs differentiated into brown adipocytes as judged by the expression of the BAT marker gene UCP1 and both PPAR{alpha} and PPAR{gamma} (Fig. 6AGo). By contrast, the preadipocytes from 30-d-old lamb PAT expressed much lower levels of UCP1, PPAR{alpha}, and PPAR{gamma} mRNA (Fig. 6AGo), indicating that increased developmental age had increased the capacity of preadipocytes to differentiate toward the WAT rather than the BAT lineage. We next examined whether this decreased capacity to differentiate into preadipocytes from 30-d-old lambs into brown adipocytes could be reversed by agonists proven to differentiate rodent preadipocytes into UCP1-expressing cells. Inclusion of the ß-agonist isoprenaline, the PPAR{alpha} agonist Wy14643, and the PPAR{gamma} agonist rosiglitazone for 24 h before harvesting cells did not further increase UCP1 mRNA in cultures prepared from 1- or 30-d-old lambs (Fig. 5AGo). The lack of effect of agonists in adipocyte cultures taken from 1-d-old lambs was consistent with the evidence that these are already maximally stimulated by the differentiation cocktail (12). Expression of PPAR{alpha} (Fig. 6BGo) and PPAR{gamma} (Fig. 6CGo) was significantly higher in the cultures prepared from 1-d-old lambs and not altered by inclusion of agonists. PGC-1{alpha} mRNA was expressed at low levels during differentiation of preadipocytes under all conditions (Fig. 6DGo). These results demonstrate that preadipocytes from 30-d-old lambs have a reduced ability to differentiate into brown adipocytes that cannot be overcome by the addition of isoprenaline, Wy14643, or rosiglitazone


Figure 6
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  		FIG. 6. Effect of adipogenic differentiation media (control), isoprenaline (Iso), Wy14643 (Wy), and rosigliatazone (Ros) on gene expression in differentiated adipocytes taken from 1-d-old PAT or 30-d-old PAT or sc adipose tissue (SCT) of neonatal lambs. Values are expressed as ratios of mRNA after 10 d of differentiation to undifferentiated (d 1) adipocytes A, UCP1 mRNA; B, PPAR{alpha} mRNA; C, PPAR{gamma} mRNA; D, PGC-1{alpha} mRNA. Results are shown as mean values for cultures prepared from three separate animals ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The characteristic ontogenic pattern of BAT development in the ovine of an increase during late fetal life and a decline during the first weeks of postnatal life was first noted by Gemmell et al. (11). These changes occurred despite animals being maintained in a cold environment and were accompanied by a loss of acute in vivo nonshivering thermogenic responses to ß-adrenergic challenge (20). Our previous studies have confirmed this earlier work by demonstrating that mitochondrial thermogenic activity and UCP1 protein content in PAT increase during the last month of fetal life, peaking at birth and then declining rapidly over the first 2 wk of neonatal life (8, 10, 21). In the present study, we have been able to confirm these changes in PAT UCP1 content and demonstrate that UCP1 mRNA rises during late fetal development and falls sharply on the first day of life even when lambs are maintained below their thermoneutral zone. We demonstrated using cell culture and in vivo responses to agonist challenges that the postnatal decline of UCP1 expression is associated with the loss of sensitivity to a sympathetic and PPAR agonists that coincide in vivo with an abrupt postnatal decline in PPAR{alpha} and PGC-1{alpha} expression. This provides evidence that the decline in BAT thermogenic function is the result of the loss of transcriptional activators during development in the ovine.

We first set out to establish the exact timing of the loss of BAT response to ß-adrenergic stimulation in the ovine and the relationship of the expression of PPAR{alpha}, PPAR{gamma}, and PGC-1{alpha} to changes in UCP1 induction during the first weeks of neonatal life. Previous studies have suggested that ruminant BAT differs from other mammals in being insensitive to the administration of ß3-adrenoreceptor agonists despite the presence of responsive ß-adrenergic receptors (9, 22). Administration of isoprenaline did not alter the high levels of UCP1 expression 24 h after birth, presumably because they were already maximally stimulated, but was able reinduce UCP1 mRNA and protein expression on d 5 even though these had sharply declined in the saline control group. This increase in UCP1 was accompanied by increases in PPAR{alpha} and PGC-1{alpha} expression. On d 7 and 21 of life, however, there were no detectable responses in UCP1, PPAR{alpha}, and PGC1{alpha} mRNA to isoprenaline administration. The timing of this loss of sensitivity to ß-adrenergic challenge occurs at the same time as the marked transformation of BAT to WAT (8) and is despite the development of systemic plasma glucose and 3-hydroxybutyrate metabolic responses to ß-adrenergic challenge over this period.

The ontogenic profile of UCP1 expression demonstrated a rise during the last month of fetal life followed by a decrease after birth even though lambs were born into an environment below their thermoneutral zone. This suggests that the isoprenaline-induced increase in UCP1 mRNA on d 5 was a response to a pharmacological rather than a physiological level of ß-adrenergic challenge. These studies in the ovine contrast with those in rodents where although BAT UCP1 expression similarly rises in late fetal life and in a thermoneutral environment falls after birth, expression can be readily induced by exposure to cold or ß-adrenergic challenge even in adult animals (19, 23).

The disappearance of ß-adrenergic sensitivity cannot be attributed to a loss of ß-adrenergic receptors since (24) demonstrated that expression of both ß1 and ß3-receptor mRNA and adenylate cyclase response to isoprenaline in ovine PAT was maintained during neonatal life, only disappearing after three months of life. Furthermore, studies in neonatal rodents have demonstrated that isoprenaline treatment invokes sensitization rather than desensitization of ß-adrenergic responses (25). We have also been able to rule out the possibility that the transition from BAT to WAT is due to apoptosis of brown adipocytes because conversion of BAT to WAT occurs without signs of apoptosis using both the DNA ladder and terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate nick end labeling methods (Finn, D., P. Trayhurn, and M. A. Lomax, unpublished). Furthermore, the conversion of ovine BAT to WAT morphological characteristics occurs without ultrastructural evidence of brown adipocyte organelle degeneration (11, 19).

Two opposing hypotheses may be advanced to account for the developmental programmed suppression of UCP1 expression in the ovine: either the loss of expression of transcriptional activators/oactivators (PPAR{alpha}, PPAR{gamma}, or PGC-1{alpha}) or the appearance of a yet unidentified corepressor. Our studies provide clear evidence to support the former hypothesis based on the decline in postnatal expression of PPAR{alpha} and PGC-1{alpha} in PAT, the low expression of UCP1, PPAR{alpha}, and PGC-1{alpha} in WAT depots in 30-d-old lambs (results not shown), and the postnatal loss of in vivo expression of these factors in response to isoprenaline challenge. We further carried out a series of cell culture experiments to establish whether developmental age altered ovine BAT differentiation response to a wider range of agonists known to stimulate BAT differentiation in rodents. A previous study demonstrated that preadipocytes taken from newborn PAT can be differentiated into the UCP1-expressing BAT phenotype, but preadipocytes from 30-d-old lambs cannot differentiate into brown adipocytes (12). We isolated preadipocytes from the stromal vascular fraction of PAT on d 1 and 30 of life and examined the effect of isoprenaline, Wy14643, or rosiglitazone, which have previously been shown to be BAT differentiation triggers in rodent cell cultures studies (26, 27, 28). Expression of UCP1 mRNA in differentiated adipocytes was 5-fold greater from newborn compared with 30-d-old preadipocyte cultures, but addition of isoprenaline or PPAR{alpha} and PPAR{gamma} agonists to the culture media failed to overcome the suppression of UCP1 expression in the adipocyte cultures from 30-d-old lambs. Ovine preadipocytes in culture clearly behave differently from those isolated from rodents, rabbits, and humans in that they fail to increase UCP1 expression in response to ß-adrenergic, PPAR{alpha}, and PPAR{gamma} agonists (29, 30, 31). Cambon et al. (30) have demonstrated that 1-month-old rabbit preadipocytes can be induced to differentiate into BAT cells simply by adding the PPAR{gamma} agonist rosiglitazone, but no agent has to date been found that can enhance UCP1 gene expression in 1-month-old lambs. There were 50- to 100-fold decreased levels of PPAR{gamma} mRNA and 3-fold decreased PPAR{alpha} mRNA in the 30-d-old ovine, adipocyte cultures relative to d-1 cultures, but there were no significant effects on PGC-1{alpha} expression. Combined, the results of our ovine adipocyte and in vivo experiments suggest that the switch from BAT to WAT development during the first month of life is a result of decreased levels of PPAR{alpha} expression.

In rodents, several studies have supported a major role for PPAR{alpha} in UCP1 expression; PPAR{alpha} is expressed in BAT but not WAT (32) and increases during cold exposure and fetal and postnatal life in parallel with increases in UCP1 mRNA (17). PPAR{alpha} is highly correlated with UCP1 in mammary adipose tissue during development (33) and also in two mouse strains differing in UCP1 expression where quantitative trait loci mapping has established a linkage between PPAR{alpha} and UCP1 gene expression (28). Studies in rodent and human cell cultures and in vivo in rodents have shown that PPAR{alpha} agonists increase UCP1 expression by binding to a promiscuous response element on the UCP1 enhancer that recognizes both PPAR{alpha} and PPAR{gamma} (27, 28, 29).

In mice, the developmental increase in PPAR{alpha}, PPAR{gamma}, and PGC-1{alpha} are relatively modest compared with the 100-fold rise in UCP1. These results have been elegantly explained by Rim et al. (17) who have demonstrated that these transcriptional factors are sequestered in the cytoplasm and translocated into the nucleus when animals experience the adrenergic challenge of a cold environment. Our results indicate a similar mechanism could be involved in the ovine because PGC-1{alpha} was predominately cytoplasmic except for d 1 of life when appreciable amounts were detected in the nucleus. This mechanism, however, does not appear to be of major importance because, in contrast to rodents, both PGC-1{alpha} and PPAR{alpha} are completely repressed by d 5 of life, suggesting that expression of these transcriptional regulators is ontogenically switched off. This mechanism may be of more relevance to PPAR{gamma}, which is expressed at relatively constant levels in BAT of both rodents (17) and ovine (this study). A previous ovine study of fetal BAT development suggested a positive relationship between PPAR{gamma} and UCP1 expression (34).

Despite the evidence for the role of PPARs in UCP1 expression, genetic ablation of either PPAR{alpha} (28) or PPAR{gamma} (35) fails to prevent UCP1 expression in BAT. However, PGC-1{alpha} knockout mice do not increase UCP1 expression in BAT in response to cold (27, 36, 37, 38). The evidence in rodents suggests that there is a complex interaction between the PPARs, the retinoid X receptor, and PGC-1{alpha} in the control of UCP1 gene expression that is capable of adapting to redundancy in one or more components, but additional studies are required to elucidate these interactions during development (26). Our experiments suggest that the ovine represents an extreme example of BAT regulation where there is a complete loss of expression and adrenergic sensitivity of two key BAT differentiation inducers, PPAR{alpha} and PGC-1{alpha}, soon after birth, resulting in the irreversible transdifferentiation of BAT to WAT. Additional mechanisms involving transcriptional repressors may be involved because studies have observed transdifferentiation of rodent brown to white adipocytes under a number of situations (e.g. activation of Wnt signaling) (13).

In conclusion, we suggest that the loss of BAT during development in the ovine is due to ontogenically programmed suppression of PPAR{alpha} and PGC-1{alpha} expression and results in the loss of sensitivity to a ß-adrenergic agonist, which causes transdifferentiation of BAT to WAT


   Acknowledgments
 
We are grateful to David Finn, John Struthers, and Margaret Wallace for expert technical assistance and the Biotechnology and Biological Sciences Research Council for funding this work.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online October 5, 2006

Abbreviations: BAT, Brown adipose tissue; PAT, perirenal adipose tissue; PGC-1{alpha}, PPAR coactivator 1{alpha}; PPAR, peroxisome proliferator-activated receptor; UCP1, uncoupling protein-1; WAT, white adipose tissue.

Received July 10, 2006.

Accepted for publication September 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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