In the present study we intended to determine how BAT (brown adipose tissue) maintained thermogenesis under treatment with OE (oleoyl-oestrone), a powerful slimming hormone that sheds off body lipid but maintains the metabolic rate. Overweight male rats were subjected to daily gavages of 10 nmol/g of OE or vehicle (control) for 10 days. A PF (pair-fed) vehicle-receiving group was used to discount the effects attributable to energy availability limitation. Interscapular BAT mass, lipid, DNA, mRNA and the RT-PCR (real-time PCR) expression of lipid and energy metabolism genes for enzymes and regulatory proteins were measured. BAT mass and lipid were decreased in OE and PF, with the latter showing a marked reduction in tissue mRNA. Maintenance of perilipin gene expression in PF and OE rats despite the loss of lipid suggests the preservation of the vacuolar interactive surface, a critical factor for thermogenic responsiveness. OE and, to a lesser extent, PF maintained the expression of genes controlling lipolysis and fatty acid oxidation, but markedly decreased the expression of those genes involved in lipogenic and acyl-glycerol synthesis. OE did not affect UCP1 (uncoupling protein 1) (decreased in PF), β3 adrenergic receptors or hormone-sensitive lipase gene mRNAs, which may translate in maintaining a full thermogenic system potential. OE rats were able to maintain a less energetically stressed BAT (probably through glucose utilization) than PF rats. These changes were not paralleled in PF rats, in which lower thermogenesis and glucose preservation resulted in a heavier toll on internal fat stores. Thus the mechanism of action of OE is more complex and tissue-specific than previously assumed.

INTRODUCTION

OE (oleoyl-oestrone) is a powerful slimming signal from adipose tissue that elicits both central and peripheral energy wasting effects [1,2], and has been postulated as a ponderostat signal [3]. OE decreases food intake without significant changes in energy expenditure [1], and the resulting energy gap is fulfilled essentially by lipid mobilization [1,3] in WAT (white adipose tissue). This lipid is mainly used by muscle, at least in obese rats [4], decreasing hyperlipidaemia and inducing a marked decrease in circulating cholesterol [5]. Under OE treatment, glycaemia is maintained in the range of normalcy, but with lower insulin and leptin levels [5].

The marked WAT energy wasting induced by OE cannot be justified by increased catecholamine sensitivity [6], or increased stimulation of expression of key regulators of lipolytic pathways [4], but essentially occurs by powerful inhibition of lipogenesis [7], which results in an unbalanced lipid equilibrium favouring the massive release of fatty acids and glycerol. This selective voiding of fat reserves is complemented by a lowered peripheral utilization of glucose [8], which reinforces the decreased food intake, partly induced by the inhibition of ghrelin expression in the stomach, and central effects on the brain [2] not mediated by neuropeptide Y. The relative availability of glucose can be attested by the accumulation of liver glycogen [1] in spite of the partially maintained hepatic conversion of glucose into triacylglycerols which, together with those from WAT, increase the liver lipoprotein output in order to fuel muscle and other peripheral tissues (M. M. Romero, J. A. Fernández-López, M. Alemany and M. Esteve, unpublished work).

BAT (brown adipose tissue) is a particular type of adipose tissue that contains all of the basic metabolic machinery of WAT, complemented by a powerful oxidative mitochondrial apparatus and the unique presence of a functional UCP1 (uncoupling protein 1) [9], as well as a tighter control of both blood flow and direct stimulation of cells by sympathetic nerves [10]. The apparent ‘quietness’ of BAT during the energy turmoil elicited by OE may be the consequence of unaltered thermogenic activity, which corresponds with energy wasting elsewhere. This peculiarity has been tested in the present study compared with a situation in which the limited energy intake (pair-feeding) theoretically exerts the same metabolic pressure on the rat body energy budget management, independently of OE treatment. The effects of OE are quite different in liver and WAT, but BAT could not be readily compared with either model because of the unique function of this tissue in thermogenesis, at least in rodents.

MATERIALS AND METHODS

Adult male Wistar rats were made overweight by cafeteria diet feeding for a limited period, as previously described [11]. The rats, initially weighing 355±5 g, were kept under standard conditions for housing and feeding. Three groups of eight rats each were randomly selected: control (C), OE and PF (pair-fed). Each day, all of the animals received an oral gavage of 0.2 ml of sunflower oil (7 kJ), which was supplemented in the OE group with 10 nmol/g of OE (OED, Barcelona, Spain). The control and OE groups had free access to pellet food (maintenance chow, Panlab, Barcelona, Spain), and the PF group were allowed only the amount of food consumed by the OE group each day; all rats had water available ad libitum. PF rats completely ate the food allotted each day. On day 10, the rats were killed and the interscapular BAT was rapidly excised, weighed, sampled, frozen and stored at −80°C until processed.

The animals were housed, handled and killed following the procedures approved by the University of Barcelona Animal Welfare and Ethics Committee, in full complement of the procedures set forth by the European Union and the Governments of Spain and Catalonia.

Tissue samples were used for the estimation of total lipid by extraction with trichloromethane/methanol [12]. DNA was measured using a standard fluorimetric method with 3,5-diaminobenzoic acid (Sigma, St Louis, MO, U.S.A.) and bovine DNA (Sigma) as a standard [13]. Tissue DNA content allowed the calculation of the approximate number of cells per gram of tissue and in the whole interscapular BAT pad, based on the assumption that, in mammals, the cell DNA content is constant; we used the genomic DNA size data [14] for somatic rat cells (5.6 pg/cell). Mean cell mass was estimated from the number of cells and the organ mass.

Total tissue RNA was extracted using Tripure reagent (Roche Applied Science, Indianapolis, IN, U.S.A.), and was quantified in a ND-100 spectrophotometer (Nanodrop Technologies, Wilmington, DE, U.S.A.). RNA samples were reverse transcribed using the MMLV (Moloney-murine-leukaemia virus) reverse transcriptase (Promega, Madison, WI, U.S.A.) and oligo-dT primers. Total tissue mRNA was determined by using the poly-(A)mRNA detection system kit (Promega).

RT-PCR (real-time PCR) amplification was carried out using reaction mixtures (10 μl final volume) containing Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, U.S.A.), the equivalent of 8 ng of reverse-transcribed RNA and 300 nM primers. Reactions were performed on an ABI PRISM 7900 HT detection system (Applied Biosystems) using a fluorescent threshold manually set to an absorbance (A) of 0.500 for all runs. The primers used for the estimation of gene expression in interscapular BAT are shown in Supplementary Table S1 (at http://www.bioscirep.org/bsr/029/bsr0290237add.htm).

A semi-quantitative approach for the estimation of the concentration of specific gene mRNAs per unit of tissue mass was performed as previously described [15]. In any case, cyclophilin was used as the control gene in all samples. The data are presented as the amount of copies (fmoles) present in the whole interscapular BAT mass, as an indication of the potential for synthesis of these specific proteins in an organ which size changes with treatment. This may allow for comparison between groups in a more direct way than a simple comparison of relative expressions.

Statistical comparison between groups was established by using the unpaired Student's t test with a limit of significance of P<0.05 using the GraphPad Prism5 program (GraphPad, La Jolla, CA, U.S.A.).

RESULTS

At the end of the study, and in relation to their initial masses, control rats gained 2.6±0.4% (not significant compared with initial values), PF rats lost 8.5±0.7% (P<0.05 compared with initial values and the gain of the control group) and OE rats lost 9.9±0.7% (P<0.05 compared with initial values and the gain of the control group). Table 1 presents the nucleic acid and cell contents of the interscapular BAT of overweight male rats treated with OE compared with control and PF rats. OE treatment resulted in the loss of approx. 40% of whole BAT lipids compared with control rats, whereas PF rats lost 57%; the differences being significant compared with control rats, but not between OE and PF groups. Both OE and PF treatments resulted in the loss of approx. one-third of the tissue mass, but with no parallel loss of cells. In any case, the RNA/DNA ratio decreased, which suggests a diminished cell function overall. However, the cells of the OE group maintained the same amount of mRNA per cell as control cells, and PF cell mRNA levels were approximately half of those of control and OE cells. The loss of tissue mass and the maintenance of cell number resulted in smaller mean cell sizes (the results include both adipocytes and non-adipocyte cells) for OE and PF rats compared with control rats.

Table 1
Nucleic acid content and cellularity in interscapular BAT of OE-treated and PF male overweight rats

The parameters represent the absolute content in the total interscapular BAT. The data are the means±S.E.M. for eight different animals. *P<0.05 compared with controls; †P<0.05 between PF and OE-treated rats.

ParameterControl groupPF groupOE group
Tissue mass (mg) 551±30 363±29* 379±21* 
Lipid content (mg per g of tissue) 569±34 374±64* 497±19 
Lipid content (mg per BAT site) 314±18 136±20* 188±16* 
DNA content (μg per g of tissue) 1.57±0.12 2.68±0.12* 2.54±0.12 
DNA content (mg per BAT site) 0.87±0.06 0.94±0.06 0.95±0.06 
Total RNA content (mg per g of tissue) 1.05±0.02 1.29±0.04*† 1.17±0.03* 
Total RNA content (mg per BAT site) 0.58±0.03 0.46±0.03* 0.44±0.02* 
RNA/DNA ratio 0.70±0.06 0.49±0.01* 0.47±0.02* 
Number of cells (×106 cells per BAT site) 155±13 168±9 170±11 
Mean cell mass (ng) 3.72±0.30 2.15±0.10* 2.25±0.11* 
Total mRNA content (μg per g of tissue) 20.7±2.3 14.5±1.9*† 28.3±2.6* 
Total mRNA content (fg/cell) 70.7±8.4 32.3±3.3*† 62.5±6.7 
ParameterControl groupPF groupOE group
Tissue mass (mg) 551±30 363±29* 379±21* 
Lipid content (mg per g of tissue) 569±34 374±64* 497±19 
Lipid content (mg per BAT site) 314±18 136±20* 188±16* 
DNA content (μg per g of tissue) 1.57±0.12 2.68±0.12* 2.54±0.12 
DNA content (mg per BAT site) 0.87±0.06 0.94±0.06 0.95±0.06 
Total RNA content (mg per g of tissue) 1.05±0.02 1.29±0.04*† 1.17±0.03* 
Total RNA content (mg per BAT site) 0.58±0.03 0.46±0.03* 0.44±0.02* 
RNA/DNA ratio 0.70±0.06 0.49±0.01* 0.47±0.02* 
Number of cells (×106 cells per BAT site) 155±13 168±9 170±11 
Mean cell mass (ng) 3.72±0.30 2.15±0.10* 2.25±0.11* 
Total mRNA content (μg per g of tissue) 20.7±2.3 14.5±1.9*† 28.3±2.6* 
Total mRNA content (fg/cell) 70.7±8.4 32.3±3.3*† 62.5±6.7 

Figure 1 shows the changes induced either by pair-feeding or OE treatment on the expression of a number of energy handling and control-related genes, presented as a percentage of the corresponding control values. OE treatment and pair-feeding induced a similar marked decrease in the expression of GLUT4 and hexokinase, and OE decreased the expression of the NADPH-producing malic enzyme. PF raised the expression of phosphoenolpyruvate carboxykinase and pyruvate dehydrogenase kinase 4 compared with both control and OE rats. Supplementary Tables S2 and S3 (at http://www.bioscirep.org/bsr/029/bsr0290237add.htm) show in absolute terms, i.e. fmoles in the whole interscapular BAT mass, expression of the genes presented in Figure 1 in order to allow for more quantitative comparisons.

Percentage of mRNA for enzyme and regulatory protein expression in the interscapular BAT mass of overweight male Wistar rats treated with OE or PF compared with control rats

Figure 1
Percentage of mRNA for enzyme and regulatory protein expression in the interscapular BAT mass of overweight male Wistar rats treated with OE or PF compared with control rats

The values represent the content of specific gene mRNA in the whole interscapular BAT mass, and are expressed as a percentage of the expression in the control rats (see Supplementary Tables S2 and S3 at http://www.bioscirep.org/bsr/029/bsr0290237add.htm), and are the means±S.E.M. for eight different animals. PF, white bars; OE, grey bars. Statistical significance of the differences between groups: stars indicate P<0.05 compared with the control group (100%), and open circles indicate P<0.05 between OE and PF groups.

Figure 1
Percentage of mRNA for enzyme and regulatory protein expression in the interscapular BAT mass of overweight male Wistar rats treated with OE or PF compared with control rats

The values represent the content of specific gene mRNA in the whole interscapular BAT mass, and are expressed as a percentage of the expression in the control rats (see Supplementary Tables S2 and S3 at http://www.bioscirep.org/bsr/029/bsr0290237add.htm), and are the means±S.E.M. for eight different animals. PF, white bars; OE, grey bars. Statistical significance of the differences between groups: stars indicate P<0.05 compared with the control group (100%), and open circles indicate P<0.05 between OE and PF groups.

OE induced a marked decrease in the expression of lipogenic enzyme genes, a decrease that was less extensive in PF rats (which even showed an increased expression of acetyl-CoA carboxylase 2 compared with control rats). OE induced a dramatic decrease in the expression of stearoyl-CoA desaturase, which was unaffected in PF rats. Glycerol-phosphate acyl-transferase gene expression was decreased in both groups compared with control rats. Fatty acid transport, translocation and binding enzyme gene expressions were not altered by either treatment, the exceptions being a decrease in expression of fatty acid transport protein in OE rats (unaffected in PF rats) and a reduction in expression of fatty acid-binding protein 3 in PF rats, which was not altered by OE. Gene expression of the β-oxidation enzyme acyl-CoA dehydrogenase isoenzyme was unaffected by OE and PF, except for decreased medium-chain dehydrogenase expression in PF rats.

With the exception of a considerable decrease in the expression of the adiponutrin gene in both OE and PF rats, gene expression of lipases was unaffected by OE, and only lipoprotein lipase expression was enhanced by pair-feeding. Perilipin expression was unchanged in both OE and PF rats compared with control rats.

The expression of the UCP1 gene was decreased in PF rats compared with control rats, but this did not occur in those treated with OE. The expression of the β1 adrenergic receptor gene was decreased in both OE and PF rats, but that of the β3 adrenergic receptor was unchanged, and the expression of the β2 adrenergic receptor was considerably higher in PF rats compared with both control and OE rats. No differences were observed in the expression of phosphodiesterase in comparison with control rats. The oestrogen receptor α gene more than doubled its expression in PF rats, but OE and control rats showed similar values. Neither treatment changed the expression of the thyroid hormone receptor gene, but both PF and OE treatment decreased the expression of thyroxine deiodinase.

OE decreased the gene expression of PPAR (peroxisome-proliferator-activated receptor) γ1 and its coactivator 1α, as well as the expression of phosphatase and tensin homologues, nuclear respiratory factor 1, ChREBP (carbohydrate-responsive element-binding protein) and the Spot 14/thyroid hormone-responsive protein. PF rats only displayed a reduction in the gene expression of ChREBP and the Spot 14/thyroid hormone-responsive protein, and displayed an increase in expression of the PPARα gene.

DISCUSSION

Under normal conditions, perilipin levels (and gene expression) are assumed to be related to the vacuole size (surface) [16] and BAT contains multiple vacuoles per cell that facilitate the rapid mobilization of fat. The lack of change in perilipin expression despite a decrease in cell size due to the observed loss of fat (and maintenance of cell numbers) resulted in the following values for perilipin mRNA (in fmol/g of tissue lipid): 148±20 (control rats), 309±22 (PF) and 207±17 (OE), the differences in PF compared with control and OE rats being significant. Expression of perilipin in PF rats was actually increased when compared with total tissue mRNA, resulting in remarkably similar expression in all of the three groups studied, irrespective of the much lower lipid content and smaller mean cell size of PF and OE. The data on fat and cell number indicate that the mean lipid vacuole size must be smaller in OE and PF rats than in control rats, despite the other differences in metabolism. However, unchanged perilipin expression is probably correlated to maintenance of perilipin levels, and to unchanged vacuolar lining surface area, roughly proportional to perilipin content [16]. Consequently, the total surface area of vacuoles could not be maintained following vacuolar shrinking, which occurs as a result of the loss of cell fat. This means that the total number of vacuoles must be increased to pack a lower volume of fat with a closely similar estimated surface area in PF and OE rats than in control rats, thus preserving the cytoplasm/fat droplet interface area and consequently the responsiveness to hormonal stimulation. The unchanged expression of the hormone-sensitive lipase gene agrees with this interpretation. The data presented indirectly hint at the maintenance of the overall interactive surface of the BAT vacuole as being an important factor in the thermogenic-response capability of the tissue.

The loss of fat (and thus postulated droplet fractionation) was more marked in PF than in OE rats, which somehow were able to maintain a much less energetically stressed tissue (probably through glucose utilization) than PF animals. This buffered effect of energy loss in OE rats is again exemplified by the marked difference in total tissue mRNA content between control rats and OE rats on one side and PF on the other, which in BAT agrees with our previous findings that the effects of OE on energy balance are not mediated simply by a decrease in dietary energy availability [17]. However, this lower mRNA content in PF rats is in full agreement with the decreased thermogenesis and BAT function [18] of food-deprived animals. Lowered mRNA levels are an index of decreased protein synthesis, in itself a normal mechanism of decreasing energy expenditure through slower protein turnover [19]. The loss of lipids in PF rats tends to be higher than in OE rats, the latter maintaining UCP levels and the full complement of mRNA (i.e. protein synthesis), which means that the flow of energy substrates to and from BAT should be different for PF and OE rats. This is supported by the differences in lipoprotein lipase gene expression, but not by the gene expression of other lipases and most fatty acid transportation/translocation enzyme genes. It should be noted, however, that the presence of the same amount of a given specific mRNA in BAT from PF rats represents a much higher ‘selective effort’ of translation than in OE or control animals, which hints at the relative importance for the tissue of the maintenance of the corresponding pathway with respect to all other pathways. This way we can observe that lipase and β-oxidation operation, as well as 3-carbon fragment preservation through activation of pyruvate dehydrogenase kinases are important pathways that seem to receive this special treatment in food restriction.

The effects of limited food availability and OE treatment on the cellularity, cell size and mass of lipid reserves of BAT were apparently similar. Analysis of the expression of the main genes controlling lipid and energy metabolism seems to confirm this initial assumption, but the effects induced by OE were deeper than those of simple food restriction (pair-feeding), since lipogenesis gene expression was more markedly inhibited in OE than in PF rats.

The postulated reduction of NADPH synthesis in OE rats (malic enzyme) suggest a lower synthetic drive [20], this is further stressed by a much decreased expression of stearoyl desaturase which is not observed in PF rats, and the differentially more marked decrease in the expression of lipogenic enzyme genes in OE rats compared with both control and PF rats. The marked decrease in adiponutrin expression in OE and PF supports a probable inhibition of lipogenesis, given the nature of adiponutrin as a marker of lipid biosynthesis [21] in addition to reflecting decreased food intake.

Glucose uptake and phosphorylation seem to be inhibited in both OE and PF animals because of the decreased expression of GLUT4 and hexokinase 2. However, 3-carbon fragment utilization through the pyruvate dehydrogenase pathway was probably increased in OE rats and decreased in PF rats (lower kinase 2 gene expression in OE rats and higher kinase 4 gene expression in PF rats).

The potential to dispose of internal fat stores remained unchanged in all three groups, as was β-oxidation (only a decrease in medium-chain acyl-CoA dehydrogenase gene expression in PF rats was shown). The possible incorporation of lipid from lipoproteins was unchanged in OE rats, since the decreased activity found in WAT [5] was not paralleled in BAT by the expression (unchanged) of lipoprotein lipase. In PF rats, lipoprotein lipase expression actually increased, which suggests that circulating lipids may continue to be taken up for energy supply under food restriction conditions [22]. The differential expression of fatty acid-binding protein 3 [23] and fatty acid-transport protein 1 genes [24] in PF and OE rats may be directly related to this different potential use of circulating lipoprotein lipid, since the latter is closely related to fatty acid transport into the cell [25].

The raised expression of the PPARα gene in PF rats compared with control rats and its lack of difference in OE rats agree with a maintainance of lipolytic activity in OE rats. The more lipogenic PPARγ [26] expression was again strongly inhibited by OE, since PPARγ activation increases both lipid and adipose tissue masses [27].

Lipid synthesis is partially controlled in adipose tissue by the insulin signalling cascade [28] through modulation of SREBP1c (sterol-regulatory-element-binding protein 1c) [29] and ChREBP [30]. Both factors have been found to play a key role in OE-stimulated liver lipogenesis [7], which is in part fuelled by glucose in spite of the generalized body lipid mobilization and decreased food intake. In the present study, however, SREBP1c remained unchanged in both PF and OE rats, even when the Spot 14/thyroid hormone-responsive protein, closely related to SREBP1c action [31] and also a marker of lipogenesis, was inhibited, as was that of ChREBP. These results suggest that OE modulation of lipid metabolism did not proceed along the same pathways in liver and BAT.

However, the possibly most far-reaching ‘metabolic’ difference between PF and OE rats was the relative maintenance of the expression of UCP1 in OE rats compared with its significant fall in PF rats. This finding is in agreement with the known maintenance of energy expenditure under OE treatment [1], in contrast with its decrease under conditions of starvation or food restriction.

The maintenance of an unchanged adrenergic pathway (β3 adrenergic receptors, phosphodiesterase, hormone-sensitive lipase and maintained vacuolar surface area) and UCP1 ensures the full operativity of the thermogenic response to eventual adrenergic stimuli in OE rats. These arrangements are less complete or probably less efficient in PF animals. Decreased adrenergic receptor expression is often a consequence of saving strategies under conditions of limited energy availability; the differential expression of UCP1 in OE and PF rats contrasts with the lack of change in β3 adrenergic receptors that control UCP1 expression [32]. A reduction in expression of UCP1 in PF rats may be related to the effects of oestrogen signalling, which reduces the sensitivity to catecholamine stimulation in BAT [33] as a way to decrease thermogenic responsiveness. However, the less marked increase in oestrogen receptor expression in OE rats may be either a consequence of increased circulating oestrogen in OE rats [34] that may help down-regulate the expression of the receptor, or another way of maintaining the ability of the BAT thermogenic system to respond to hormonal signals.

The data presented on gene expression suggest that, in PF rats, the BAT energy needs are low, and thus limited glucose availability (very low 3-carbon fragment conversion into acetyl-CoA) was translated into decreased fat stores despite external lipid utilization. However, in OE rats, this same overall energy scarcity was compounded by the need to fuel thermogenesis, which poses an additional burden for BAT, and can explain why glucose utilization (in spite of assumedly limited entry) may be increased, and why lipid synthesis is strongly limited in contrast with β-oxidation, which is only slightly decreased. The success of this OE-driven strategy is proven by both the minor change in metabolic orientation, maintenance of thermogenesis and the lower overall loss of lipid. Since the main difference between PF and OE rats seems to be in the regulation of pyruvate dehydrogenase (and a stronger inhibition of lipid synthesis in OE) through differential gene expression, it can be speculated that the continuous availability of glucose in OE rats [5,8] may be a key factor in the preservation of part of the BAT lipid and the full operativity of thermogenesis.

Pair-feeding changed the expression of growth and differentiation factors little compared with control rats, in contrast with OE-treated rats. The expression of the phosphatase and tensin homologue, which is implicated in cell survival [35], as well as the PPARγ coactivator 1α [36] and the nuclear respiratory factor 1 genes [37] related to mitochondriogenesis, were inhibited by OE, but not by pair-feeding. However, the gene expression of neither the vascular endothelial growth factor A, an angiogenic factor [38], nor the mitochondriogenic transcriptional factor A [39] were altered by OE (or by PF), suggesting few changes in tissue architecture in parallel with the loss of tissue mass and fat (but not cells). In any case, the effects of OE were more marked than those of pair-feeding. The better maintenance of expression of cell development factors by PF rats disagrees with the decrease in UCP1 expression, which is presumably an index of thermogenic capability. The cell changes cannot be attributed directly to a change in thyroid receptor regulation, in spite of the important control of BAT development and function by the thyroid. The decrease in thyroxine deiodinase expression may be a direct consequence of energy scarcity [40].

In spite of some differences in the control of tissue factors, the limits set for the synthesis of fat, management of its stores, the use of glucose and the maintenance of thermogenic potential conform with the bulk of the changes in gene expression induced by OE on BAT. These changes were not paralleled in PF rats, which used a lower thermogenesis and glucose-preservation strategy which takes a heavier toll on internal fat stores. The effects of OE on BAT energy metabolism are, thus, fairly different from those observed in the liver, which suggests that the mechanism of action of OE is more complex and tissue-specific than previously assumed.

Abbreviations

     
  • BAT

    brown adipose tissue

  •  
  • ChREBP

    carbohydrate-responsive element-binding protein

  •  
  • OE

    oleoyl-oestrone

  •  
  • PF

    pair-fed

  •  
  • PPAR

    peroxisome-proliferator-activated receptor

  •  
  • SREBP1c

    sterol-regulatory-element-binding protein 1c

  •  
  • UCP

    uncoupling protein

  •  
  • WAT

    white adipose tissue

We thank OED SL (Barcelona, Spain) for supplying oleoyl-oestrone and for use of instruments.

FUNDING

This work was supported by the Fondo de Investigaciones Sanitarias [grant number PI052179]; the Plan Nacional de Investigación en Biomedicina [grant number SAF2006-05134] (both from the Government of Spain); OED SL; and the Centros de Investigación Biomédica en Red Fisiopatología de la Obesidad y Nutrición of the Spanish Institute of Health Carlos III.

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Supplementary data