PPAR-␥2 Expression in Response to Cafeteria Diet: Gender- and Depot-Specific Effects Enrique Rodrı´guez, Joan Ribot, Ana M. Rodrı´guez, and Andreu Palou
Abstract RODRI´GUEZ, ENRIQUE, JOAN RIBOT, ANA M. RODRI´GUEZ, AND ANDREU PALOU. PPAR-␥2 expression in response to cafeteria diet: gender- and depotspecific effects. Obes Res. 2004;12:1455–1463. Objective: To investigate the effects of short-term cafeteria (CAF) diet feeding on the expression of adipogenic transcription factors and their association with adiposity. Research Methods and Procedures: Four-week-old male and female Wistar rats were fed CAF diet or standard chow for 2 weeks. Body weight, energy intake, tissue weights, and serum parameters were determined. Peroxisome proliferator-activated receptor (PPAR)-␥2, PPAR␣, CCAAT enhancer-binding protein-␣, and adipocyte differentiation and determination factor 1 mRNAs in gonadal white adipose tissue (gWAT) (visceral depot) and inguinal white adipose tissue (iWAT) (subcutaneous depot) and in interscapular brown adipose tissue were measured by reverse transcription-polymerase chain reaction. Results: Short-term CAF diet feeding resulted in increases in body weight, adipose tissue weights, and lipid serum levels. Increased adiposity was more related to an increase in visceral fat than an increase in subcutaneous fat. This difference was associated with a higher expression of key adipogenic transcription factors (mainly PPAR␥2 and CCAAT enhancer-binding protein-␣) in gWAT when compared with iWAT. Higher hypertrophy of gWAT was found in females, whereas males showed a higher hypertrophy of iWAT. Differential gender and depot response to CAF diet could be explained by depot and gender differential expres-
Received for review November 19, 2003. Accepted in final form July 1, 2004. The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Laboratori de Biologia Molecular, Nutricio´ i Biotecnologia, Departament de Biologia Fonamental i Cie`ncies de la Salut, Universitat de les Illes Balears, Cra. Valldemossa Km 7.5, E-07122 Palma de Mallorca, Spain. Address correspondence to Andreu Palou, Laboratori de Biologia Molecular, Nutricio´ i Biotecnologia, Departament de Biologia Fonamental i Cie`ncies de la Salut, Universitat de les Illes Balears, Cra Valldemossa, Km 7.5. E-07122, Palma de Mallorca, Spain. E-mail:
[email protected] Copyright © 2004 NAASO
sion of key adipogenic transcription factors, especially PPAR␥2. Hence, reduced hypertrophy of female iWAT and defective thermogenesis in interscapular brown adipose tissue in response to CAF diet were related to decreased PPAR␥2 mRNA levels, whereas increased hypertrophy in male iWAT and gWAT and in female gWAT was related to a tendency toward increased PPAR␥2 mRNA levels in response to overfeeding. Discussion: Our results suggest the involvement of PPAR␥2 in gender- and depot-specific effects of CAF diet on development and function in adipose tissues. Key words: adipose tissues, adipogenesis, sexual dimorphism
Introduction Body weight is a balance between energy intake and energy expenditure. Several molecular mechanisms are involved in the regulation of these two processes, and adipose tissues play an important and active role. White adipose tissue (WAT)1 stores triacylglycerides and releases fatty acids according to energy requirements, as well as secreting a variety of adipocytokines that have important roles in the regulation of energy intake, lipid metabolism, and glucose homeostasis. Moreover, in mammals, WAT is distributed in different anatomical locations that have different physiological and biochemical properties (1). On the other hand, brown adipose tissue (BAT) dissipates its fat stores, producing heat (adaptive thermogenesis) as a consequence of uncoupling the mitochondrial oxidative phosphorylation (2). Obesity is a disorder that results from excess and defective adipose tissue and is associated with life-threatening pathologies, including cardiovascular diseases and diabetes
1 Nonstandard abbreviations: WAT, white adipose tissue; BAT, brown adipose tissue; PPAR, peroxisome proliferator-activated receptor; C/EBP, CCAAT enhancer-binding protein; ADD1, adipocyte differentiation and determination factor 1; CAF, cafeteria; UCP, uncoupling protein; iBAT, interscapular brown adipose; gWAT, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; RT, reverse transcription; PCR, polymerase chain reaction.
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(3). Alterations in adipose tissue mass result from changes in adipocyte size and/or number. Adipocyte growth and differentiation are complex processes characterized by multiple changes in cell morphology, hormone sensitivity, and gene expression that eventually lead to the phenotype of the mature adipocyte expressing the genes that control lipid metabolism and thermogenesis (4 – 6). Several transcription factors act cooperatively and sequentially to trigger the terminal differentiation program (7,8). These include members of the peroxisome proliferator-activated receptor (PPAR) family, a lipid-activated subgroup of the nuclear hormone receptors, the CCAAT enhancer-binding proteins (C/EBPs), and the adipocyte differentiation and determination factor 1 (ADD1), a member of the sterol regulatory element binding proteins. Cafeteria (CAF) diet has been reported to induce increased body weight and increased adipose mass in rats even after a short period of time (9 –11). Several models of high-fat diets have been widely used to study the effects of such diets on body metabolism and body weight. Nevertheless, CAF-fed rats represent a useful model for human obesity studies because the CAF diet is a palatable hypercaloric and hyperlipidic diet that induces voluntary hyperphagia and fast body weight gain (2,10,11). To maintain energy balance and counteract the high fat intake, CAF-fed rats increase both lipolytic activity in WAT and uncoupling protein (UCP) expression in BAT and muscle to avoid weight gain. Moreover, the response to overfeeding is sex-dependent. In females, CAF diet feeding induces a higher body weight, which cannot be attributed to a higher energy intake with respect to their male counterparts but would be more in accordance with decreased lipolytic activity in WAT and less effective thermogenesis in BAT (10,11). In addition to genderrelated differences found in the expression of different adrenoceptors (10,11), the activation of PPAR␥2 could constitute an important part of the mechanism behind the adipogenic effect of high-fat diets (12,13), taking into account that other transcription factors such as C/EBP␣ and ADD1 could also play an important role. Gender differences in the expression of adipogenic transcription factors in response to CAF diet feeding and compared expression between visceral and subcutaneous fat have not been assessed at this time. The present study was taken to compare visceral and subcutaneous fat response to a short-term (15 days) overfeeding with CAF diet, to gain insight into the modulation of adipogenic transcription factors by a high-fat diet, and to study whether the gender dimorphism previously reported by our group could be related to a differential expression of key adipogenic transcription factors in both males and females. Our results provide evidence that visceral fat is more sensitive to CAF diet-induced hypertrophy than subcutaneous fat and that such a difference can be due to a differential pattern of expression of several adipogenic transcription 1456
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factors in both depots, especially PPAR␥2. In addition, gender differential response to short-term overfeeding can be explained, at least in part, by differential mRNA levels of PPAR␥2.
Research Methods and Procedures Animals, Diets, and Tissue Collection All experimental procedures were performed according to the national and institutional guidelines for animal care and use at the University of Illes Balears. Twenty-four animals at 4 weeks of age were obtained from CRIFFA (Barcelona, Spain) and were housed three per cage at 22 °C with a 12-hour light/dark cycle (8 AM to 8 PM). Animals were gender divided and assigned into two dietary groups: control and CAF-fed rats. Both groups had ad libitum free access to drinking water and standard chow (type A03; Panlab, Barcelona, Spain), and after a 4-day adaptation period to this diet and housing conditions, one-half of the animals (six females and six males) were also given a CAF diet (CAF group) in addition to the standard chow. The CAF diet used included the following foodstuffs: cookies with liver paˆ te´ and sobrassada (a typical Majorcan sausage), candies, fresh bacon, biscuits, chocolate, salted peanuts, cheese, milk containing 20% (w/v) sucrose, and ensaı¨mada (a typical Majorcan pastry) (14). Animals were fed for a short term (15 days) with control diet or CAF diet, and their body weight and energy intake were monitored throughout the experiment. At the end of the dietary treatment, control and CAF-fed animals were killed (in a fed condition) by decapitation at the start of the light cycle. Total interscapular BAT (iBAT), gonadal WAT (gWAT) (epididymal in males and ovaric in females), and subcutaneous inguinal WAT (iWAT; from inguinal depot) were excised, weighed, and frozen in N2 liquid and finally stored at ⫺70 °C until analysis. Serum Measurements Serum levels of several parameters were measured enzymatically using commercial kits and the following standard procedures: triacylglycerides (procedure no. 336; Sigma Diagnostics, St. Louis, MO), glucose (R-Biopharm; Roche, Darmstadt, Germany), insulin (DRG Instruments, Marburg, Germany), and total cholesterol (procedure no. 352; Sigma Diagnostics). RNA Isolation and Semiquantification of Adipogenic Transcription Factors Using Reverse Transcription (RT)-Polymerase Chain Reaction (PCR) Total RNA was isolated from 100 to 200 mg of iBAT, gWAT, and iWAT using Tripure Reagent (Roche, Barcelona, Spain) and according to the instructions of the manufacturer. To semiquantify the levels of PPAR␥2, ADD1, and C/EBP␣ mRNAs (plus PPAR␣ in iBAT), we
Role of PPAR␥2 in Cafeteria Diet Effects, Rodrı´guez et al.
Table 1. Oligonucleotides used for RT-PCR Gene
Primer sequence
Product size (base pairs)
PPAR␣
S: 5⬘-CCCTGCCTTCCCTGTGAACTGAC-3⬘ A: 5⬘-GGGACTCATCTGTACTGGTGGGGAC-3⬘ S: 5⬘-GGTGAAACTCTGGGAGATCC-3⬘ A:5⬘-TGAGGGAGTTTGAAGACTCTTC-3⬘ S: 5⬘-AAGGCCAAGAAGTCGGTGGA-3⬘ A: 5⬘-CAGTTCGCGGCTCAGCTGTT-3⬘ S:5⬘-AGCCATGGATTGCACATTTG-3⬘ A:5⬘-GGTACATCTTTACAGCAGTG-3⬘ S: 5⬘-ACGGGCATTGTGATGGACTC-3⬘ A: 5⬘-GTGGTGGTGAAGCTGTAGCC-3⬘
386
PPAR␥2 C/EBP␣ ADD1
-actin
400 189 260 164
S, sense primer; A, antisense primer.
developed an RT-PCR assay using the housekeeping gene -actin as an internal control as previously described (15). In brief, 0.5 g of total RNA was denatured at 65 °C for 10 minutes and reverse transcribed in the presence of 50 pmol random primers using MuLV reverse transcriptase (Perkin-Elmer, Madrid, Spain) at 42 °C for 30 minutes in a Perkin-Elmer 2400 Thermal Cycler. After the reaction, the RT medium (10%) was added to a PCR mix containing Taq DNA polymerase (Promega, Lyon, France) and 1 pmol -actin and 10 pmol (for PPAR␥2), or 3 pmol -actin and 10 pmol (for ADD1), or 5 pmol -actin and 10 pmol (for C/EBP␣), or 1.5 pmol -actin and 10 pmol (for PPAR␣) of specific primers (for sequences, see Table 1). The reaction mixture was first heated to 95 °C for 2 minutes to denature the cDNA. This was followed by 25 to 30 cycles of denaturation at 95 °C for 15 seconds, annealing at 58 to 60 °C for 15 seconds, and extension at 72 °C for 30 seconds, with an additional extension at 72 °C for 7 minutes after the last cycle. The PCR products were separated in 2% or 3.5% agarose gel (MS-8; Pronadisa, Madrid, Spain) in 0.5⫻ Tris-borate EDTA buffer stained with ethidium bromide and visualized using an image recording system (Gelprinter; TDI, Madrid, Spain). The densities of the target bands were then quantified using an image processing and analyzing program (1 day Image Analysis; Kodak, New Haven, CT). Statistical Analysis Data were expressed as means ⫾ SE. The effect of the diet (D), gender (G), and interaction (D ⫻ G) were tested using two-way ANOVA. Contrasts between means were assessed by Student’s t test. The analyses were performed with SPSS for Windows (SPSS, Chicago, IL).
Results Effect of Short-Term CAF Diet Feeding on Body Weight, Energy Intake, and Adiposity Index of Male and Female Rats As shown in Table 2, body weight was higher in both male and female rats fed a CAF diet than in rats fed a standard chow. Gender differences were observed in the body weight excess attained after a 15-day feeding period: 6% in males and 14% in females when compared with their respective controls. In both sexes, the energy intake of the CAF diet-fed rats was 3 times that of standard chow-fed animals throughout the period studied. Indeed, body weight gain resulted from voluntary hyperphagia and from a preference for fat intake (lipids accounted for 60% of the total energy intake in CAF diet-fed rats but only 12% in controls), in accordance with previous results (9,11). CAF diet feeding promoted adipose tissue hypertrophy in both genders. The increase in the adiposity index (sum of gWAT, iWAT, and iBAT weights expressed as a percentage of body weight) (16) was 53% in males and 62% in females when compared with their controls (Table 2). No changes in liver weight were observed; however, a differential response depending on the regional localization was found in the fat depots analyzed. Thus, gWAT (a visceral adipose depot) showed a higher increase (95%) in response to overfeeding than subcutaneous depots (33% for iWAT and 25% for iBAT), indicating a higher sensitivity of visceral fat to hypertrophy in response to CAF diet feeding when compared with that of subcutaneous fat. Only males showed a significant increase in iWAT (43% in males and 24% in females). In addition to that, iBAT of males fed a CAF diet showed a higher tendency to hypertrophy than that of females. OBESITY RESEARCH Vol. 12 No. 9 September 2004
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Table 2. Effect of short-term cafeteria diet feeding on body weight, liver weight, iBAT, iWAT, and gWAT expressed as a percentage of body weight as well as adiposity index (measured as the sum of iWAT, gWAT, and iBAT weights expressed as a percentage of body weight) Male
Female
Parameters
Control
Cafeteria
Control
Cafeteria
Body weight (g) BAT weight (%) iWAT weight (%) gWAT weight (%) Liver weight (%) Adiposity index (%) Energy intake (kcal/d/kg)
261 ⫾ 3 0.17 ⫾ 0.02 1.02 ⫾ 0.12 0.74 ⫾ 0.05 4.74 ⫾ 0.13 1.93 294 ⫾ 31
276 ⫾ 5* 0.24 ⫾ 0.04 1.46 ⫾ 0.08* 1.25 ⫾ 0.10* 4.49 ⫾ 0.26 2.95 1021 ⫾ 129*
194 ⫾ 4† 0.17 ⫾ 0.01 0.82 ⫾ 0.07 0.75 ⫾ 0.19 4.38 ⫾ 0.07† 1.74 253 ⫾ 42
221 ⫾ 5*† 0.19 ⫾ 0.01 1.02 ⫾ 0.08† 1.61 ⫾ 0.29* 4.70 ⫾ 0.17 2.82 1073 ⫾ 138*
ANOVA D, G D, G D
D
The data represent the means ⫾ SE of six animals per group. Significant differences were tested by ANOVA (p ⬍ 0.05): G, gender effect; D, diet effect. Student’s t test (p ⬍ 0.05). * Cafeteria vs. control. † Females vs. males.
Effect of Short-Term CAF Diet Feeding on Insulin, Glucose, Triacylglycerides, and Cholesterol Serum Levels of Male and Female Rats A gender dimorphism was found in glucose and triacylglyceride serum levels, with lower levels in female than in male rats (see Table 3). No significant differences in insulin serum levels (see Table 3) were found in response to shortterm overfeeding, but female rats showed a tendency to have higher insulin serum levels. Nevertheless, male rats showed significantly decreased serum levels of glucose in response to short-term CAF diet feeding. Higher serum levels of triacylglycerides and total cholesterol (see Table 3) were found in rats fed a CAF diet when
compared with rats fed a standard chow. Moreover, male rats showed a higher increase in serum triacylglycerides in response to CAF diet than females. Effect of Short-Term CAF Diet Feeding on mRNA Levels of PPAR␥2, C/EBP␣, and ADD1 in WAT of Male and Female Rats Figure 1, A and B, show the mRNA levels for the above-mentioned adipogenic transcription factors in gWAT and iWAT, respectively. A differential gender pattern for C/EBP␣ and ADD1 mRNA levels was showed in gWAT (visceral adipose depot) and for PPAR␥2 in iWAT (subcutaneous adipose depot). In all cases, females showed lower
Table 3. Effect of short-term cafeteria diet feeding on serum insulin, glucose, triacylglycerides, and total cholesterol levels Male Parameters Insulin (pM) Glucose (mM) Triacylglycerides (mM) Cholesterol (mM)
Female
Control
Cafeteria
Control
Cafeteria
51.41 ⫾ 10.54 6.61 ⫾ 0.58
52.75 ⫾ 10.12 4.77 ⫾ 0.11*
44.68 ⫾ 4.76 4.67 ⫾ 0.47†
69.51 ⫾ 14.34 4.85 ⫾ 0.61
1.73 ⫾ 0.20 2.22 ⫾ 0.07
3.19 ⫾ 0.03* 2.91 ⫾ 0.08*
1.11 ⫾ 0.15† 1.99 ⫾ 0.16
1.73 ⫾ 0.21*† 2.66 ⫾ 0.07*
ANOVA
D, G, D ⫻ G D
The data represent the means ⫾ SE of six animals per group. Significant differences were tested by ANOVA (p ⬍ 0.05): G, gender effect; D, diet effect; D ⫻ G, interaction of diet and gender. Student’s t test (p ⬍ 0.05). * Cafeteria vs. control. † Females vs. males.
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Figure 1: Effect of short-term (15 days) CAF diet feeding on mRNA levels of key adipogenic transcription factors (PPAR␥2, C/EBP␣, and ADD1) in gWAT (A) and iWAT (B). Levels for those mRNA were analyzed by RT-PCR and normalized to the expression of -actin mRNA. Data represent means ⫾ SE of at least four animals per group. Significant differences were tested by ANOVA (p ⬍ 0.05): G, gender effect; D ⫻ G, interaction of diet and gender. Student’s t test (p ⬍ 0.05): †, females vs. males. Representative RT-PCR images are shown below each quantification analysis. Upper bands belong to the adipogenic transcription factor amplified, whereas lower bands belong in all cases to -actin.
levels. Moreover, the mRNA levels of PPAR␥2, C/EBP␣, and ADD1 in iWAT were significantly lower (53%, 57%, and 26%, respectively, in control male rats) than those in gWAT (see Figure 2). In response to short-term overfeeding, we also found gender- and depot-related differences in the mRNA levels of PPAR␥2. Hence, male CAF diet-fed rats showed higher PPAR␥2 mRNA levels (⫹62%) in iWAT when compared with their controls, whereas females showed reduced PPAR␥2 mRNA levels (⫺41%) compared with control animals over the same experimental time period. In contrast, in gWAT, both males and females showed a general tendency for increased expression levels of the PPAR␥2 after CAF diet feeding, although they did not reach statistical significance. Effect of Short-Term CAF Diet Feeding on mRNA Levels of PPAR␥2, PPAR␣, C/EBP␣, and ADD1 in BAT of Male and Female Rats Figure 3 shows PPAR␥2, PPAR␣, C/EBP␣, and ADD1 mRNA levels in iBAT for the different experimental groups. Control male and female rats showed similar levels of the studied adipogenic transcription factors. The CAF
diet-fed rats showed lower levels of PPAR␥2 mRNA, a reduction that was greater in females.
Discussion In this study, we found gender- and depot-related differences in the expression of the key adipogenic transcription factor PPAR␥2 in the adipose tissue of rats fed with a CAF diet, differences that were correlated with changes in adiposity, in circulating levels of lipids and body weight. Several models of high-fat diets have been widely used to study the effects of such diets on body metabolism and body weight. In this way, CAF-fed rats represent a useful model for human obesity studies because the CAF diet is a hypercaloric and hyperlipidic diet that induces voluntary hyperphagia and obesity (2,9 –11). Short-term CAF diet feeding (15 days) was enough to produce a significant increase in adiposity in both male and female Wistar rats (53% and 62%, respectively) that was associated with an increase in body weight in both genders (6% in males and 14% in females). Interestingly, the higher adiposity found in response to CAF diet is related more to the increase in the amount of visceral fat (⬃95% increase in gWAT) OBESITY RESEARCH Vol. 12 No. 9 September 2004
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Figure 2: Representative RT-PCR comparing mRNA levels of PPAR␥2, C/EBP␣, and ADD1 in visceral (gWAT) and subcutaneous (iWAT) fat depots in control male rats. Relative mRNA levels for each transcription factor are shown as percentage and compared with gWAT (100%). Significant differences were tested using Student’s t test (p ⬍ 0.05): ‡, iWAT vs. gWAT.
than an increase in subcutaneous depots (around a 30% increase in iWAT and iBAT). Adipose tissue mass is determined by two distinct processes: the formation of new adipocytes from precursor cells (adipocyte differentiation) and the increase in adipocyte size due to fat storage (adipocyte hypertrophy). PPAR␥2 is the first identified adipocyte transcription factor that appears to promote differentiation and to control the expression of most fat-specific genes. Previous studies have reported a relationship between the induction of PPAR␥2 and the development of high fat-induced obesity (17–19). Differences between visceral and subcutaneous fat depots have been widely reported by several studies that show that visceral fat seems to be more metabolically active and have a higher lipolytic ratio than subcutaneous fat (20 –22). Taking into account that visceral depots are more easily mobilized than subcutaneous ones, our results point out that visceral fat has a higher sensitivity to hypertrophy than subcutaneous depots in response to overfeeding in male and female rats. In addition, our experiment shows that the higher adiposity in gWAT in response to overfeeding is clearly associated with significantly higher basal mRNA levels of key adipogenic transcription factors (PPAR␥2, C/EBP␣, and ADD1) in this tissue, when compared with iWAT (see Figure 2). This could explain, at least in part, the higher tendency for gWAT hypertrophy in response to CAF 1460
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Figure 3: Effect of short-term (15 days) CAF diet feeding on mRNA levels of key adipogenic transcription factors in iBAT PPARg2, PPAR␣, C/EBP␣, and ADD1. mRNA levels were analyzed by RT-PCR and normalized to the expression of -actin mRNA. Data represent means ⫾ SE of at least four animals per group. Significant differences were tested by ANOVA (p ⬍ 0.05): D, diet effect. Student’s t test (p ⬍ 0.05): *, CAF vs. control. Representative RT-PCR images are shown below each quantitation analysis. Upper bands belong to the adipogenic transcription factor amplified, whereas lower bands belong in all cases to -actin.
diet feeding, especially when taking into consideration that these transcription factors have been reported to be of great importance in the adipose differentiation process (7,23–25) and that the amount of these factors, principally of PPAR␥2, plays a critical role in adipocyte hypertrophy and development of insulin resistance under a high-fat diet (18). Although we have studied the expression of these factors only at the mRNA level, this level of gene expression is of great importance because different studies have correlated adipogenic transcription factor mRNA expression with changes in adipogenesis, adiposity, and lipid metabolism (7,15,19), indicating that transcription is the main regulatory step of these factors, although without discarding other control steps. A differential, gender response to overfeeding in Wistar rats has been reported in previous studies from our group, which showed a higher body weight in females in response to both short- and long-term CAF diet feeding, and this was associated with a less effective thermogenic capacity and an impaired adipose tissue lipolytic activity in female compared with male rats (9 –11). The present study is in agreement with these previous results but also provides new data
Role of PPAR␥2 in Cafeteria Diet Effects, Rodrı´guez et al.
on fat development, indicating a gender-specific implication of each regional fat depot; specifically, increased body weight in female rats in response to overfeeding is due mainly to visceral fat (⫹117% increase in gWAT weight vs. ⫹24% in iWAT), whereas in male rats increased adiposity is shared in a similar proportion by both visceral and subcutaneous fat, with just a slightly greater increase of gWAT (⫹68% increase in weight vs. ⫹40% in iWAT). This could have important health implications because visceral fat seems to play a more important role in the development of the metabolic disorders associated with the development of obesity (26 –30). In addition, we found that female rats showed a tendency to develop insulin resistance, and other authors have found increases in serum glucose after CAF diet feeding only in female rats (17,19). iWAT and gWAT gender differential responses to CAF diet found in our study could be explained by depot and gender differential expression of key adipogenic transcription factors. Hence, reduced hypertrophy of female iWAT in response to CAF diet could be related to the decreased PPAR␥2 mRNA levels in response to the high-fat diet (see Figure 1B), which was in contrast to what happened in males, who clearly showed increased levels. Moreover, hypertrophy of gWAT in response to CAF diet could be explained by a tendency toward increased PPAR␥2 mRNA levels. Our results agree with previous studies that report time-dependent increases in PPAR␥2 mRNA levels in abdominal WAT of female Wistar rats (17) and in epididymal WAT of male Wistar rats (19) in response to a short period of feeding with CAF diet, thus indicating a higher capability for both adipogenesis and lipogenesis. In addition to this enhanced expression of PPAR␥2 and the reported increase of retinoid X receptor-␣ expression in female Wistar rats after the consumption of a high-fat diet (31), the increased formation and activity of the PPAR:retinoid X receptor heterodimer could also help to explain the hypertrophy of WAT depots as a consequence of the increased availability of circulating lipids (the endogenous ligand of PPAR moiety is thought to be a product of fat metabolism) in CAF diet-fed animals. Moreover, site-specific effects on adipose metabolism and lipid deposition have been shown in animals treated with PPAR␥ agonists (32). It should also be pointed out that previous studies showing a decreased total lipolytic capacity in gWAT isolated adipocytes from female Wistar rats, when compared with males (11), should be taken into account to explain the higher hypertrophy in females of gWAT and the lower circulating triacylglyceride levels (see Table 2) in response to overfeeding. Lipolysis is achieved mainly by the hormone-sensitive lipase, which catalyzes the rate-limiting step in the breakdown of adipocyte triacylglycerides (33,34). Moreover, access to the lipid droplet constitutes another potential mechanism for the control of lipolysis, so perilipins—a family of lipid droplet-associated phosphoproteins—
regulate the magnitude of lipolysis and, thus, levels of stored lipids (35,36). In both cases, in addition to the shortterm modulation of activity by phosphorylation, variations in gene expression are associated with changes in lipolytic capacity (37– 41). It is of note that females showed lower mRNA levels of C/EBP␣ and ADD1 and that both transcription factors are known to be involved in the induction of the adipose differentiation process (7,23–25), which is essential for the lipolytic function of adipocytes. Thus, they could be responsible for the differential gender response in gWAT hypertrophy reported in our study. In regards to the iBAT response to short-term overfeeding, previous studies from our group have reported a defective thermogenic capacity in female rats fed a CAF diet for a short (15 day) or a long (100 day) period of time (9,10). Hence, females showed a lower iBAT hypertrophy and a decreased induction of UCP1 levels in response to the hypercaloric diet. In this sense, it has been reported that activated PPAR␥2 is an important transactivator of the ucp1 gene (42) and a potent inductor of BAT differentiation (43). Thus, our present study suggests that the defective iBAT thermogenesis in females found in those previous studies could be due to significantly lower PPAR␥2 mRNA levels in iBAT (see Figure 3), which could contribute, at least in part, to the decreased UCP1 expression and defective thermogenic capacity in response to overfeeding in female iBAT. In addition, the higher plasma triacylglycerides found in CAF diet-fed males (see Table 2) could indicate a recirculation to the iBAT, where fatty acids would be burnt by thermogenesis and would enhance male thermogenic capacity. Nevertheless, it should be also taken into account that other mechanisms such as regional and gender differential - and ␣-adrenergic receptor distribution can also contribute to the differential response to overfeeding (10). In summary, key adipogenic transcription factors such as C/EBP␣, ADD1, and especially PPAR␥2 seem to be important in the differential sensitivity of visceral and subcutaneous fat depots to overfeeding and in the gender differential response to CAF diet feeding reported in previous studies.
Acknowledgments This work was supported by the Spanish Government (Direccio´ n General de Investigacio´ n Grant BFI2000-0988C06-01 and Ministerio de Sanidad y Consumo Grants FIS 01/1379 and G03/028) and by the European Union (COST Action 918 and DLARFID Project QLRT-2001-00183). E.R. was the recipient of a doctoral fellowship from the Spanish Government (Ministerio de Educacio´ n, Cultura y Deportes). References 1. Pond CM, Mattacks CA. The effects of noradrenaline and insulin on lipolysis in adipocytes isolated from nine different adipose depots of guinea-pigs. Int J Obes. 1991;15:609 –18. OBESITY RESEARCH Vol. 12 No. 9 September 2004
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