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Endocrinology Vol. 148, No. 4 1539-1549
Copyright © 2007 by The Endocrine Society

Overexpression of Adiponectin Targeted to Adipose Tissue in Transgenic Mice: Impaired Adipocyte Differentiation

Isabelle B. Bauche1, Samira Ait El Mkadem1, Anne-Marie Pottier, Maximin Senou, Marie-Christine Many, René Rezsohazy, Luc Penicaud, Norikazu Maeda, Tohru Funahashi and Sonia M. Brichard

Endocrinology and Metabolism Unit (I.B.B., S.A.E.M., A.-M.P., S.M.B.), Experimental Morphology Unit (M.S., M.-C.M.), University of Louvain, Faculty of Medicine, 1200 Brussels, Belgium; Unit of Veterinary Sciences (R.R.), Institut des Sciences de la Vie, University of Louvain, 1348 Louvain-la-Neuve, Belgium; Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche 5018 (L.P.), CNRS and Paul Sabatier University, 31062 Toulouse, France; and Department of Internal Medicine and Molecular Science (N.M., T.F.), Graduate School of Medicine, Osaka University, Osaka 560-0043, Japan

Address all correspondence and requests for reprints to: S. M. Brichard, Unité d’Endocrinologie et Métabolisme, University of Louvain/Endocrinology 5530, Avenue Hippocrate, 55, B-1200 Brussels, Belgium. E-mail: brichard{at}endo.ucl.ac.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adiponectin (ApN) is an adipokine whose expression and plasma levels are inversely related to obesity and insulin-resistant states. Chronic repercussions of ApN treatment or overexpression on adiposity and body weight are still controversial. Here, we generated a transgenic (Tg) mouse model allowing persistent and moderate overexpression of native full-length ApN targeted to white adipose tissue. Adipose mass and adipocyte size of Tg mice were reduced despite preserved calorie intake. This reduction resulted from increased energy expenditure and up-regulation of uncoupling proteins, and from abrogation of the adipocyte differentiation program, as shown by the loss of a key lipogenic enzyme and of adipocyte markers. Adipose mass remodeling favors enhanced insulin sensitivity and improved lipid profile of Tg mice. Alteration of the adipocyte phenotype was likely to result from increased expression of the preadipocyte factor-1 and from down-regulation of the transcription factor, CCAAT/enhancer binding protein-{alpha}, which orchestrates adipocyte differentiation. We further found that recombinant ApN directly stimulated pre- adipocyte factor-1 mRNA and attenuated CCAAT/enhancer binding protein-{alpha} expression in cultured 3T3-F442A cells. Conversely, opposite changes in the expression of these genes were observed in white fat of ApN-deficient mice. Thus, besides enhanced energy expenditure, our work shows that impairment of adipocyte differentiation contributes to the anti-adiposity effect of ApN.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADIPONECTIN (ApN) IS AN adipokine, specifically secreted by adipocytes, that circulates at relatively high concentrations in the bloodstream. It plays a fundamental role in energy homeostasis and inflammation (1). This 30-kDa protein is composed of an N-terminal collagenous domain and a C-terminal globular domain. The latter fragment [globular ApN (gApN)] generated by proteolysis may exert some activity (2). Two types of ApN receptor mediate most effects of the hormone via stimulation of AMP kinase, peroxisome proliferator-activated receptor (PPAR) {alpha}, and p38 MAPK. ApN receptor 1 (AdipoR1), which is a high-affinity receptor for gApN, is most abundantly expressed in skeletal muscle, whereas ApN receptor 2 (AdipoR2), which serves as a moderate-affinity receptor for both forms of ApN, is predominant in liver (3). Both receptor types are present in adipocytes, which suggests that ApN may act on these cells in an autocrine or paracrine manner (4, 5).

Unlike most adipocytokines, circulating ApN is decreased in human obesity and related disorders (type 2 diabetes or cardiovascular disease, which are components of the metabolic syndrome) (6, 7, 8, 9). ApN supplementation could carry significant therapeutic potential in these adverse metabolic events, as shown in mice. ApN treatment plays a protective role against atherosclerosis in apolipoprotein E-deficient mice (10). The adipocytokine is also a potent enhancer of insulin action in mouse models of obesity, lipoatrophy, or diabetes (11, 12). Its administration induces insulin-dependent suppression of hepatic gluconeogenesis, thereby lowering plasma glucose concentrations (11). It also increases glucose uptake by muscles (13, 14). ApN alters lipid metabolism as well, by increasing fatty-acid oxidation in several tissues including liver and muscle, thereby accelerating the clearance of plasma free fatty acids (2, 13, 14, 15). Concomitantly, ApN stimulates the expression of uncoupling proteins (UCPs) in various organs and enhances thermogenesis (12, 16, 17).

Our understanding of ApN action on adipose tissue is still poor. In vitro, ApN acutely stimulated glucose uptake by isolated rat adipocytes (18), and suppressed IL-6 and TNF{alpha} expression in pig adipocytes (19). Recently, marked overexpression of ApN in stably transduced 3T3-L1 cells has been shown to result in adipocyte differentiation and lipid accumulation (20). The in vivo effects of ApN treatment on mouse adiposity and body weight remain controversial: some studies reported a decrease in fat mass (2, 16, 17), whereas others reported no change or even an increase (12, 21). Moreover, ApN was usually delivered by infection with a recombinant adenovirus or injection of purified recombinant protein, and experiments were conducted on a short or middle term basis (from a few days up to 3 wk). A good way to investigate the repercussions of chronic and early ApN supplement on adipose tissue is to generate appropriate Tg mouse models. It has been extremely difficult to elevate native ApN in adipose tissue of Tg mice because of a negative feedback exerted on endogenous production (21, 22). Two Tg models overexpressing ApN have been generated so far. In the first model, it was necessary to develop a particular Tg strategy using a deletion mutant of ApN to circumvent this problem. This mutant of full-length ApN cDNA was then placed under control of the adipocyte promoter (aP2) (21). In the second model, gApN cDNA was placed under control of a liver promoter (23). In the former case, an unexpected obese phenotype was observed, but the physiological relevance of this model bearing a deletion in the collagenous domain of ApN may not be straightforward (21). In the latter case, effects on body weight were variable (unchanged or decreased), but circulating gApN was very high and influenced other organs, so that repercussions on adipose tissue could be masked or indirectly mediated (23).

We generated a heterozygous Tg mouse line overexpressing native ApN specifically in white adipose tissue (WAT) to study the chronic effects of a local, additional but homotopic expression of ApN in vivo. This line carried approximately 100 copies of a transgene, in which native full-length ApN was placed under control of aP2. Previously characterized mouse lines that carried only six copies of this transgene failed to overexpress ApN (22). In the present line carrying a larger number of copies, ApN overexpression masks the down-regulation of the endogenous gene. Thanks to this high-expression line, we more particularly examined the chronic repercussions of ApN on body weight and fat accumulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of Tg mice
A fusion gene was designed, comprising the 5.4-kb aP2 promoter fragment (kindly provided by B. M. Spiegelman, Dana-Farber Cancer Institute, Boston, MA) (24), the 1276-bp mouse full-length adiponectin (ApN) cDNA [gift of P. E. Scherer, Department of Cell Biology and Diabetes Research and Training Center, Albert Einstein College of Medicine, Bronx, New York (25)] and the simian virus 40 polyadenylation signal (Fig. 1AGo). The integrity of this 8-kb construct was confirmed by mapping with restriction endonucleases and DNA sequencing of crucial regions (aP2-ApN junction and ApN cDNA). The transgene was purified and microinjected into the pronucleus of fertilized FVB mice eggs (Eurogentec, Liège, Belgium).


Figure 1
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  		FIG. 1. Generation of mice carrying 100 copies of the ApN transgene under control of the adipocyte-specific aP2 promoter. A, Schematic representation of the aP2 promoter/mouse ApN cDNA fusion gene. The bar represents the DNA probe (fragment of exon 3) used for Southern blotting. B, Southern blot analysis of HindIII-digested mouse genomic DNA from WT and Tg mice hybridized with the cDNA probe shown in panel A. The 6.7-kb band corresponds to the transgene and the 2.7-kb band to the endogenous gene. C, ApN gene expression in several non-WATs of WT and Tg male mice. ApN mRNA levels were measured in BAT and in nonfat tissues. Data from 40-ng total RNA equivalents were quantified by RTQ-PCR, normalized to the levels of cyclophilin, and then presented as relative expression compared with the values obtained in WAT (inguinal depot) of WT mice (dotted line). Results are the mean ± SEM for six to eight mice per group.

 
Tg mice were screened by PCR amplification of tail genomic DNA with aP2 promoter-specific (sense, 5'-AGTCAAAACAGGAACCTTTAAAATACTC-3') and ApN cDNA-specific (antisense, 5'-AGAAAGCCAGTAAATGTAGAG-3') oligonucleotides, which give rise to a 1065-bp product. Identification of Tg founder F0 mice and quantification of transgene copies were further achieved by Southern blot analysis of HindIII-digested genomic DNA hybridized with a PCR probe amplified from the exon 3 of ApN (Fig. 1AGo). For this experiment, the filters were exposed to autoradiographic films after hybridization with the radiolabeled probe. ODs of the transgene band on the blots were quantified by scanning densitometry (Image Master TotalLab; Amersham Pharmacia, Little Chalfont, Buckinghamshire, UK) and expressed relative to the OD of the band corresponding to the endogenous locus.

Eight F0 mice had integrated the transgene, but only three of these were fertile. Two carried a low copy number of the transgene, which did not allow overexpression of ApN (22). The third one carried approximately 100 copies. The reported analysis focused on the progeny of this last line. Tg mice were maintained on a pure (C57/Bl6J) background and used as heterozygotes.

Animal care, sampling, and tests
Tg mice and their wild-type (WT) littermates were housed in groups of two to five in filter-top cages with a fixed 12-h light, 12-h dark cycle. They received from weaning a high-sucrose diet that promotes insulin resistance. This diet was composed of (% of total gross energy): 68 carbohydrate (cornstarch/sucrose, 1:3.5), 11 fat (milk fat/soybean oil, 3:1), 21 protein (casein) (TD00220; Harlan, Horst, The Netherlands). In an additional experiment, some ApN-deficient mice, which are characterized by a lack of ApN mRNA in fat and ApN protein in plasma, were obtained from Maeda et al. (26); these animals and their WT controls were housed as described above and received a common laboratory chow (RO3-10; Safe, Villemoisson-sur-Orge, France).

Food intake and body weight were measured daily. On several occasions, tail vein blood collected from fed animals (between 0800 and 0900 h). Some mice underwent an oral glucose tolerance test (OGTT) or an insulin tolerance test after an overnight fast. The tests started at 0800 h. For the OGTT, glucose (30% in water) was introduced into the stomach through a fine gastric catheter at a dose of 2 g/kg body weight. For the insulin tolerance test, ActrapidR (Novo Nordisk A/S, Bagsvaerd, Denmark) was injected ip at a dose of 0.75 U/kg body weight. Blood samples were obtained at 0, 30, 60, 120, and 180 min. At the age of 5 months, mice were killed by decapitation (between 0800 and 1000 h). Pairs of white fat pads from several depots [sc (inguinal), gonadal (epididymal and parametrial), and prerectal-ip (also refereed to elsewhere as "retrovesical") (22, 27)] as well as interscapular brown adipose tissue (BAT) were immediately removed, weighed, frozen in liquid nitrogen, and stored at –80 C.

The University Animal Care Committee has approved all procedures.

Culture of 3T3-F442A adipocytes
Mouse 3T3-F442A preadipocytes were grown at 37 C in 5% CO2 in basal medium [i.e. DMEM with 1 g/liter glucose containing 10% fetal calf serum, 8 mg/liter biotin and 1% (vol/vol) of a commercial mixture containing penicillin and streptomycin (Invitrogen Life Technologies, Merelbeke, Belgium)], as described (27). Two days after confluence (d 0), adipocyte differentiation was initiated by addition of 17 nM insulin, 2 nM T3, 100 nM dexamethasone, and 100 µM isobutylmethylxanthine for 48 h. Then, cells were refed by basal medium containing 17 nM insulin and 2 nM T3 (27). The medium was changed every 24 h. At d 3, more than 90% of the cells had changed their morphology and more than 70% had accumulated fat droplets as evidenced by Oil Red O staining. These cells were cultured with or without mouse recombinant ApN [full-length form (22)] added to the medium at a final concentration of 1 µg/ml from d 0–3.

Real-time quantitative PCR (RTQ-PCR)
RNA was isolated from mouse tissues and cultured cells by TriPure Isolation Reagent (Roche Diagnostics, Vilvoorde, Belgium) (28). mRNAs were quantified by RTQ-PCR (27). Two micrograms of total RNA were reverse transcripted using oligo (deoxythymidine) primers and Superscript II Rnase H– Reverse Transcriptase (Invitrogen Life Technologies). RTQ-PCR primers were designed (Primer Express Software, Applied Biosystems) for ApN (these primers were ApN cDNA-specific and recognized both endogenous ApN and transgene-derived ApN), AdipoR1, AdipoR2, aP2, fatty acid synthase (FAS), UCP1, 2, 3, acyl-coenzyme A oxidase (ACO) PPAR{alpha} and {gamma}, sterol regulatory element-binding protein (SREBP)-1, GLUT4, TNF{alpha}, leptin, preadipocyte factor (Pref)-1, CCAAT/enhancer binding protein-{alpha} (C/EBP-{alpha}) and cyclophilin (cyclo) (Table 1Go). Forty nanograms of total RNA equivalents were amplified with iQ Syber Green Supermix (Bio-Rad Laboratories, Nazareth, Belgium) containing 300 nM of each specific primer, using iCycler iQ Real Time PCR Detection System (Bio-Rad). PCR efficiency was approximately 1. The threshold cycles (Ct) were measured in separate tubes and in duplicate. The identity and purity of the amplified product were checked by electrophoresis on agarose mini-gels and analysis of the melting curve carried out at the end of amplification. To ensure the quality of the measurements, each plate included a negative control for each gene. The {Delta}Ct values were calculated in every sample for each gene of interest as followed: Ctgene of interest – Ctreporter gene with cyclophilin whose mRNA levels did not differ between control and test groups, as the reporter gene. Relative changes in the expression level of one specific gene ({Delta}{Delta}Ct) were calculated as {Delta}Ct of the test group minus {Delta}Ct of the control group, and then presented as 2–{Delta}{Delta}Ct.


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  		TABLE 1. Murine gene sequences (5'–3') used as forward and reverse primers for RTQ-PCR

 
Indirect calorimetry
Twenty-four-hour energy expenditure was measured by indirect calorimetry (Oxylet, Panlab-Bioseb, Chaville, France). The mice were housed in individual metabolic chambers, in which they were accustomed for at least 12 h before starting the measurements. They had free access to food and water. The temperature in the metabolic cage (23 ± 1 C) was stable and controlled throughout the experiment. The following metabolic parameters were measured: oxygen consumption (VO2), carbon dioxide production (VCO2), energy expenditure (calculated according to the following formula = (3.815 + 1.232 x VO2/VCO2) x VO2), and locomotor activity. Oxygen consumption and carbon dioxide production were recorded at 5-min intervals using a computer-assisted data acquisition program (Chart 5.2; AD Instruments, Sydney, Australia) over a 24-h period and data were averaged for each mouse. Physical activities of the mice were monitored by an infrared photocell beam-interruption method (Panlab-Bioseb, Chaville France). Computer-assisted processing of respiratory exchanges and spontaneous activity signals made it possible to compute that part of the total metabolic rate devoted to fuelling the energy cost of activity. Thus, by continuously extracting the energy expended with activity, it was possible to compute the resting metabolism of these free-moving mice.

Immunohistochemistry
Adipose tissue was fixed in 10% formaldehyde for 24 h and embedded in paraffin. The 5 µm-thick sections were stained with hematoxylin-eosin-safran (HES). For immunohistochemistry, sections were processed as previously described (29) using rabbit polyclonal antibodies directed against ApN (Chemicon, Biognast, Hesle, Belgium) and caspase-6 (Santa Cruz Biotechnology, Tebu-Bio, Baeckout, Belgium). Binding of antibodies was detected by applying for 30 min at room temperature a second (goat antirabbit) antibody conjugated to peroxidase-labeled polymer (En vision +; Dako, Copenhagen, Denmark). Staining was specific because there was no labeling when adipose tissue was incubated with preimmune serum used as control (not shown).

Morphometry
The mean relative proportion of adipocytes was estimated by a point-counting technique (30) on paraffin-embedded, HES counterstained sections of inguinal tissue from Tg mice and their littermates. The number of adipocytes per microscopical field (density) was determined, at magnification x500, on 20 fields for each mouse. Adipocytes (500–800) were counted for each section. The mean diameter of the adipocytes (in micrometers) was calculated as {surd}(number of fields/ number of adipocytes) x D, with D being the diameter of the microscopical field (220 µm for the objective 50).

Quantification of total plasma ApN and determination of oligomers
Total plasma ApN concentrations were measured by a commercially available RIA kit (RIA mouse ApN kit; Linco Research, St. Charles, MO) as described (31). Samples (0.5 µl) were run in duplicate.

For separation of plasma ApN complexes, samples (0.33 µl) were solubilized in Laemmli buffer and subjected to SDS-PAGE under nonreducing and non-heat-denaturating conditions. Alternatively, samples were heated at 100 C for 5 min in the presence of 5% 2-mercaptoethanol for complete reduction and heat denaturation. Immunoblotting was performed using a rabbit polyclonal antibody directed against murine ApN (BioVendor Laboratory Medicine, Heidelberg, Germany; final concentration 1 µg/ml). After reaction with a secondary antibody (antirabbit IgG-hoseradish peroxidase), the blots were treated with enhanced chemiluminescence (SuperSignal West Dura Extended Duration Substrate, Pierce Science, Antwerpen Belgium) and analyzed using the Kodak Image Station system (Analis, Suarlee, Belgium).

Other analytical procedures
Blood glucose was measured using a glucometer (Medisense Precision Xtra Plus, Abott-Medisense, Louvain-la-Neuve, Belgium) and plasma insulin by RIA (kit from Linco Research). The insulin resistance indices were calculated as the insulin and glucose products derived from values obtained at the end of the OGTT (t180) (32). Plasma lipids [total cholesterol, nonesterified free fatty acids (NEFA), and triglycerides] were measured as reported (27).

Statistical analysis
Results are the means ± SEM for the indicated numbers of individual mice (in vivo study) or separate experiments (in vitro study). Comparisons between the two groups of mice or two different culture conditions were made using two-tailed unpaired Student’s t test. Differences were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of mice carrying the aP2-ApN-Tg
We generated a heterozygous Tg mouse line to investigate the chronic repercussions of ApN overexpression on adiposity. Native full-length ApN was placed under the control of aP2, so that the hormone expression should be targeted mainly to adipose tissue (Fig. 1AGo). We obtained one line, which carried 100 copies of the transgene as assessed by Southern blotting (Fig. 1BGo). The 2.7-kb band corresponded to the endogenous gene and the 6.7-kb band to the aP2-ApN transgene. In this line, ApN gene overexpression was restricted to WAT. Indeed, ApN mRNA levels did not significantly differ in BAT of WT and Tg mice and were similarly low (≤ 0.05) in the other tissues of the two groups, when compared with the amount found in WAT of WT mice (Fig. 1CGo).

Overexpression of ApN in Tg mice
As expected, ApN mRNA levels increased by 50–150% in almost every white fat depot of Tg males and females when compared with WT littermates (Fig. 2AGo). Accordingly, immunohistochemistry clearly demonstrated that ApN protein labeling was augmented in white fat of Tg mice. Immunostaining of adipose tissue sections showed that ApN was localized in the cytoplasmic edging with the periphery of the adipocytes and that labeling was much more intense in Tg animals (Fig. 2BGo; inguinal fat of males as an example).


Figure 2
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  		FIG. 2. Gene expression and immunodetection of ApN in adipose tissue, and levels of ApN in plasma of Tg mice. Five-month-old Tg and WT mice were used. A, ApN mRNA levels were measured in several fat depots by RTQ-PCR as described in Fig. 1Go and are presented as relative expression compared with respective WT values. Due to limited amounts of prerectal fat in males (see Fig. 4BGo), ApN mRNAs of this depot were measured in females only. B, Representative sections of ApN immunodetection in inguinal adipose tissue are shown (at magnification, x500). C, Total plasma ApN concentrations were measured by RIA and expressed as micrograms per milliliter. D, Representative Western blots showing HMM multimers migrating above 300 kDa and LMM hexamers migrating at approximately 150 kDa under nonreducing and non-heat-denaturating SDS-PAGE conditions. Separate gels were performed for males and females. Values are the mean ± SEM for eight mice per group (a and c). °, P = 0.09; *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. WT mice.

 
In agreement with adipose tissue content, total plasma ApN levels rose 2- to 10-fold in Tg mice. The largest increase was observed in Tg females (Fig. 2CGo). ApN multimers were analyzed by SDS-PAGE under nonreducing and non-heat-denaturating conditions, which allows adequate separation of high molecular mass (HMM) and low molecular mass (LMM) complexes (33). As described (33), ApN circulates as a HMM form greater than 300 kDa, corresponding to a multimeric assembly of 12–16 molecules, and a LMM hexamer of approximately 150 kDa (Fig. 2DGo). Under reducing and heat-denaturating conditions, the multimeric circulating forms were converted to a 30-kDa monomer (Fig. 2DGo). Although the absolute levels of both HMM and LMM multimers increased in Tg mice, there was no major change in the multimer distribution between Tg and respective WT animals (Fig. 2DGo).

Glucose homeostasis and circulating lipid profile
Because ApN exerts antidiabetic properties, we examined glucose homeostasis. In the basal state, fed blood glucose levels were reduced by approximately 5–15% in Tg mice of both genders, and fed plasma insulin were reduced as well (Fig. 3Go, A and B). During the OGTT, blood glucose and plasma insulin levels were also lower in Tg than in WT mice at some time points (Fig. 3Go, C and D). The insulin resistance indices derived from values obtained at the end of the OGTT were decreased by approximately 35% in Tg mice [Fig. 3CGo, inset for females; similar data obtained for males (data not shown)]. An insulin tolerance test confirmed the enhanced insulin sensitivity (data not shown).


Figure 3
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  		FIG. 3. Glucose homeostasis of Tg mice. A and B, Basal blood glucose and plasma insulin were sampled in the fed state in 3- and 5-month-old animals (only 3 months old for insulin). C and D, Blood glucose (G) and plasma insulin levels during an OGTT performed in 3-month-old mice after an overnight fast. Inset, IR (insulin resistance) indices [I (ng/ml) x G (mg/dl)] derived from the OGTT. Values are the mean ± SEM for eight to nine mice per group. °, P = 0.08; *, P < 0.05 vs. WT mice.

 
The plasma lipid profile was also modified in Tg mice (Table 2Go). In agreement with other reports (2, 21, 23), plasma levels of triglycerides, cholesterol, and NEFA were significantly reduced in Tg animals.


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  		TABLE 2. Plasma lipid levels in Tg mice

 
Body weight, adiposity, and energy intake and expenditure in Tg mice
As shown in Fig. 4AGo, body weight decreased significantly in Tg mice of both genders from 3 months of age onwards. At 5 months, white fat was sampled in several depots. Weight of every fat pad was reduced in Tg mice, with the largest reduction being observed in the inguinal depot (~–55%) (Fig. 4BGo).


Figure 4
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  		FIG. 4. Body weight, fat pad weights, and histological sections of adipose tissue in Tg mice. A, Body weight of Tg and WT mice was monitored throughout the study. B and C, Fat pad weights and HES staining of inguinal adipose tissue sections (magnification, x500) were examined in 5-month-old mice. Values are the mean ± SEM for 10–15 mice per group (A and B) and for five mice per group (C); representative sections are shown for C. *, P < 0.05; ***, P < 0.001 vs. WT mice.

 
Surprisingly, daily food consumption was unaltered in Tg mice or even slightly elevated in females (by 10%) (Table 3Go). As a result, feed efficiency was decreased in Tg animals of both genders. A potential explanation for this finding could be an increase in the metabolic rate. To test this hypothesis, we measured 24-h energy expenditure by indirect calorimetry in 5-month-old mice. As shown in Table 3Go, energy expenditure was significantly higher in Tg than in WT mice. Locomotor activity was not influenced by the presence of the transgene (not shown).


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  		TABLE 3. Food consumption and indirect calorimetry in Tg mice

 
Characteristics of fat in Tg mice: morphometry and expression of key molecules involved in adipose tissue metabolism and development
Histological examination of inguinal adipose tissue sections after HES staining showed a higher density of smaller adipocytes in Tg mice (Fig. 4CGo). Morphometric analysis confirmed that adipocyte size (diameter in micrometers) was halved in Tg animals compared with WT littermates (39 ± 0.6 vs. 61.3 ± 1.4 for males; 35.6 ± 0.4 vs. 60.2 ± 1.2 for females; n = 5 per group; P < 0.05 or less). In addition, adipocyte number per microscopical field (objective 50) was ~2.5-fold higher in Tg than in WT mice (33.5 ± 0.8 vs. 14.5 ± 0.7 for males; 39.6 ± 0.9 vs.15.2 ± 0.5 for females; P < 0.0001 for each comparison). However, because fat mass was reduced in Tg mice (Fig. 4Go), the estimated number of total adipocytes per animal was unlikely to be markedly altered in this model.

Gene expression of molecules involved in adipose tissue metabolism and development was next quantified in fat depots of 5-month-old Tg males. Because of increased energy expenditure, we measured the expression of UCPs, molecules involved in energy dissipation. Abundance of UCP 2 mRNA, the isoform expressed at a high level in white fat, doubled in the inguinal depot of Tg mice (Fig. 5Go, left panel). UCP 1 mRNAs did not change in this depot, but tended to rise in epididymal fat (by 80%; P = 0.08) and BAT as well (by 40%; P = 0.07). UCP 3 expression, which predominates in skeletal muscle, was not significantly increased in tibialis anterior muscle (data not shown). Concomitantly with the increase of UCP2 expression, mRNA levels of FAS, a key enzyme of lipogenesis and of TNF{alpha}, an adipokine implicated in insulin resistance were decreased by 50 and 60% in inguinal fat of Tg mice. Leptin mRNAs were also reduced (Fig. 5Go, left panel). The expression of other messengers involved in glucose or lipid metabolism (Glut 4, aP2, and ACO) did not change (data not shown).


Figure 5
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  		FIG. 5. Expression of several genes implicated in adipose tissue metabolism and development of Tg mice. Inguinal fat was sampled in 5-month-old in WT and Tg mice. mRNA levels of key molecules playing a role in adipose tissue metabolism (UCP 2, FAS, TNF{alpha}, leptin, and AdipoR1 and R2) as well as of transcription/growth factors involved in adipose tissue development (Pref-1, C/EBP{alpha}, SREBP-1, PPAR {alpha} and {gamma}) were quantified by RTQ-PCR as described in Fig. 1Go, and are presented as relative expression compared with respective WT values. Results are the mean ± SEM for eight mice per group. °, P = 0.06; *, P < 0.05 vs. WT mice.

 
We measured the expression of ApN receptors in inguinal fat of Tg mice as well. AdipoR1 mRNAs did not vary, whereas AdipoR2 mRNAs increased 3-fold when compared with WT mice (Fig. 5Go, left panel).

Because the adipose tissue of Tg mice was characterized by younger and smaller adipocytes, we searched for changes in transcription/growth factors orchestrating adipose tissue development (Fig. 5Go, right panel). The expression of Pref-1, an epidermal growth factor implicated in the maintenance of the preadipose state (34), was increased in inguinal fat of Tg mice, whereas mRNA levels of C/EBP{alpha}, a factor inducing adipocyte gene expression and differentiation (35), tended to be decreased (but this trend did reach statistical significance; P = 0.06). The presence of the transgene did not affect the expression of other transcription factors also implicated in adipose tissue development, such as SREBP, PPAR{alpha}, and PPAR{gamma} (36).

Eventually, reduced fatness in Tg mice did not result from a higher rate of apoptosis in white fat. Immunostaining of caspase-6, a marker of apoptosis, was similar in inguinal adipose tissue sections of Tg and WT mice (not shown).

Effects of ApN on Pref-1 and C/EBP{alpha} expression in 3T3-F442A cells
We next examined whether ApN could directly modulate the expression of the transcription factors, which were modified in vivo. To this end, we added recombinant full-length ApN to the culture medium of differentiating 3T3-F442A adipocytes for 72 h. At the end of the experiments, we measured mRNA levels of Pref-1 and C/EBP{alpha}. When compared with control conditions, recombinant ApN doubled the abundance of Pref-1 mRNA and decreased by approximately 60% C/EBP{alpha} mRNAs (Fig. 6Go).


Figure 6
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  		FIG. 6. Expression of Pref-1 and C/EBP{alpha} in 3T3-F442A. Differentiating adipocytes were cultured in the presence or in the absence of mouse recombinant ApN (full-length form; 1 µg/ml) for 3 d. mRNA levels of Pref-1 and C/EBP{alpha} were quantified by RTQ-PCR and are presented as relative expression compared with control values (i.e. in the absence of ApN). Results are the mean ± SEM for 12 experiments obtained from three independent cultures. *, P < 0.05; ***, P < 0.001 vs. respective controls.

 
Pref-1 and C/EBP{alpha} expression in white fat of ApN-deficient mice
We tested a contrario the hypothesis that ApN modulates the expression of adipose growth/transcription factors and examined whether the lack of ApN could induce changes in their abundance in vivo. To this end, we measured the expression of Pref-1 and C/EBP{alpha} in inguinal adipose tissue of 5-month-old ApN–/– mice, where no circulating ApN is detectable (26). The expression of Pref-1 was reduced by approximately 80% in inguinal fat of ApN-deficient mice, whereas mRNA levels of C/EBP{alpha} were increased by approximately 50% (Fig. 7Go).


Figure 7
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  		FIG. 7. Expression of Pref-1 and C/EBP{alpha} in white fat of ApN-deficient mice. Inguinal fat was sampled in 5-month-old WT and ApN-deficient mice. mRNA levels of Pref-1 and C/EBP{alpha} were quantified by RTQ-PCR and are presented as relative expression compared with WT values. Results are the mean ± SEM for six mice per group. *, P < 0.05; **, P < 0.01 vs. WT mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here, we generated a Tg mouse model allowing persistent and moderate overexpression of native full-length ApN targeted to WAT. Adipose mass and adipocyte size of Tg mice were reduced despite preserved calorie intake. This resulted from increased energy expenditure and up-regulation of UCP, and from abrogation of the adipocyte differentiation program, as shown by the stimulation of Pref-1 and the down-regulation of C/EBP{alpha}. We further found that recombinant ApN stimulated Pref-1 mRNA and attenuated C/EBP{alpha} expression in 3T3-F442A cells, whereas opposite changes in the expression of these genes were observed in white fat of ApN-deficient mice. Thus, besides enhanced energy expenditure, our work shows that impairment of adipocyte differentiation contributes to the antiobesity effect of ApN.

The average 2-fold level of ApN overexpression in WAT of aP2-ApN Tg mice most likely represents an increase that is physiologically relevant (37). This was accompanied by a similar increase of circulating ApN levels in males but by a higher increment in females. This higher increment was only observed in females fed the high-sucrose (not high-fat) diet and may actually reflect a paradoxical and diet-specific decrease of plasma ApN in control females rather than marked ApN elevation in Tg ones (this study and data not shown). Importantly, there was no change in circulating ApN multimer distribution in plasma of Tg mice compared with respective controls.

Chronic effects of ApN on body weight and adipose mass are still debated. Tg mouse models overexpressing ApN could be useful tools to address this issue. Two models have been generated so far (21, 23). In the first one, full-length ApN cDNA was mutated then placed under control of the aP2 (21); in the second one, gApN cDNA was placed under control of a liver promoter (23). In the first case, an unusual obese phenotype with bilateral exophthalmia and expansion of interscapular tissue was observed (21). In the latter case, effects on body weight were variable (unchanged or decreased), but circulating gApN was very high and influenced primarily other organs than adipose tissue (23). Our present study in Tg mice with moderate overexpression of native ApN targeted to white fat unambiguously shows that chronic and early ApN reduced fat mass and adipocyte size. These data fit a contrario with those obtained in our mouse lines carrying a low copy number of the same transgene, but exhibiting a decrease in ApN parameters (due to a negative feedback on endogenous production) (see introductory text) (22). The phenotype of these mice was characteristic of partial ApN deficiency (moderate increase in adiposity and insulin resistance). It is still unclear why ApN–/– mice, with a complete lack of ApN only exhibited insulin resistance and not obesity in response to a high-fat diet (26, 38). One may raise the possibility that, in case of complete loss of ApN, some compensatory mechanisms may operate and actually mask the effects on adipose tissue. The data of the present study also directly fit with other reports on acute or short-term effects of ApN administration (2, 16, 17).

The mechanisms underlying reduction of body fat in response to ApN supplementation are also disputed and have been ascribed either to increased energy expenditure (16, 17) or to decreased food intake (39). Here, we clearly show that reduced adiposity was not due to anorexia but rather to enhanced energy expenditure as illustrated by decreased feed efficiency and indirect calorimetric measurements. Accordingly, the expression of UCPs, molecules involved in energy dissipation (UCP2 and possibly UCP1 in this study) was augmented, in agreement with other acute or short-term reports on ApN administration (12, 16, 17).

In addition to increased energy expenditure, impaired adipocyte differentiation (34, 40) is a novel mechanism, which could contribute to reduce fat mass. Forced expression of ApN to white fat resulted in smaller (younger) adipocytes, whereas down-regulating some adipocyte markers such FAS, leptin, and TNF{alpha}. The decreased expression of the key lipogenic enzyme, FAS may play a role in the lower lipid accumulation within adipocytes. This down-regulation was associated with altered expression of transcription/growth factors known to modulate adipocyte differentiation. Thus, the preadipocyte marker, Pref-1, an epidermal growth factor, which functions in the maintenance of the preadipose state by inhibiting differentiation into adipocytes was elevated in fat tissue of Tg mice (41). Pref-1-deficient mice displayed obesity and increased serum lipid parameters (42), whereas mice overexpressing the soluble form of this growth factor showed decreased adipose tissue mass and adipocyte marker expression (41). On the other hand, the expression of the pleiotropic transcriptional activator, C/EBP{alpha} that coordinately stimulates the expression of the adipocyte genes giving rise to the adipocyte phenotype (35), was down-regulated in fat of our Tg animals. Taken together, these results suggest that adipocyte differentiation was impaired in Tg mice possibly due to altered expression of two key transcription/growth factors that orchestrate this stage. We next provided evidence that recombinant ApN induced direct changes of these key regulators in 3T3-F442A cells. Thus, at variance with another report on 3T3-L1 cells infected by a recombinant lentivirus that produced very high concentrations of the adipokine (20), our in vitro results support the hypothesis that ApN directly prevents the appearance of the adipocyte phenotype. Eventually, we showed that opposite modifications of Pref-1 and C/EBP{alpha} mRNA occurred in adipose tissue of ApN-deficient mice. Hence, targeting ApN to adipocyte may mitigate lipid accumulation and alter phenotype of adipocyte. This alteration of the adipocyte phenotype suggests molecular targets for the treatment of morbid obesity (43).

ApN causes adipose tissue remodeling and leads to a higher density of smaller adipocytes. These smaller adipocytes may further contribute to the metabolic action of the adipokine. Indeed, smaller adipocytes are known to better retain free fatty acids than larger ones and to release fewer inflammatory cytokines, such as TNF{alpha} that impair insulin signaling (44, 45). These cells may therefore play a role in the increased insulin sensitivity and in the hypolipemic effect of ApN.

ApN exerts its metabolic effects via two types of receptors: AdipoR1 and AdipoR2. Both types are expressed in adipocytes (5). AdipoR2 expression was up-regulated in fat of Tg mice. Because ApN may directly inhibit AdipoR2 in cultured adipocytes (20, 22), the up-regulation of this receptor isoform was rather explained by an indirect mechanism such as attenuation of TNF{alpha} expression. Indeed, TNF{alpha} mRNA was reduced in adipose tissue of Tg mice, a finding a contrario consonant with the enhanced expression of this cytokine observed in fat of mice with partial or complete ApN deficiency (22, 26). Taken together, these data reinforce the suggestion that ApN and TNF{alpha} exert negative reciprocal interactions on their local production in adipose tissue (26). Because TNF{alpha} has been found to decrease AdipoR2 mRNA in primary cultures of pig adipocytes (46), the local reduction of TNF{alpha} may explain why AdipoR2 mRNA was up-regulated in fat of our Tg mice. Thus, despite ApN excess, there was no down-regulation, but rather an up-regulation of ApN receptor expression in these mice. This suggests that the potency of ApN produced by these Tg mice was preserved and even possibly enhanced.

In conclusion, mice overexpressing ApN specifically in white fat showed a clear reduction in adiposity due to increased energy expenditure and to impaired adipocyte differentiation. Adipose tissue remodeling results in smaller and less mature adipocytes characterized by altered expression of lipogenic enzymes and adipocyte markers, and increased expression of UCPs and a preadipocyte marker. This prevention of the adipocyte phenotype could theoretically induce a lower rate of relapse after conventional treatment of obesity by caloric restriction (43). It also suggests molecular targets for pharmacologic treatment of morbid obesity.


   Acknowledgments
 
We are grateful to Pr. C. Remacle (University of Louvain) for discussion, to the Tg facilities provided by the University of Louvain, to C. Diepart (University of Louvain) for skillful assistance, and to L. Montbrun (Paul Sabatier University) from the Physiopathological Exploration platform of the Toulouse Genopole for performing the calorimetric experiments.


   Footnotes
 
This work was supported by grants from the Foundation of Scientific and Medical Research (3.4580.05), from the Fonds National de la Recherche Scientifique (1.5.189.04), the Fund for Scientific Development (University of Louvain), and Grant Action de recherche concertée 05/10-328 from the General Division of Scientific Research. I.B.B. has a fellowship from the Fonds pour la formation à la recherche dans l’industrie et dans l’agriculture (Belgium).

First Published Online January 4, 2007

1 I.B.B. and S.A.E.M. contributed equally to this work. Back

Abbreviations: ACO, Acyl-coenzyme A oxidase; aP2, adipocyte P2; ApN, adiponectin; AdipoR1, ApN receptor 1; AdipoR2, ApN receptor 2; BAT, brown adipose tissue; C/EBP-{alpha}, CCAAT/enhancer binding protein-{alpha}; cyclo, cyclophilin; Ct, cycle threshold; FAS, fatty acid synthase; gApN, globular ApN; GLUT4, insulin-sensitive glucose transporter; HES, hematoxylin-eosin-safran; HMM, high molecular mass; LMM, low molecular mass; NEFA, nonesterified free fatty acids, OGTT, oral glucose tolerance test; Pref-1, preadipocyte factor-1; PPAR, peroxisome proliferator-activated receptor; RTQ-PCR, real-time quantitative PCR; SREBP-1, sterol regulatory element-binding protein-1; Tg, transgenic or transgene; UCP1, 2, 3, uncoupling protein 1, 2, 3; WAT, white adipose tissue; WT, wild type.

Received June 20, 2006.

Accepted for publication December 27, 2006.


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