The inhibition of fat cell proliferation by n-3 fatty acids in dietary obese mice
© Hensler et al; licensee BioMed Central Ltd. 2011
Received: 21 June 2011
Accepted: 2 August 2011
Published: 2 August 2011
Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) of marine origin exert multiple beneficial effects on health. Our previous study in mice showed that reduction of adiposity by LC n-3 PUFA was associated with both, a shift in adipose tissue metabolism and a decrease in tissue cellularity. The aim of this study was to further characterize the effects of LC n-3 PUFA on fat cell proliferation and differentiation in obese mice.
A model of inducible and reversible lipoatrophy (aP2-Cre-ERT2 PPARγL2/L2 mice) was used, in which the death of mature adipocytes could be achieved by a selective ablation of peroxisome proliferator-activated receptor γ in response to i.p. injection of tamoxifen. Before the injection, obesity was induced in male mice by 8-week-feeding a corn oil-based high-fat diet (cHF) and, subsequently, mice were randomly assigned (day 0) to one of the following groups: (i) mice injected by corn-oil-vehicle only, i.e."control" mice, and fed cHF; (ii) mice injected by tamoxifen in corn oil, i.e. "mutant" mice, fed cHF; (iii) control mice fed cHF diet with15% of dietary lipids replaced by LC n-3 PUFA concentrate (cHF+F); and (iv) mutant mice fed cHF+F. Blood and tissue samples were collected at days 14 and 42.
Mutant mice achieved a maximum weight loss within 10 days post-injection, followed by a compensatory body weight gain, which was significantly faster in the cHF as compared with the cHF+F mutant mice. Also in control mice, body weight gain was depressed in response to dietary LC n-3 PUFA. At day 42, body weights in all groups stabilized, with no significant differences in adipocyte size between the groups, although body weight and adiposity was lower in the cHF+F as compared with the cHF mice, with a stronger effect in the mutant than in control mice. Gene expression analysis documented depression of adipocyte maturation during the reconstitution of adipose tissue in the cHF+F mutant mice.
Dietary LC n-3 PUFA could reduce both hypertrophy and hyperplasia of fat cells in vivo. Results are in agreement with the involvement of fat cell turnover in control of adiposity.
KeywordsDHA and EPA fish oil fat cell turnover
Adipose tissue and its secreted products, adipokines, have a major role in the development of obesity-associated metabolic disarrangement including dyslipidaemia and insulin resistance (i.e. the components of metabolic syndrome). Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA), namely eicosapentaenoic acid (EPA; 20:5 n-3) and docosahexaenoic acid (DHA; 22:6 n-3) act as natural hypolipidemics, reduce risk of cardiovascular disease and could prevent development of obesity and insulin resistance in humans . Also our experiments on mice have demonstrated that substitution of 15% lipids in a corn oil-based high fat diet (cHF) by LC n-3 PUFA concentrate (i.e. feeding cHF+F diet, see Methods) prevented dietary induced obesity and associated metabolic disorders [2–4]. The preferential decrease in abdominal adipose tissue growth resulted not only from modulation of metabolism in response to LC n-3 PUFA , but probably also in part from the inhibition of fat cell proliferation. Quantification of adipose tissue DNA revealed that the reduction of epididymal fat was associated with 34-50% depression of tissue cellularity . In vitro, both EPA and DHA inhibited adipocyte differentiation and lipid droplet formation [6–8] and DHA induced apoptosis in postconfluent preadipocytes . To further characterize inhibitory effect of LC n-3 PUFA on adipose cell proliferation and differentiation in vivo, and to learn more about the role of fat cell turnover in the control of adiposity , we used mouse transgenic model of inducible and reversible lipoatrophy (aP2-Cre-ERT2 PPARγL2/L2). In this model, death of mature brown and white adipocytes is achieved by selective ablation of peroxisome proliferator-activated receptor γ (PPARγ) using the tamoxifen-dependent Cre-ERT2 recombination system . PPARγ is essential for survival of mature adipocyte and deletion of PPARγ causes adipocyte death, triggers an inflammatory reaction and promotes proliferation and differentiation of preadipocytes into new adipocyte expressing PPARγ . Our results document that LC n-3 PUFA slow down compensatory adipose tissue growth and adipocyte proliferation after the transgenically-induced transient lipoatrophy and they also support the notion  that fat cell turnover is involved in the control of adipose tissue mass.
Animals and experimental design
Histological analysis of adipose tissue
Epididymal white fat samples were fixed in 4% formaldehyde embedded in paraffin and stained with hematoxylin/eosin. Morphometry of adipocytes was performed as before , using NIS-Elements 3.0 AR morphometric software (Laboratory Imaging, Prague, Czech Republic). The morphometry data are based on more than 800 cells taken randomly from six different areas per animal.
Quantitative real time PCR
Gene specific forward and reverse primer sequences used for qRT-PCR
NCBI accession number
Adiponectin levels were determined using Western blotting as before . Tris-acetate gradient gel (NuPAGE 3-8%, Invitrogen, Life Technologies, Carlsbad, CA, USA) was used for division of multimeric forms of adiponectin in plasma. Primary rabbit anti-mouse polyclonal antibodies (BioVendor, Brno, Czech Republic), followed by secondary donkey anti-rabbit IgG infrared dye conjugated antibodies (IR Dye 800, Rockland, Gilbertsville, PA, USA) were using. Membranes were scanned using Odyssey IR imager (Li-Cor Biosciences, Lincoln, NE, USA).
All values are presented as mean ± SE. Logarithmic transformation was used to stabilize variance in cells when necessary. Data were analyzed by one-way ANOVA with Holm-Sidak posthoc test using SigmaStat 3.5 statistical software. Comparisons were judged to be significant at p ≤ 0.05.
Body weight, fat depots weight, adipocyte size and plasma adiponectin
Final body weight (g)
44.1 ± 2.1
42.2 ± 1.8
44.9 ± 1.9
41.5 ± 2c
Epididymal fat (mg)
1607 ± 210
1369 ± 165
1169 ± 184a
1180 ± 28a
2934 ± 292
2391 ± 191a
2892 ± 354
2378 ± 261a,c
Size of adipocytes (μm2)
4397 ± 518
3381 ± 285a
3471 ± 422a
3126 ± 323a,b
3232 ± 267
2653 ± 139
3182 ± 323
2964 ± 273
Subcutaneous fat (mg)
479 ± 59
395 ± 39
319 ± 37a
332 ± 37a
686 ± 64
542 ± 59
656 ± 73
463 ± 74a,c
Interscapular brown fat (mg)
147 ± 17
124 ± 11
123 ± 13a
121 ± 8a
190 ± 23
159 ± 13
179 ± 15
150 ± 3
0.70 ± 0.06
0.79 ± 0.06
0.52 ± 0.03a
0.50 ± 0.04a,b
0.79 ± 0.04
0.82 ± 0.03
0.51 ± 0.04a
0.57 ± 0.02b
0.01 ± 0.00
0.01 ± 0.00
0.01 ± 0.00
0.01 ± 0.00
1.49 ± 0.10
1.62 ± 0.08
1.04 ± 0.06a.b
1.12 ± 0.06a,b
0.54 ± 0.04
0.96 ± 0.10a
0.66 ± 0.07
1.06 ± 0.11a,c
0.53 ± 0.02
0.66 ± 0.05
0.58 ± 0.04
0.75 ± 0.05a,c
0.01 ± 0..0
0.02 ± 0.0
0.02 ± 0.0
0.02 ± 0.0
1.09 ± 0.06
1.64 ± 0.15a
1.25 ± 0.10
1.82 ± 0.14a,c
Histological and morphometric analysis of epididymal fat was performed to characterize effect of PPARγ ablation on tissue morphology in the model of dietary obese mice. At day 14, mean size of adipocytes was smaller in mutant as compared with control mice, and it was decreased further in response to dietary LC n-3 PUFA, resulting in the smallest adipocytes in the cHF+F mutant mice (Table 2). Importantly, at day 42, mean size of adipocytes was similar in all the groups (Table 2), and also distribution of fat cell sizes was not significantly affected by either diet, or the transient genetic ablation of PPARγ (Figure 1B).
Plasma levels of total adiponectin and of its biological active high molecular weight (HMW) form, which is implicated in enhancement of insulin sensitivity , were lower in mutant as compared with control mice at day 14. Latter on, total and HMW adiponectin level were similar in both control and mutant mice, with a tendency to be higher in the mutant mice (Table 2). In accordance with our previous findings showing induction of adiponectin by LC n-3 PUFA [3, 13], adiponectin levels were higher in the cHF+F as compared with the cHF mice, in both control and mutant mice, even at day 14 (Table 2).
Adipose tissue belongs to the most flexible tissues in the body, because it is rapidly remodeled by hyperplasia and hypertrophy of adipocytes, depending on energy fluxes. Fat cell turnover might contribute to determination of adiposity . However, the mechanisms, which are involved in setting the turnover and their links to energy balance, remain to be established. Enormous plasticity of adipose tissue was also documented by our results. In agreement with the first study on this transgenic model , as well as other model of reversible lipoatrophy , 6 weeks after tamoxifen injection, adipose tissue from mutant mice appeared histologically identical to untreated mice, indicating complete tissue regeneration. The global, robust return of fat mass was confirmed by progressive body weight gain, especially in mutant mice fed cHF. The death fat cells are replaced by newly differentiated adipocytes originated from endogenous preadipocytes. The histological changes of epididymal fat depot from mice fed cHF were mirrored by changes of transcript levels for genes encoding Scd-1, Cidec (FSP27), and Cox3. As already described, the above genes are activated during adipocyte differentiation and maturation [15–17]. Similarly, plasma level of adiponectin was associated with the reduction of fat mass in the mutant mice compared to controls at day 14. Thus, similarly as in the transgenic mice fed chow diet , also in the dietary obese mice studied here, the transgenically-induced lipoatrophy was fully reversible within approximately 6 weeks after the PPARγ ablation.
Based on our previous results suggesting that the anti-obesity effect LC n-3 PUFA could be, at least in part, explained by the prevention of fat cell proliferation during high-fat diet-feeding, we have challenged both control and the transgenic mice by dietary LC n-3 PUFA. Indeed, the cHF+F diet treatment decreased body weight gain in both control and mutant mice. At both time points analyzed (day 14 and day 42), total body weight, as well as weights of adipose depots were the lowest in the mutant mice fed cHF+F diet. Nevertheless, when body weights stabilized in a group-specific manner, by the end of the treatment at 6 weeks, body weights were affected more by the diet than by the transient genetic ablation. Equal size of adipocytes in all the groups at the end of the treatment, suggest (i) similar number of adipocytes in epididymal fat within the same type of diet; (ii) existence of mechanisms, which tend to stabilize fat cell number via controlling fat cell turnover; and (iii) tuning of these hypothetical mechanisms by LC n-3 PUFA or their metabolites (see below).
In accordance with our already published data showing stimulation of mitochondrial biogenesis and β-oxidation  in white adipose tissue in response to dietary LC n-3 PUFA, feeding cHF+F diet induced Pparα and Cox3 expression in control mice. At day 14, tamoxifen-induced-lipoatrophy in mutant mice was associated with lower expression of the above genes and interfered also with the LC n-3 PUFA effects. At day 42, expression of Pparα and Cox3 was strongly induced by dietary LC n-3 PUFA while no effect of tamoxifen injection was found. Furthermore, qRT-PCR analysis shown identical pattern of Cidec expression, i.e. significant reduction by tamoxifen treatment in control mice at day 14 and marked induction by LC n-3 PUFA at day 42 with the highest level in the mutant cHF+F mice.
Our results document that dietary LC n-3 PUFA could decrease adiposity in obese mice by a mechanism, which depends on counteraction of both, differentiation and proliferation of adipose cells. One possible mechanism involves the changes in fatty acid composition of cellular membranes, and hence, altered formation of PUFA-derived active metabolites like as eicosanoids [1, 6, 18, 19]. The anti-proliferative effect may be involved in the decreased adiposity of pups born to rat dams that fed diets supplemented by n-3 fatty acids during gestation and sucking (reviewed in ref. ). It has been hypothesized that LC n-3 PUFA are involved in the anti-obesity effect of breast-feeding . Importantly, our results support the notion  that adiposity is closely linked to the control of fat cell turnover and that mechanisms could exist, which control fat cell proliferation independent of energy balance.
corn oil-based high fat diet
cHF diet supplemented with LC n-3 PUFA concentrate (15% of dietary lipids)
- PPAR-α (-γ):
peroxisome proliferator activated receptor-α (-γ)
- LC n-3 PUFA long-chain n-3:
polyunsaturated fatty acids.
This work was supported by the Czech Science Foundation (301/11/0226). MH was also supported by CSF PhD project (305/08/H037). The authors declare no conflict of interest.
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