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. 2004 Mar 30;101(13):4543-7.
doi: 10.1073/pnas.0400356101. Epub 2004 Mar 16.

Peroxisome proliferator-activated receptor gamma is required in mature white and brown adipocytes for their survival in the mouse

Takeshi Imai  1 , Reiko Takakuwa, Sandra Marchand, Emilie Dentz, Jean-Marc Bornert, Nadia Messaddeq, Olivia Wendling, Manuel Mark, Béatrice Desvergne, Walter Wahli, Pierre Chambon, Daniel Metzger
Affiliations

Peroxisome proliferator-activated receptor gamma is required in mature white and brown adipocytes for their survival in the mouse

Takeshi Imai et al. Proc Natl Acad Sci U S A. .

Abstract

The peroxisome proliferator-activated receptor gamma (PPARgamma) mediates the activity of the insulin-sensitizing thiazolidinediones and plays an important role in adipocyte differentiation and fat accretion. The analysis of PPARgamma functions in mature adipocytes is precluded by lethality of PPARgamma(-/-) fetuses and tetraploid-rescued pups. Therefore we have selectively ablated PPARgamma in adipocytes of adult mice by using the tamoxifen-dependent Cre-ER(T2) recombination system. We show that mature PPARgamma-null white and brown adipocytes die within a few days and are replaced by newly formed PPARgamma-positive adipocytes, demonstrating that PPARgamma is essential for the in vivo survival of mature adipocytes, in addition to its well established requirement for their differentiation. Our data suggest that potent PPARgamma antagonists could be used to acutely reduce obesity.

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Figures

Fig. 1.
Fig. 1.
Selective Tam-induced PPARγ gene disruption in adipocytes of WAT and BAT of adult mice. (A) Diagram of the WT PPARγ genomic locus (+), the floxed PPARγ L2 allele, and the PPARγ L–-null allele obtained after Cre-mediated excision of exons 1 and 2. Black boxes and arrows indicate exons and PCR primers, respectively. The probe O7 location is shown. (B) Timing of Tam administration and phenotypic analysis. (C) Adipocyte-selective Tam-induced generation of PPARγ L– alleles. PCR analysis of DNA from the indicated cell types and tissues of 8-week-old aP2-Cre-ERT2(tg/0)/PPARγL2/L2 mice at D4 (lanes 6–11), from purified epididymal adipocytes and iBAT isolated from (i) aP2-Cre-ERT2(tg/0)/PPARγL2/L2 mice at D0, D4, D14, and D42 (lanes 12–15 and lanes 19–22, respectively) and (ii) control aP2-Cre-ERT2(tg/0)/PPARγL2/+ mice at D0, D4, and D42 (lanes 16–18 and lanes 23–25, respectively). Control PCR on genomic DNA from of PPARγL–/+, PPARγL2/+, PPARγL2/L2, and PPARγ+/+ mice and from 9.5 days postconception PPARγL–/L– embryos are presented in lanes 1–5; PCR fragments corresponding to the PPARγ L2, L–, and + alleles are displayed. Macrophage: i.p. cells containing ≈30% macrophages. (D) Epididymal fat pad (epiWAT) and iBAT weight of CT and of MT mice at D7. Values are expressed as the mean ± SEM (n = 5). *, P < 0.05. (E) Body fat content evaluated by DEXA scanning. CT and PPARγad–/– premutant mice (MT) were analyzed before and for 7 weeks after Tam treatment. The percentage of fat content in mutant mice relative to CT mice for each time point is shown. Values (black squares for CT and open circles for MT) are expressed as the mean ± SEM (n = 7). The body fat content of CT mice for each time point was set to 100. *, P < 0.05.
Fig. 2.
Fig. 2.
Histological analysis of WAT of Tam-treated PPARγad–/– premutant mice. Histological sections from paraffin-embedded epididymal WAT from CT (aP2-Cre-ERT2(0/0)/PPARγL2/L2) mice at D4 (A) and D2 (F) and MT mice at D2 (G), D4 (H and I), D7 (B), D14 (J), and D42 (C) (Tam injection at D0), and D67 (D) and D116 (E), after a second Tam injection at D56. (A–I) Hematoxylin/eosin staining. (J) Trichrome staining. Ultrastructure of adipose tissue of CT (aP2-Cre-ERT2(0/0)/PPARγL2/L2) at D4 (K) and MT mice at D 4 (L) and D7 (M–O). Blue arrows point to clusters of infiltrating cells (B and D); black arrows point to adipocytes with abnormal profiles (G); white arrows point to small lipid droplets in adipocytes (G, M, and N) and to phagocytosed lipid droplets (O); green arrows point to neutrophils (H and I); red arrows point to lymphocytes (I and N); yellow arrows point to macrophages (I, N, and O); orange arrows point to fibroblast-like cells (J and N); and black and gray arrows point to disrupted adipocyte cell membrane and cell debris (L). Lp, lipid droplet; Nc, nucleus. (Scales are indicated in each image.)
Fig. 3.
Fig. 3.
Expression of Pref-1 and genes involved in respiratory chain function in Tam-treated PPARγad–/– premutant mice. (A) Pref-1 expression was analyzed by RT-PCR performed on RNA extracted from epididymal WAT of CT (lanes 2, 4, 6, and 8) and MT (lanes 3, 5, 7, and 9) mice at D0, D7, D14, and D42. Hypoxanthine phosphoribosyltransferase was used as an internal control. (B) Transcript levels of the indicated genes analyzed by quantitative RT-PCR on RNA isolated from BAT of CT (filled bars) and MT (open bars) mice at D4. Values are expressed as mean ± SEM (relative to CT). n = 5. *, P < 0.05; **, P < 0.005; #, no statistically significant difference.
Fig. 4.
Fig. 4.
Histological analysis of BAT of Tam-treated PPARγad–/– premutant mice. Histological sections from paraffin-embedded interscapular BAT from CT (aP2-Cre-ERT2(0/0)/PPARγL2/+) mice at D4 (A and F) and MT mice (B–E and G–J) at D4 (B and G), D7 (C and H), D14 (D, I, and J), and D42 (E). (A–I) Hematoxylin and eosin staining. (J) Trichrome staining. (K–O) EM. (K) CT (aP2-Cre-ERT2(0/0)/PPARγL2/+) at D4. (Inset) Typical mitochondria. (L–N) MT at D4. (O) mutant at D14. White arrows point to fused lipid droplets (B and G) and small lipid droplets (O); blue arrows point to necrotic area (C and H) and clumped chromatin (L); green arrows point to detached nuclear membrane (L); red arrows point to lymphocytes (D and I); orange arrows point to fibroblast-like cells (J); purple arrows point to glycogen (O). Lp, lipid droplet; Nc, nucleus; m, mitochondria; hm and sm, hypertrophic and swollen mitochondria, respectively. (Scales are indicated in each image.)
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