Lsd1 prevents age-programed loss of beige adipocytes - PubMed (original) (raw)

Lsd1 prevents age-programed loss of beige adipocytes

Delphine Duteil et al. Proc Natl Acad Sci U S A. 2017.

Abstract

Aging is accompanied by major changes in adipose tissue distribution and function. In particular, with time, thermogenic-competent beige adipocytes progressively gain a white adipocyte morphology. However, the mechanisms controlling the age-related transition of beige adipocytes to white adipocytes remain unclear. Lysine-specific demethylase 1 (Lsd1) is an epigenetic eraser enzyme positively regulating differentiation and function of adipocytes. Here we show that Lsd1 levels decrease in aging inguinal white adipose tissue concomitantly with beige fat cell decline. Accordingly, adipocyte-specific increase of Lsd1 expression is sufficient to rescue the age-related transition of beige adipocytes to white adipocytes in vivo, whereas loss of Lsd1 precipitates it. Lsd1 maintains beige adipocytes by controlling the expression of peroxisome proliferator-activated receptor α (Ppara), and treatment with a Ppara agonist is sufficient to rescue the loss of beige adipocytes caused by Lsd1 ablation. In summary, our data provide insights into the mechanism controlling the age-related beige-to-white adipocyte transition and identify Lsd1 as a regulator of beige fat cell maintenance.

Keywords: Lsd1; Ppara; adipocyte; aging; beige fat.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Lsd1 prevents the age-programmed loss of beige adipocytes. (A) qRT-PCR analysis showing relative Lsd1 mRNA levels in epWAT and ingWAT and in liver of 10- and 30-wk-old WT mice (mean ± SEM; ***P < 0.001; 10-wk-old mice, n = 9; 30-wk-old mice, n = 12). (B) Western blot analysis of Lsd1 protein levels in ingWAT of 10- and 30-wk-old WT mice. β-Tubulin was used as a loading control. (C) H&E staining and (D) immunohistochemical detection of Ucp1 on representative sections of ingWAT from control (Ctrl) and adipose tissue-specific Lsd1 overexpressing (Lsd1cTg) mice at 10 and 30 wk of age. (Scale bars: 200 µm.) (E) qRT-PCR analysis showing relative mRNA levels of indicated genes in ingWAT of control and Lsd1cTg mice at 10 and 30 wk of age (mean ± SEM; *P < 0.05; n = 12).

Fig. S1.

Fig. S1.

Lsd1 prevents the age-programmed loss of beige adipocytes (Fig. 1). (A) Western blot analysis of Lsd1 protein levels in epWAT and liver of 10- and 30-wk-old WT mice. β-Tubulin was used as a loading control. (B) Scheme depicting generation of adipocyte-specific Lsd1-overexpressing mice (Lsd1cTg). To confirm expression of the Lsd1 transgene, we performed immunoprecipitations (IP) in ingWAT of control (Ctrl) and Lsd1cTg mice with anti-Flag antibody followed by Western blot analysis using anti-Lsd1 antibody. (C) Western blot analysis of Lsd1 and Ucp1 protein levels in ingWAT of control and Lsd1cTg mice at 10 and 30 wk of age. β-Tubulin was used as a loading control. (D) Rabbit IgG (rIgG) control for Ucp1 immunohistochemical detection shown in Fig. 1_C_ performed on representative sections of ingWAT of 10- and 30-wk-old control and Lsd1cTg mice. (E) qRT-PCR analysis showing relative mRNA levels of indicated genes in ingWAT of control and Lsd1cTg mice at 10 and 30 wk of age (mean ± SEM; *P < 0.05; n = 12).

Fig. 2.

Fig. 2.

Loss of Lsd1 in adipocytes accelerates the beige-to-white transition of ingWAT. (A) Immunohistochemical detection of Ucp1 on ingWAT from control (Ctrl) and adipocyte-specific Lsd1 KO (Lsd1cKO) mice at 6, 10, and 30 wk of age. (Scale bars: 200 µm.) (B) qRT-PCR analysis showing relative mRNA levels of indicated genes in ingWAT of control and Lsd1cKO mice at 10 and 30 wk of age (mean ± SEM; *P < 0.05; n = 10). (C) Immunofluorescence detection using anti-GFP and anti-perilipin (Plin1) antibodies on representative sections of ingWAT of control and Lsd1cKO-GFP mice containing Ucp1-Dtr-GFP fusion construct, which enables detection of membrane-localized GFP protein in Ucp1-positive cells and their descendants. (Scale bars: 100 µm.)

Fig. S2.

Fig. S2.

Loss of Lsd1 in adipocytes accelerates the beige-to-white transition of ingWAT (Fig. 2). (A) qRT-PCR analysis showing relative Lsd1 mRNA levels in BAT, epWAT, ingWAT, and liver of control (Ctrl) and adipocyte-specific Lsd1cKO mice (mean ± SEM; **P < 0.01 and ***P < 0.001; n = 10). (B) Western blot analysis of Lsd1 and Ucp1 in ingWAT of 10 wk-old control and Lsd1cKO mice. β-Tubulin was used as a loading control. (C) H&E staining and (D) rabbit IgG (rIgG) control for the Ucp1 immunohistochemical detection shown in Fig. 2_A_ performed on representative sections of ingWAT of 10- and 30-wk-old control and Lsd1cKO mice. (Scale bars: 200 μm.) (E) qRT-PCR analysis showing relative mRNA levels of indicated genes in ingWAT of control and Lsd1cKO mice at 10 and 30 wk of age (mean ± SEM; *P < 0.05; n = 10). (F) Western blot analysis of Parp and Casp3 in ingWAT of 10- and 30-wk-old control and Lsd1cKO mice. β-Tubulin was used as a loading control. (G) Cartoon depicting the Ucp1-Dtr-GFP knock-in construct, Adipoq-CreERT2 transgene, and conditional and deleted Lsd1 alleles. Lsd1iKO-GFP mice were obtained after Tam injection. (H) Genotyping of mouse BAT (marked “B”), ingWAT (“W”), and liver (“L”) biopsies of control and inducible adipose-specific Lsd1iKO-GFP mice for the presence of Lsd1 conditional (“p”) allele (1), Adipoq-CreERT2 recombinase (Cre) (2), or Lsd1 recombined (“d”) allele (3) by semiquantitative PCR. (I) qRT-PCR and (J) Western blot analyses showing Lsd1 mRNA and protein levels in ingWAT of 10-week-old control and Lsd1iKO-GFP mice. β-Tubulin was used as a loading control (G) (mean ± SEM; ***P < 0.001; n = 10). (K) H&E staining and immunohistochemical detection of Ucp1 on ingWAT from Lsd1p/p control and inducible adipocyte-specific Lsd1 KO mice (Lsd1iKO-GFP), both containing the Ucp1-Dtr-GFP reporter. construct. (Scale bars: 200 μm.) (L and M) Immunofluorescence detection using (L) anti-GFP and anti-Plin1 or (M) anti-GFP and anti-Lsd1 antibodies on representative sections of ingWAT from control and Lsd1iKO-GFP mice. (Scale bars: L, 100 μm; M, 50 μm.) (M) Arrows indicate the GFP-positive and Lsd1-negative adipocytes.

Fig. 3.

Fig. 3.

Lsd1 is required for development and maintenance of cold-induced beige adipocytes. (A) H&E staining and (B) immunofluorescence detection using anti-GFP and anti-Plin1 antibodies on representative sections of ingWAT of control (Ctrl) and Lsd1iKO-GFP mice in the absence or presence of Tam treatment. Time scale indicates age of the mice and start and end of Tam and cold treatment. Dagger indicates the time point at which mice were killed. (Scale bars: 200 µm.) (C) qRT-PCR analysis showing relative mRNA levels of indicated genes in ingWAT extracts from control and Lsd1iKO-GFP mice treated as described in A (mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; n = 7).

Fig. S3.

Fig. S3.

Lsd1 is required for development and maintenance of cold-induced beige adipocytes (Fig. 3). (A) H&E staining and (B) immunofluorescence detection using anti-GFP and anti-Plin1 antibodies on representative sections of ingWAT of Lsd1p/p/Ucp1-Dtr-GFP control (Ctrl) and Lsd1iKO-GFP mice treated with Tam for 5 d and maintained at 24 °C or 10 °C for 10 d. Time scale indicates age of the mice and start and end of Tam and cold treatment. Dagger indicates the time point at which mice were killed (Scale bars: 200 μm.) (C) Mitochondrial respiration measurement performed on ingWAT extracts from control and Lsd1iKO-GFP mice maintained at 24 °C or 10 °C for 10 d (mean ± SEM; *P < 0.05 and **P < 0.01; n = 9). (D) qRT-PCR analysis showing relative mRNA levels of indicated genes in ingWAT of control and Lsd1iKO-GFP mice maintained at 24 °C or 10 °C for 10 d (mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; n = 9). (E) Mitochondrial respiration measurement in ingWAT extracts from control and Lsd1iKO-GFP mice treated as described in Fig. 3_A_ (mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; n = 7). (F and G) Scheme illustrating the role of Lsd1 in the development and maintenance of beige adipocytes. (F) Beige adipocytes prevail in ingWAT of young rodents. In contrast, upon Lsd1 ablation in Lsd1iKO-GFP mice, beige adipocytes are not maintained and undergo a beige-to-white adipocyte transition. ingWAT of control mice responds to cold exposure by the formation of additional, inducible beige adipocytes, whereas, in ingWAT from Lsd1iKO-GFP mice, no beige adipocytes emerge upon cold stimulation. (G) Mice were preexposed to cold for 10 d, which results in the emergence of inducible beige adipocytes. While the cold exposure was continued, Lsd1 ablation was induced by Tam. ingWAT of control mice responds to prolonged cold stimulation by increased appearance of inducible beige adipocytes. In contrast, upon Lsd1 ablation, cold-induced beige adipocytes cannot be maintained and transition to a white adipocyte morphology despite the presence of the cold stimulus. Time scale indicates age of the mice and the start and end of Tam and cold treatment. Dagger indicates the time point at which mice were killed.

Fig. S4.

Fig. S4.

Lsd1 targets Ppara to maintain beige adipocytes (Fig. 4). (A) Pie chart depicting the ratio of differentially expressed up- and down-regulated genes obtained from RNA-seq of ingWAT from control (Ctrl) and Lsd1cKO mice at 6 wk of age. (B) Pathway enrichment analysis of down-regulated genes from RNA-seq of ingWAT from control and Lsd1cKO mice at 6 wk of age. (C and D) qRT-PCR analysis showing relative mRNA levels of indicated genes in differentiated (C and D) immortalized adipocytes and (D) C3H-10T1/2 cells induced for white or beige adipogenesis and treated with vehicle or Lsd1-specific inhibitor QC6688 [Lsd1(i)] (mean + SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; n = 3). (E) ChIP-qPCR analysis of the Ppara promotor (−801 to −632 bp) performed with anti-Lsd1, anti-p300, anti-PolII, anti-H3K27ac, anti-H3K4me2, anti-H3K4me3, H3K9me2, H3K9me3, and anti-H3 antibodies, and rabbit (rIgG) or mouse IgG (mIgG) in differentiated immortalized adipocytes induced for white or beige adipogenesis. The precipitated chromatin was quantified by qPCR by using primers flanking the −801 to −632 bp region of the Ppara gene (mean ± SEM; *P < 0.05 and **P < 0.01; n = 3). (F and G) ChIP analysis performed with anti-Lsd1, anti-p300, anti-PolII, anti-H3K27ac, anti-H3K4me2, anti-H3K4me3, H3K9me2, H3K9me3, and anti-H3 antibodies, and rabbit (rIgG) or mouse IgG (mIgG) in differentiated (F) C3H-10T1/2 adipocytes or (G) immortalized adipocytes induced for white or beige adipogenesis and treated with vehicle or Lsd1(i). The precipitated chromatin was quantified by qPCR by using primers flanking an unrelated region (mean ± SEM; n = 3). (H) Western blot analysis of Lsd1 and Ppara protein levels in ingWAT of 10- and 30 wk-old WT mice. β-Actin was used as a loading control. (I) H&E staining and (J) immunohistochemical detection of Ucp1 on representative sections and (K) qRT-PCR analysis showing relative mRNA levels of indicated genes performed on ingWAT of control and Lsd1cKO mice at 10 wk of age treated for 3 d with vehicle or the Ppara agonist GW9578 (mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; n = 6). (Scale bars: Left, 200 μm; Right, 50 μm.)

Fig. 4.

Fig. 4.

Lsd1 targets Ppara to maintain beige adipocytes. (A) ChIP-qPCR analysis of the Ppara promotor (−801 to −632 bp) performed with anti-Lsd1, anti-p300, anti-PolII, anti-H3K27ac, anti-H3K4me2, anti-H3K4me3, H3K9me2, H3K9me3, anti-H3 antibodies, and rabbit (rIgG) or mouse IgG (mIgG) in differentiated C3H-10T1/2 adipocytes treated with Lsd1-specific inhibitor QC6688 [Lsd1(i)] or vehicle. The precipitated chromatin was quantified by qPCR using primers flanking the −801 to −632 bp region of the Ppara gene (mean ± SEM; *P < 0.05 and **P < 0.01; n = 3). (B) H&E staining and (C) immunohistochemical detection of Ucp1 on representative sections and (D) qRT-PCR analysis showing relative mRNA levels of indicated genes in ingWAT of control (Ctrl) and Lsd1cTg mice at 30 wk of age treated for 3 d with vehicle or the Ppara antagonist GW6471 (D) (mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; n = 6). (Scale bars: 200 µm.)

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