Inhibition of PPARγ, adipogenesis and insulin sensitivity by MAGED1

in Journal of Endocrinology
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Peroxisome proliferator-activated receptor-γ (PPARγ) is a master regulator of adipogenesis and a target of the thiazolidinedione (TZD) class of antidiabetic drugs; therefore, identifying novel regulators of PPARγ action in adipocytes is essential for the future development of therapeutics for diabetes. MAGE family member D1 (MAGED1), by acting as an adaptor for ubiquitin-dependent degradation pathways and a co-factor for transcription, plays an important role in neural development, cell differentiation and circadian rhythm. Here, we showed that MAGED1 expression was downregulated during adipogenesis and loss of MAGED1 promoted preadipocyte proliferation and differentiation in vitro. MAGED1 bound to PPARγ and suppressed the stability and transcriptional activity of PPARγ. Compared to WT littermates, MAGED1-deficient mice showed increased levels of PPARγ protein and its target genes, more CD29+CD34+Sca-1+ adipocyte precursors and hyperplasia of white adipose tissues (WATs). Moreover, MAGED1-deficient mice developed late-onset obesity as a result of decreased energy expenditure and physical activity. However, these mice were metabolically healthy as shown by improved glucose clearance and insulin sensitivity, normal levels of serum lipids and enhanced secretion of adipokines such as leptin and adiponectin. Taken together, our data identify MAGED1 as a novel negative regulator of PPARγ activity, adipogenesis and insulin sensitivity in mice. MAGED1 might therefore serve as a novel pharmaceutical target to treat obesity-associated insulin resistance.

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  • Figure S1. MAGED1 inhibits the transcriptional activity of PPAR. (A) Schematic view of protein domains of MAGED1 and locations of two LXXLL motifs. (B) PPRE-luciferase assay in NIH/3T3 cells (n = 4-8). (C) Pparg1 and Pparg2 promoter-driver luciferase assay in the absence/presence of MAGED1 in NIH/3T3 cells (n = 3). (D) Assays of luciferase reporters containing -677bp or -2275bp proximal promoter fragments of Pparg2 in NIH/3T3 cells (n = 3). (E) Pparg2 (-677bp) luciferase assay with various amounts of MAGED1 in NIH/3T3 cells (n = 6). (F) Pparg2 (-2275bp) luciferase assay with various amounts of MAGED1 in NIH/3T3 cells (n = 6). (G) Pparg2 luciferase assay in the presence of MAGED1 siRNA in NIH/3T3 cells (n = 6). Data presented as mean  SEM. *P<0.05, **P<0.01, and ***P<0.001 by One-way ANOVA with Tukey’s multiple comparisons test (B, E, and F) and two-tailed t-test (C, D, and G).
  • Figure S2. Normal BAT in MAGED1 KO mice. (A) Representative images of H&E staining of BAT from 8-month-old male WT and KO mice. (B) UCP1 protein expression in BAT of 3-, 6-, and 11-month-old WT and KO mice, determined by western blotting.
  • Figure S3. MAGED1 deficiency does not alter live gluconeogenesis and islet function. (A) Pyruvate tolerance test with 2g/kg BW of sodium pyruvate in 7 months old mice (n = 7-8). (B) Levels of G6pc and Pck1 gene expression in the liver of 7 months old mice (n = 3). (C) Serum levels of insulin after 7 months old mice were injected with a bolus of glucose (n = 4-5). (D) Glucose-stimulated insulin secretion of isolated islets from 7 months old WT and KO mice (n = 3-4). Data presented as mean  SEM.

 

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    MAGED1 controls adipogenesis in vitro. (A) Expression levels of Maged1 during the adipogenic induction of 3T3-L1 cells (n = 6). (B) Expression of MAGED1 and PPARγ proteins during 3T3-L1 adipocyte differentiation. (C) mRNA levels of Maged1 in gWAT from mice fed with NC or HFD (n = 4–5). (D) WT and KO MEFs were differentiated into adipocytes by MDI or MDI plus T3 and stained with Oil-Red O. The staining was quantified by spectrophotometry at OD 500 of the dye extracted from cells (n = 3). (E) SVF cells from gWAT of WT and KO mice were differentiated into adipocytes and Oil-Red O staining was quantified (n = 9). (F, G and H) Relative mRNA levels of total Pparg, Pparg2 and Adipoq in differentiated WT and KO SVFs (n = 6–7). (I) Expression of PPARγ protein in differentiated WT and KO SVFs. Data presented as mean ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test (A) and two-tailed t-test (C, D, E, F, G and H).

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    MAGED1 inhibits PPARγ stability and activity. (A) FLAG-tagged MAGED1 was co-expressed with MYC-tagged PPARγ1 or PPARγ2 in HEK 293 cells and their interactions were determined by reciprocal immunoprecipitation with anti-MYC and anti-FLAG antibodies. Normal IgG was used for immunoprecipitation in the last lane as negative controls. (B and C) PPARγ1 (B) and PPARγ2 (C) were co-transfected with vector or MAGED1 in HEK 293 cells and then treated with CHX for indicated times. Expression of PPARγ was determined by immunoblotting. Relative levels of PPARγ protein were shown below the Myc blots. (D) PPRE-luciferase assay in 3T3-L1 cells (n = 4). (E) Pparg1 and Pparg2 promoter-driver luciferase assay in the absence/presence of MAGED1 in 3T3-L1 cells (n = 4). Data presented as mean ± s.e.m. *P < 0.05, **P < 0.01, and ***P < 0.001 by One-way ANOVA with Tukey’s multiple comparisons test (D) and two-tailed t-test (E).

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    Increased adiposity in young MAGED1-KO mice. (A) Body weight of 2–4 weeks old WT and KO mice (n = 7–13). (B, C and D) Weight of different fat depots in 2-week-old (B, n = 7–13), 3-week-old (C, n = 6–11) and 4-week-old (D, n = 5–16) mice. (E and F) Adipocyte numbers (E) and cell sizes (F) in gWAT (n = 20). (G) Representative H&E images of gWAT. (H) Frequency distribution of adipocyte sizes in gWAT (n = 20). Data presented as mean ± s.e.m. or scatter dot blot with lines at mean and s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001 by two-tailed t-test.

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    Enhancement of adipogenesis in MAGED1 KO mice. (A) Expression of PPARγ in gWAT shown by Western blotting. (B) Quantitative RT-PCR of PPARγ-target genes in gWAT in 4-week-old mice (n = 5). (C) MTT assay of cultured preadipocytes (n = 11–14). (D) Expression of Cyclin D1 and A2 in culture SVF cells. (E) Immunostaining of BrdU in gWAT from mice injected with BrdU from postnatal day 7 to 13. (F) Quantification of the percentage of BrdU+ adipocytes (n = 7). (G) Frequency of CD29+CD34+Sca-1+ adipocyte precursors in gonadal SVF cells (n = 3). Data presented as mean ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001 by two-tailed t-test.

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    Late-onset obesity in MAGED1-deficient mice. (A) Growth curve of male WT and KO mice (n = 9). (B) Body composition of 7 months old male WT and KO mice (n = 9). (C) Adipose weight of 1-year-old male WT and KO mice (n = 4–5). (D) Growth curve of female WT and KO mice (n = 10–11). (E and F) Serum levels of leptin in fed (E) and fasted (F) mice (n = 3–6). (G and H) Serum levels of adiponectin in fed (G, n = 6–9) and fasted (H, n = 6–7) mice. (I, J and K) Serum levels of triglyceride (I), cholesterol (J) and free fatty acid (K) (n = 5–10). Data presented as mean ± s.e.m. or scatter dot blot with lines at mean and s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001 by two-tailed t-test.

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    Energy metabolism in MAGED1 mice. (A, B, C and D) Metabolic cage studies of 6-week-old male WT and KO mice (n = 7–9) showing food intake (A), RER (B), heat production (C) and physical activity (D). (E, F, G and H) Metabolic cage studies of body weight-matched 4-month-old male WT and KO mice (n = 8) showing food intake (E), RER (F), heat production (G) and physical activity (H). Absolute levels during the day and night shown at the top and average levels shown at the bottom. Data presented as mean ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001 by two-tailed t-test.

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    Glucose metabolism in MAGED1 mice. (A and B) Glucose tolerance tests with a dose of 2 g/kg BW glucose in 3 months (A, n = 11–13) and 6-month-old (B, n = 5–7) mice. Area under curves shown to the right. (C and D) Insulin tolerance tests with a dose of 0.75 U/kg BW insulin in 3-month (C, n = 12–14) and 6-month (D, n = 7)-old mice. Area under curves shown to the right. (E) Blood glucose levels of overnight-fasted mice at different ages (n = 4–13). (F, G, H and I) 15.5 months old mice were fasted overnight and levels of blood glucose (F), serum insulin (G), HOMA-IR (H) and serum glucagon (I) were determined (n = 4–5). Data presented as mean ± s.e.m. or scatter dot blot with lines at mean and s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001 by two-tailed t-test.

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    MAGED1 controls insulin sensitivity. (A and B) Immortalized WT and KO MEFs were treated with various concentrations of insulin for 5 min (A) or 100 nM insulin for various times (B). Total proteins were run for immunoblotting with indicated antibodies. (C) gWAT proteins from ad libitum-fed 8-month-old WT and KO mice were subjected to immunoblotting (n = 5). (D) Expression of Il6 and Tnfa genes in gWAT (n = 5). (E) Serum levels of TNFα in WT and KO mice at different ages (n = 3–8). Data presented as mean ± s.e.m. or scatter dot blot with lines at mean and s.e.m.

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