1 State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, Jiangsu, China
2 Laboratory Animal Center, Nantong University, Nantong, Jiangsu, China
3 School of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing, Jiangsu, China
4 Laboratory of Gastrointestinal Microbiology, Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, China
5 Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, Minnesota, USA
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.
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.