Article Title: WldS Enhances Insulin Transcription and Secretion via a SIRT1-Dependent Pathway and Improves Glucose Homeostasis
Figure Lengend Snippet: Wld S downregulates UCP2 expression and upregulates ATP levels through SIRT1. A : Wld S colocalized with SIRT1 in MIN6 cells. The stable cell lines expressing EGFP-Wld S were transiently transfected with pCMV-myc-SIRT1 and stained with anti-Myc antibody and DAPI. Scale bar, 5 μm. B : Wld S was coimmunoprecipitated with SIRT1 from the pancreatic lysates of Wld S mice. C : Wld S and its enzyme-dead mutant Wld S -H112A coimmunoprecipitated with SIRT1. The MIN6 cells stably expressing EGFP, EGFP-Wld S , or EGFP-Wld S -H112A were used for immunoprecipitation. UCP2 protein levels were also detected by Western blot. D and E : Liquid chromatography-tandem mass spectrometry analysis of NMN, NADP, NADPH, NAD, NADH, NA, and NAM extracted from pancreas of 9-week-old wild-type (WT) ( D ) or Wld S mice ( E ). *With significant difference. F : Quantification of the small molecules corresponding to D and E showed that NAD and NMN levels were upregulated in the pancreas of Wld S mice ( n = 4 for each genotype). G : Wld S repressed UCP2 promoter activity like SIRT1. UCP2 promoter activity was measured by luciferase assay in 293T cells transfected with pGL3-UCP2-Promoter and the indicated plasmids. H : Wld S downregulated UCP2 mRNA levels dependent on its enzyme activity. UCP2 mRNA level was measured by real-time PCR with MIN6 cell lines stably expressing EGFP, EGFP-Wld S , and EGFP-Wld S -H112A. I : Wld S downregulated UCP2 mRNA levels via SIRT1. UCP2 mRNA levels were determined by real-time PCR using islets isolated from mice with the indicated genotype ( n = 4 for each genotype). J : Wld S downregulated UCP2 protein levels via SIRT1. The protein levels in brown fat tissue with the indicated genotype were detected with SIRT1, Wld S , UCP2, and tubulin antibodies ( n = 3 for each genotype). K : Quantification of the UCP2 protein levels corresponding to J . L : Wld S increased ATP levels in primary cultured islets at the indicated glucose concentration ( n = 3). M : Wld S upregulated ATP level in islets via SIRT1. ATP levels were measured in islets with indicated genotypes at 2 mmol/L or 20 mmol/L glucose ( n = 3). * P
Article Snippet: The samples were then analyzed using a liquid chromatography-tandem mass spectrometry with an Agilent 1200 series high-performance liquid chromatography system and a 4000 Q Trap tandem mass spectrometer (Applied Biosystems).
Techniques: Expressing, Stable Transfection, Transfection, Staining, Mouse Assay, Mutagenesis, Immunoprecipitation, Western Blot, Liquid Chromatography, Mass Spectrometry, Activity Assay, Luciferase, Real-time Polymerase Chain Reaction, Isolation, Cell Culture, Concentration Assay