BACKGROUND & AIMS: Vesicular glutamate transporter (VGLUT) has been reported to be involved in glucose-induced insulin secretion. It has been shown that glucose stimulates the expression of VGLUT isoform 2 (VGLUT2) in beta cells via transcriptional mechanism. In this study, we identified the mouse VGLUT2 (mVGLUT2) promoter and characterized the transcriptional mechanism of glucose-stimulated mVGLUT2 expression in beta-cells. METHODS: A promoter region of mVGLUT2 was cloned by genomic polymerase chain reaction. The mechanism of Sp1 in glucose-induced transactivation of mVGLUT2 was investigated by luciferase assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, and Western blot analysis. RESULTS: A promoter containing 2133 base pairs of upstream sequence of the 5'-flanking region of mVGLUT2 complementary DNA was cloned. Transient transfection of various 5'-end deletion constructs of the mVGLUT2 promoter/luciferase reporter indicated that the region between -96 to +68 base pair contains the basal promoter for mVGLUT2. Mutational analysis and electromobility shift assay showed an important role for the transcription factor Sp1 in both basal and glucose-induced mVGLUT2 transcription. The interaction between Sp1 and mVGLUT2 was confirmed by chromatin immunoprecipitation assays. Glucose stimulates the phosphorylation of Sp1 via mitogen-activated protein kinase P38 and P44/42. This leads to increased binding activity of Sp1 to the mVGLUT2 promoter and results in activation of the gene. CONCLUSIONS: We cloned the mouse VGLUT2 promoter and showed a novel molecular mechanism of glucose-induced mVGLUT2 transcription.
BACKGROUND & AIMS: Vesicular glutamate transporter (VGLUT) has been reported to be involved in glucose-induced insulin secretion. It has been shown that glucose stimulates the expression of VGLUT isoform 2 (VGLUT2) in beta cells via transcriptional mechanism. In this study, we identified the mouseVGLUT2 (mVGLUT2) promoter and characterized the transcriptional mechanism of glucose-stimulated mVGLUT2 expression in beta-cells. METHODS: A promoter region of mVGLUT2 was cloned by genomic polymerase chain reaction. The mechanism of Sp1 in glucose-induced transactivation of mVGLUT2 was investigated by luciferase assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, and Western blot analysis. RESULTS: A promoter containing 2133 base pairs of upstream sequence of the 5'-flanking region of mVGLUT2 complementary DNA was cloned. Transient transfection of various 5'-end deletion constructs of the mVGLUT2 promoter/luciferase reporter indicated that the region between -96 to +68 base pair contains the basal promoter for mVGLUT2. Mutational analysis and electromobility shift assay showed an important role for the transcription factor Sp1 in both basal and glucose-induced mVGLUT2 transcription. The interaction between Sp1 and mVGLUT2 was confirmed by chromatin immunoprecipitation assays. Glucose stimulates the phosphorylation of Sp1 via mitogen-activated protein kinase P38 and P44/42. This leads to increased binding activity of Sp1 to the mVGLUT2 promoter and results in activation of the gene. CONCLUSIONS: We cloned the mouseVGLUT2 promoter and showed a novel molecular mechanism of glucose-induced mVGLUT2 transcription.
Authors: M Hayashi; M Otsuka; R Morimoto; S Hirota; S Yatsushiro; J Takeda; A Yamamoto; Y Moriyama Journal: J Biol Chem Date: 2001-09-10 Impact factor: 5.157
Authors: Mark P Keller; Mary E Rabaglia; Kathryn L Schueler; Donnie S Stapleton; Daniel M Gatti; Matthew Vincent; Kelly A Mitok; Ziyue Wang; Takanao Ishimura; Shane P Simonett; Christopher H Emfinger; Rahul Das; Tim Beck; Christina Kendziorski; Karl W Broman; Brian S Yandell; Gary A Churchill; Alan D Attie Journal: J Clin Invest Date: 2019-07-25 Impact factor: 14.808
Authors: Carlos V Melo; Miranda Mele; Michele Curcio; Diogo Comprido; Carla G Silva; Carlos B Duarte Journal: PLoS One Date: 2013-01-11 Impact factor: 3.240