Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 1. Effects of 3-O-acetyloleanolic acid isolated from Forsythiae Fructus on the secretion of FGF21 in C2C12 myotubes. (A) Outline of the extraction and isolation of 3-O-acetyloleanolic acid from Forsythiae Fructus. (B) Structure of 3-O-acetyloleanolic acid. (C) The secretion of FGF21 was increased by 3-O-acetyloleanolic acid isolated from Forsythiae Fructus in C2C12 myotubes. Each value is the mean ± SEM of four separate experiments. The double asterisk indicates P < 0.01.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Isolation, Extraction

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 2. 3-O-Acetyloleanolic acid up- regulates FGF21 expression in C2C12 myotubes. (A) The secretion of FGF21 was increased by 3-O-acetyloleanolic acid in a concentration-dependent manner in C2C12 myotubes. Cells were treated with the indicated concentration of 3-O-acetyloleanolic acid for 20 h, then the media were collected and pro cessed for ELISA. Each value represents the mean ± SEM of five separate ex periments. **P < 0.01, comparison with vehicle control. (B) FGF21 mRNA expression was up-regulated by 3-O- acetyloleanolic acid in C2C12 myo tubes. Cells were treated with 30 µM 3- O-acetyloleanolic acid for 2 and 5 h, harvested, and then processed for real- time qRT-PCR. Each value represents the mean ± SEM of five separate ex periments. *P < 0.01; **P < 0.01, com parison with vehicle control.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison, Control, Quantitative RT-PCR

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 3. 3-O-Acetyloleanolic acid stimulates FGF21 expression in mice. (A) Effect of the intraperitoneal administration of 3-O-acetyloleanolic acid at 30 mg/kg on FGF21 mRNA levels in soleus muscle. Total RNA was isolated from the soleus muscle of saline-treated and 3-O-acetyloleanolic acid-treated mice. FGF21 mRNA was determined by qRT-PCR using specific primers. Relative mRNA levels were normalized to 18S. Each value is the mean ± SEM of five mice. The single asterisk indicates P < 0.05. (B) 3-O-acetyloleanolic acid at 30 mg/kg increased circulating FGF21. The plasma FGF21 concentration was determined by FGF21-specific ELISA after the intraperitoneal administration of 3-O-acetyloleanolic acid at 30 mg/kg. Each value represents the mean ± SEM of five mice. The double asterisk in dicates P < 0.01.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Expressing, Isolation, Saline, Quantitative RT-PCR, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 5. Role of p38 MAPK in the up-regulation of FGF21 secretion caused by 3-O-acetyloleanolic acid in C2C12 cells. (A) Cells were treated with 30 µM 3-O-ace tyloleanolic acid for 10 min, harvested, and then processed for western blotting analysis of the phosphorylated and total p38 levels in whole cell lysate. Western blots were quantified using ImageJ software (National Institutes of Health). Each value represents the mean ± SEM of four to five separate experiments. **P < 0.01, comparison with vehicle control. (B) Relative luciferase activity of the FGF21 promoter construct (mFgf21pro1-luc) in C2C12 cells following treatment with 3-O- acetyloleanolic acid and co-transfection of the TGR5-expression plasmid. The total amount of DNA transfected was standardized with an empty vector. After transfection, the cells were preincubated with the indicated concentrations of SB203580 for 20 min and then stimulated with 30 μM 3-O-acetyloleanolic acid for 8 h. Luciferase activity was normalized by β-gal and expressed relative to that in vehicle-treated cells. Each value is the mean ± SEM of five independent experiments. **P < 0.01, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, comparison of SB203580 concentrations. (C) Cells were incubated for 20 h with 3-O- acetyloleanolic acid and SB203580, and FGF21 production was determined by ELISA. Each value represents the mean ± SEM of five separate experiments. **P < 0.01, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, comparison of SB203580 concentrations. (D) Cells were incubated for 5 h in 3-O- acetyloleanolic acid and SB203580, and Fgf21 mRNA levels were determined by qRT-PCR. Fgf21 mRNA levels were normalized relative to 18S mRNA levels. Each value represents the mean ± SEM of five experiments. **P < 0.01, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, comparison of SB203580 concentrations.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Western Blot, Software, Comparison, Control, Luciferase, Activity Assay, Construct, Cotransfection, Expressing, Plasmid Preparation, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 6. 3-O-acetyloleanolic acid up-regulates FGF21 expression concomitant with the up-regulation of Nr4a1 in C2C12 myotubes. (A) Nr4a1 mRNA expression was up-regulated by 3-O-acetyloleanolic acid in C2C12 myotubes. Cells were treated with 30 µM 3-O-acetyloleanolic acid for 1, 2 and 5 h, harvested, and then processed for real-time qRT-PCR. Each value represents the mean ± SEM of four separate experiments. *P < 0.05, comparison with vehicle control. (B) Cells were incubated for 5 h with 3-O-acetyloleanolic acid and SB203580, and Nr4a1 mRNA levels were determined by qRT-PCR. Nr4a1 mRNA levels were normalized relative to 18S mRNA levels. Each value represents the mean ± SEM of four separate experiments. *P < 0.05, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, com parison of SB203580 concentrations. (C) Cells were transfected with a wild-type FGF21 promoter construct (mFgf21pro1-luc) or NBRE-mutated version (NBRE-mut- mFgf21pro1-luc), and relative luciferase activity was measured. Data are means ± SEM of four separate experiments. **P < 0.01, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, comparison of constructs.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Expressing, Quantitative RT-PCR, Comparison, Control, Incubation, Transfection, Construct, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: Paradoxical Regulation of Human FGF21 by Both Fasting and Feeding Signals: Is FGF21 a Nutritional Adaptation Factor?

doi: 10.1371/journal.pone.0022976

Figure Lengend Snippet: A: Changes in FGF21 gene expression stimulated by the indicated dose of glucose for 6 h in HepG2. B: Changes in FGF21 protein abundance in HepG2 cells stimulated by indicated dose of glucose for 24 h. C: Changes in FGF21 , L-pk , and Fasn gene expression stimulated by 10 mM glucose (Glu), xylitol (Xyl) or mannitol (Man) for 6 h in mouse primary hepatocytes. The relative mRNA amount for each gene treated with 5 mM of glucose (A), or NT (non-treatment) (C) was set as 1.0. D: Changes in FGF21 concentration in the cultured media of mouse primary hepatocytes stimulated by 3 mM glucose (G3) or 25 mM glucose (G25) for indicated time. Data represent mean ± SE (n = 3). †: Concentration-dependent effects were observed by regression analysis, p<0.05. *: p<0.05 as compared to NT, mannitol and G3 stimulation.

Article Snippet: Luciferase gene constructs containing −1672 to +230 bp of the human FGF21 promoter (pFGF21-1.6k) reporter vector and a series of 5′-deletion mutant vectors (pFGF21-1k, pFGF21-555, pFGF21-443, pFGF21-289 and pFGF21+11) were prepared from human genomic DNA and pGL4.12 vectors (Promega).

Techniques: Expressing, Concentration Assay, Cell Culture

Journal: PLoS ONE

Article Title: Paradoxical Regulation of Human FGF21 by Both Fasting and Feeding Signals: Is FGF21 a Nutritional Adaptation Factor?

doi: 10.1371/journal.pone.0022976

Figure Lengend Snippet: A: Changes in relative luciferase activity of a pFGF21-1.6k reporter vector containing the −1672 to +230 bp region of the human FGF21 promoter simulated by indicated dose of wy-14643 for 6 h in mouse primary hepatocytes. B: Schematic representation of 5′-deletion mutants of the human FGF21 promoter and changes in relative luciferase activity after stimulation with or without 10 µM wy-14643 for 6 h. C: Changes in relative luciferase activity of the pFGF21-1.6k reporter vector stimulated by 10 −7 M glucagon or 10 −6 M forskolin (FSK) for 6 h in mouse primary hepatocytes. D: Schematic representation of 5′-deletion mutants of the human FGF21 promoter and changes in relative luciferase activity after stimulation with or without 10 −7 M glucagon. The relative luciferase activity treated with DMSO (A), or NT (non-treatment) (C) was set as 1.0. For deletion experiments (B and D), the relative luciferase activity of each deletion constructs treated with DMSO (B) or control (D) was set as 1.0. Data represent mean ± SE (n = 3). †: Concentration-dependent effects were observed by regression analysis, p<0.05. *: p<0.05 as compared to DMSO or NT.

Article Snippet: Luciferase gene constructs containing −1672 to +230 bp of the human FGF21 promoter (pFGF21-1.6k) reporter vector and a series of 5′-deletion mutant vectors (pFGF21-1k, pFGF21-555, pFGF21-443, pFGF21-289 and pFGF21+11) were prepared from human genomic DNA and pGL4.12 vectors (Promega).

Techniques: Luciferase, Activity Assay, Plasmid Preparation, Construct, Concentration Assay

Journal: PLoS ONE

Article Title: Paradoxical Regulation of Human FGF21 by Both Fasting and Feeding Signals: Is FGF21 a Nutritional Adaptation Factor?

doi: 10.1371/journal.pone.0022976

Figure Lengend Snippet: A: Changes in relative luciferase activity of the pFGF21-1.6k reporter vector stimulated by indicated dose of glucose for 24 h in HepG2 cells. B: Basal luciferase activity of the pFGF21-1.6k reporter vector in mouse primary hepatocytes (Mpl) and HepG2 cells. C: Changes in relative luciferase activity of the pFGF21-1.6k reporter vector stimulated by 10 mM glucose (Glu), xylitol (Xyl) or mannitol (Man) for 6 h in mouse primary hepatocytes. D: Changes in relative luciferase activity of the pFGF21-1.6k reporter vector stimulated by the indicated dose of xylitol for 6 h in mouse primary hepatocytes. E, F: Schematic representation of 5′-deletion mutants of the human FGF21 promoter and changes in relative luciferase activity after stimulation with or without 20 mM xylitol (E) or 20 mM glucose (F). G, H: Basal luciferase activities of 5′-deletion mutants of the human FGF21 promoter in mouse primary hepatocytes (G) and HepG2 cells (H). The relative luciferase activity for C through F was expressed as described in the . For basal luciferase activity analysis (G and H), the relative luciferase activity of -1672 bp was set as 1.0. Data represent mean ± SE (n = 3). †: Concentration-dependent effects were observed by regression analysis, p<0.05. *: p<0.05 as compared to the control.

Article Snippet: Luciferase gene constructs containing −1672 to +230 bp of the human FGF21 promoter (pFGF21-1.6k) reporter vector and a series of 5′-deletion mutant vectors (pFGF21-1k, pFGF21-555, pFGF21-443, pFGF21-289 and pFGF21+11) were prepared from human genomic DNA and pGL4.12 vectors (Promega).

Techniques: Luciferase, Activity Assay, Plasmid Preparation, Concentration Assay

Journal: PLoS ONE

Article Title: Paradoxical Regulation of Human FGF21 by Both Fasting and Feeding Signals: Is FGF21 a Nutritional Adaptation Factor?

doi: 10.1371/journal.pone.0022976

Figure Lengend Snippet: A, B: Changes in ChREBP protein abundance in cytosolic and nuclear fractions from mouse primary hepatocytes stimulated by 10 mM xylitol for 6 h. C: Putative carbohydrate responsive element (ChoRE) in the human FGF21 promoter. D: An electrophoretic mobility shift assay was performed with the mouse primary hepatocyte nuclear fraction stimulated by xylitol (X) or not (NT). We used a putative ChoRE (−380 to −366 bp; lanes 1–5). Factor C is a competitor ( L-pk ; −142 to −124 bp) and Ab is ChREBP antibody. Arrow indicates DNA and protein complex. Data represent mean ± SE (n = 3). *: p<0.05 as compared to NT (non-treatment).

Article Snippet: Luciferase gene constructs containing −1672 to +230 bp of the human FGF21 promoter (pFGF21-1.6k) reporter vector and a series of 5′-deletion mutant vectors (pFGF21-1k, pFGF21-555, pFGF21-443, pFGF21-289 and pFGF21+11) were prepared from human genomic DNA and pGL4.12 vectors (Promega).

Techniques: Electrophoretic Mobility Shift Assay

Journal: PLoS ONE

Article Title: Paradoxical Regulation of Human FGF21 by Both Fasting and Feeding Signals: Is FGF21 a Nutritional Adaptation Factor?

doi: 10.1371/journal.pone.0022976

Figure Lengend Snippet: A, B: Changes in relative ChREBP gene expression and protein abundance at 48 h after transfection of either shLacZ or shChREBP vector in mouse primary hepatocytes. C: Changes in relative luciferase activity of the human −1.6 kb FGF21 reporter vector stimulated by 20 mM glucose 48 h after transfection of either shLacZ or shChREBP vector in mouse primary hepatocytes. The relative mRNA expression and protein abundance of ChREBP transfected with shLacZ vector was set as 1.0. The relative luciferase activity of NT (non-treatment) was 1.0. Data represent mean ± SE (n = 3). * p<0.05 as compared to shLacZ or NT.

Article Snippet: Luciferase gene constructs containing −1672 to +230 bp of the human FGF21 promoter (pFGF21-1.6k) reporter vector and a series of 5′-deletion mutant vectors (pFGF21-1k, pFGF21-555, pFGF21-443, pFGF21-289 and pFGF21+11) were prepared from human genomic DNA and pGL4.12 vectors (Promega).

Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: Paradoxical Regulation of Human FGF21 by Both Fasting and Feeding Signals: Is FGF21 a Nutritional Adaptation Factor?

doi: 10.1371/journal.pone.0022976

Figure Lengend Snippet: A: Both fasting (PPARα and glucagon) and over feeding (glucose) signals activate human FGF21 gene transcription. B: Human FGF21 gene expression responds to nutritional crisis including fasting, starvation, over feeding and obesity.

Article Snippet: Luciferase gene constructs containing −1672 to +230 bp of the human FGF21 promoter (pFGF21-1.6k) reporter vector and a series of 5′-deletion mutant vectors (pFGF21-1k, pFGF21-555, pFGF21-443, pFGF21-289 and pFGF21+11) were prepared from human genomic DNA and pGL4.12 vectors (Promega).

Techniques: Expressing