Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Moxibustion-Simulating Bipolar Radiofrequency Suppresses Weight Gain and Induces Adipose Tissue Browning via Activation of UCP1 and FGF21 in a Mouse Model of Diet-Induced Obesity

doi: 10.1155/2018/4737515

Figure Lengend Snippet: M-RF stimulation increases the level of FGF21 in serum and WAT of DIO mice. (a) ELISA for serum FGF21 in control ( n = 5), RF-L ( n = 5), RF-M ( n = 5), and RF-H ( n = 5). (b) Representative Western blotting for FGF21 in WATs of DIO mice in control ( n = 5), RF-L ( n = 5), RF-M ( n = 5), and RF-H ( n = 5). (c) Quantification of (b) and calculated in terms of ratio relative to control. Note that all three levels of M-RF stimulation significantly increased the level of FGF21 in serum and WAT compared to the control. Results are presented as the mean ± SD. ∗ , P < 0.05 versus control. ∗∗ , P < 0.01 versus control.

Article Snippet: To quantify FGF21 in mouse serum, an enzyme-linked immunosorbent assay (ELISA) was performed using the mouse FGF21 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot

Journal: Molecular medicine reports

Article Title: Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration.

doi: 10.3892/mmr.2016.4942

Figure Lengend Snippet: Figure 2. Expression patterns of FGF21 and β‑klotho in skin following wounding. (A) Reverse‑transcription polymerase chain reaction analysis was used to analyze the expression of FGF21 and β‑klotho prior to and 3 h after wounding. Expression levels of (B) FGF21 and (C) β‑klotho were normalized against those of GAPDH. The experiments were repeated at least three times and values are expressed as the mean ± standard error of three replicates. ***P<0.001 vs. Con. Con, control.

Article Snippet: Subsequently, cells were cultured for 72 h with addition of 0.8% methanol every 24 h. Proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime Institute of Biotechnology, Shanghai, China) and western blot analysis was performed using mouse anti-FGF21 monoclonal antibody (1:1,000; MAB25371; R&D Systems, Inc., Minneapolis, MN, USA) (18).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Molecular medicine reports

Article Title: Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration.

doi: 10.3892/mmr.2016.4942

Figure Lengend Snippet: Figure 1. FGF21 expression patterns in a variety of mouse tissues. (A) Expression patterns of FGF21 were analyzed by reverse‑transcription polymerase chain reaction analysis. (B) FGF21 levels in A were normal ized against those of GAPDH. The experiments were repeated at least three times and values are expressed as the mean ± standard deviation of three replicates. **P<0.01; ***P<0.001 vs. heart FGF21 levels.

Article Snippet: Subsequently, cells were cultured for 72 h with addition of 0.8% methanol every 24 h. Proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime Institute of Biotechnology, Shanghai, China) and western blot analysis was performed using mouse anti-FGF21 monoclonal antibody (1:1,000; MAB25371; R&D Systems, Inc., Minneapolis, MN, USA) (18).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal: Molecular medicine reports

Article Title: Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration.

doi: 10.3892/mmr.2016.4942

Figure Lengend Snippet: Figure 4. Polymerase chain reaction analysis confirmed that the FGF21 frag ment was successfully integrated into SMD1168 colonies. Lanes 1‑10 are colonies selected from a minimal dextrose plate. Yeast cells transfected with empty vector was used as a negative control. M, marker.

Article Snippet: Subsequently, cells were cultured for 72 h with addition of 0.8% methanol every 24 h. Proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime Institute of Biotechnology, Shanghai, China) and western blot analysis was performed using mouse anti-FGF21 monoclonal antibody (1:1,000; MAB25371; R&D Systems, Inc., Minneapolis, MN, USA) (18).

Techniques: Polymerase Chain Reaction, Transfection, Plasmid Preparation, Negative Control, Marker

Journal: Molecular medicine reports

Article Title: Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration.

doi: 10.3892/mmr.2016.4942

Figure Lengend Snippet: Figure 3. Gene synthesis of FGF21. PCR products of the gene FGF21. Lanes: M, 2000 DNA marker; 1, PCR reaction with primer pair P1/RP1; 2, PCR reaction with primer pair P2/RP2 using the PCR product from the previous cycle as a template; 3‑7, PCR reactions with primer pairs P3/RP3‑P7/RP7, respectively, using the PCR product from the previous round as a template. PCR, polymerase chain reaction.

Article Snippet: Subsequently, cells were cultured for 72 h with addition of 0.8% methanol every 24 h. Proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime Institute of Biotechnology, Shanghai, China) and western blot analysis was performed using mouse anti-FGF21 monoclonal antibody (1:1,000; MAB25371; R&D Systems, Inc., Minneapolis, MN, USA) (18).

Techniques: Marker, Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration.

doi: 10.3892/mmr.2016.4942

Figure Lengend Snippet: Figure 6. Effects of exogenous FGF21 treatment on the activation of cell migration and JNK phosphorylation levels. (A) Wound healing assay was performed to analyze the effects of FGF21 (100 ng/ml) in human fibroblasts under low‑glucose (5.5 mM) conditions. Scale bar, 0.05 µm. Magnification, 40x. (B) The cell migra tion distance was quantified from A. (C and D) Following 6 h culture, 100 ng/ml FGF21 was added to the culture medium and gently shaken. Phosphorylation levels of JNK were analyzed after 30 min of FGF21 stimulation. All experiments were performed after incubation with 5 µg/ml mitomycin‑C, an inhibitor of cell proliferation, for one day. Images were captured using an ImageQuant LAS 4000. Values are expressed as the mean ± standard error (n=10 for B and n=3 for D). *P<0.05 vs. control. p‑JNK, phosphorylated c‑Jun N‑terminal kinase; t‑JNK, total c‑Jun N‑terminal kinase; FGF, fibroblast growth factor; Con, control.

Article Snippet: Subsequently, cells were cultured for 72 h with addition of 0.8% methanol every 24 h. Proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime Institute of Biotechnology, Shanghai, China) and western blot analysis was performed using mouse anti-FGF21 monoclonal antibody (1:1,000; MAB25371; R&D Systems, Inc., Minneapolis, MN, USA) (18).

Techniques: Activation Assay, Migration, Phospho-proteomics, Wound Healing Assay, Incubation, Control

Journal: Molecular medicine reports

Article Title: Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration.

doi: 10.3892/mmr.2016.4942

Figure Lengend Snippet: Figure 5. Protein expression and purification of FGF21. (A) SDS‑PAGE analysis of supernatant of engineered P. pastoris cells and purified FGF21 protein. Lanes: 1, marker; 2, supernatant without transfection with the FGF21 overexpression vector; 3, supernatant after transfection; 4, purified protein. (B) Purified FGF21 protein was analyzed by western blotting using an Epson Perfection V700 photo scanner. Lanes: 1, protein in the supernatant after transfection; 2, purified protein. (C) The purity of recombinant FGF21 was analyzed by high‑performance liquid chromatography. FGF, fibroblast growth factor.

Article Snippet: Subsequently, cells were cultured for 72 h with addition of 0.8% methanol every 24 h. Proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime Institute of Biotechnology, Shanghai, China) and western blot analysis was performed using mouse anti-FGF21 monoclonal antibody (1:1,000; MAB25371; R&D Systems, Inc., Minneapolis, MN, USA) (18).

Techniques: Expressing, Purification, Marker, Transfection, Over Expression, Plasmid Preparation, Western Blot, Recombinant, High Performance Liquid Chromatography

Journal: Cell Metabolism

Article Title: GDF15 Provides an Endocrine Signal of Nutritional Stress in Mice and Humans

doi: 10.1016/j.cmet.2018.12.016

Figure Lengend Snippet: GDF15 Expression Is Regulated by the Cellular ISR Pathway (A and C) GDF15 mRNA expression (A) and immunoblot analysis (C) of ISR components in wild-type (WT) mouse embryonic fibroblasts (MEFs) treated with vehicle control (Con), cobalt chloride (CoCl2, 625 μM), thapsigargin (Tg, 1 μM), tunicamycin (Tn 5 μg/mL), or L-Histidinol (His, 1 mM) for 6 h. (B) GDF15 mRNA expression in human cell lines (HeLA, HuH7, and A549) treated with Tn (5 μg/mL) for 6 h. (D) GDF15 mRNA expression in WT MEFs pre-treated for 1 h either with the PERK inhibitor GSK2606414 (GSK, 200 nM) or eIF2α inhibitor ISRIB (ISR, 100 nM), then co-treated with Tn (5 μg/mL) for a further 6 h. (E–G) GDF15 mRNA expression (E) in EIF2α Ser51 (SS) or phospho mutant (AA) MEFs or (F) in ATF4 wild-type (WT) or ATF4 knockout (KO) MEFs and (G) in control siRNA and CHOP siRNA transfected WT MEFs treated with Tn (5 μg/mL) for 6 h. (H) Diagram outlining pathway by which GDF15 and FGF21 expression is regulated by TN. mRNA expression is presented as fold expression relative to its respective control treatment for each cell type (set at 1) or TN-treated samples (set as 100) with normalization to HPRT gene expression in MEFs and GAPDH in human cells. Data are expressed as mean ± SD from at least three independent experiments. ∗∗∗ p < 0.001 versus control (con) for (A) and (B), and versus TN stimulated for (D)–(G) by two-tailed Student’s t test. Blots shown are representative of three independent experiments with Calnexin used as a loading control. See also Figures S3 and .

Article Snippet: Mouse FGF21 was analyzed by FGF21 Quantakine ELISA (R&D Systems) following the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Control, Mutagenesis, Knock-Out, Transfection, Gene Expression, Two Tailed Test

Journal: Cell Metabolism

Article Title: GDF15 Provides an Endocrine Signal of Nutritional Stress in Mice and Humans

doi: 10.1016/j.cmet.2018.12.016

Figure Lengend Snippet:

Article Snippet: Mouse FGF21 was analyzed by FGF21 Quantakine ELISA (R&D Systems) following the manufacturer’s instructions.

Techniques: Recombinant, SYBR Green Assay, Protease Inhibitor, Reverse Transcription, Western Blot, Injection, Enzyme-linked Immunosorbent Assay, Control, TaqMan Assay, Software, Sterility, Electrophoresis, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor 21 (FGF21) Protects against High Fat Diet Induced Inflammation and Islet Hyperplasia in Pancreas

doi: 10.1371/journal.pone.0148252

Figure Lengend Snippet: FGF21 is regulated by the nutritional status in fed, fasted and diet-induced obesity (DIO). Fasting reduced FGF21 mRNA in whole pancreas (A). β-klotho, the co-receptor of FGF21, is expressed in the pancreas and is inversely regulated by fasting (B). FGF21 protein was only detected in pancreas compared to liver and inguinal white adipose tissue (IWAT) (C). Pancreas FGF21 protein level was reduced by fasting in both chow fed and high fat diet fed conditions (D, E). Separation of pancreatic fractions to evaluate the individual contribution of islets and acinar tissue on FGF21 expression show that acinar pancreas contributes to the majority of FGF21 levels while islet cells have very low FGF21 levels and are not nutritionally regulated (F). Acinar expression of FGF21 is regulated with fasting (F, G). n = 6 per group. Each western blot lane represents an individual animal.

Article Snippet: Antibodies were purchased from commercial vendors: FGF21 (AF3057; R&D systems, Minneapolis, MN); Insulin (ab7842; Abcam, Cambridge, MA); β-actin (ab8226; Abcam); α -tubulin (ab7291; Abcam); pERK1/2 (4370; Cell Signaling Technology, Denver, MA); ERK1/2 (9107; Cell Signaling Technology); glucagon (2654; Sigma Aldrich, St. Louis, MO); somatostatin (sc7819; Santa Cruz, Dallas, TX); CD3 (MA1-90582; Thermo Scientific, Waltham, MA).

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor 21 (FGF21) Protects against High Fat Diet Induced Inflammation and Islet Hyperplasia in Pancreas

doi: 10.1371/journal.pone.0148252

Figure Lengend Snippet: Acute FGF21 exposure induces ERK1/2 signaling events therefore phosphorylation of ERK1/2 (pERK1/2) has been chosen as a marker of FGF21 signaling. Up to 50% of acinar cells show strong nuclear pERK1/2 staining (brown) after FGF21 administration (A i, iii). Islets represent a very unique pattern with pERK1/2 only on the peripheral cells (A ii, iv). Percentage of pERK1/2-labeled acinar cells with saline and FGF21 administration (B). Western blot of pERK1/2 and total ERK1/2 from the same pancreas samples (C). Epididymal white adipose tissue (EWAT) is a positive control for FGF21 signaling (C). S = saline and F = FGF21. Confocal photomicrographs show FGF21-induced pERK1/2 immunoreactivity in islets (D). Absence of pERK1/2 staining (green) after saline treatment (Di; Inset , islet outline is indicated by insulin-immunoreactivity shown in red). FGF21 did not elicit pERK1/2 in insulin (red)-producing β-cells (D ii). Merged images show pERK1/2 co-localization in glucagon-positive α cells (D iii) and somatostatin-positive δ cell (D iv). Split panels show glucagon (iii a ) and pERK (iii b ) labeling or somatostatin (iv a ) and pERK (iv b ) labeling in single cells. A robust and specific pERK1/2 staining was observed in almost 10% of total islet nuclei mostly at the periphery. Outlined region (dashed line) in the inset indicates the region represented in each panel. Representative pERK1/2 labelled cells are indicated by white arrows. n = 4 per group. Scale: 200 μm, A; 20 μm, D.

Article Snippet: Antibodies were purchased from commercial vendors: FGF21 (AF3057; R&D systems, Minneapolis, MN); Insulin (ab7842; Abcam, Cambridge, MA); β-actin (ab8226; Abcam); α -tubulin (ab7291; Abcam); pERK1/2 (4370; Cell Signaling Technology, Denver, MA); ERK1/2 (9107; Cell Signaling Technology); glucagon (2654; Sigma Aldrich, St. Louis, MO); somatostatin (sc7819; Santa Cruz, Dallas, TX); CD3 (MA1-90582; Thermo Scientific, Waltham, MA).

Techniques: Phospho-proteomics, Marker, Staining, Labeling, Saline, Western Blot, Positive Control

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor 21 (FGF21) Protects against High Fat Diet Induced Inflammation and Islet Hyperplasia in Pancreas

doi: 10.1371/journal.pone.0148252

Figure Lengend Snippet: Chronic FGF21 treatment for 3 days by mini osmotic pumps led to the reduction in FGF21 expression in pancreas (A). FGF21 infusion down-regulated insulin but glucagon, somatostatin or amylase expression. While FGF21 infusion did not alter serum glucose levels (B), it reduced the serum levels of pancreatic hormones, insulin (C) and glucagon (D) without affecting the amylase activity (E). n = 10 per group.

Article Snippet: Antibodies were purchased from commercial vendors: FGF21 (AF3057; R&D systems, Minneapolis, MN); Insulin (ab7842; Abcam, Cambridge, MA); β-actin (ab8226; Abcam); α -tubulin (ab7291; Abcam); pERK1/2 (4370; Cell Signaling Technology, Denver, MA); ERK1/2 (9107; Cell Signaling Technology); glucagon (2654; Sigma Aldrich, St. Louis, MO); somatostatin (sc7819; Santa Cruz, Dallas, TX); CD3 (MA1-90582; Thermo Scientific, Waltham, MA).

Techniques: Expressing, Activity Assay

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor 21 (FGF21) Protects against High Fat Diet Induced Inflammation and Islet Hyperplasia in Pancreas

doi: 10.1371/journal.pone.0148252

Figure Lengend Snippet: WT and FGF21 KO mice on chow diet have similar body weight (A), glucose tolerance (B) and insulin secretion in response to glucose (C), despite notable differences in their pancreatic morphology (D i, iii). After consuming high fat diet for 16 weeks, FGF21 KO mice (D iv) had increased islet hyperplasia compared with WT mice (D ii). Islet cell surface area was measured and represented (E). Islet cell proliferation markers were substantially over expressed in obese FGF21 KO animals (F). Islet hyperplasia leads to increased serum insulin (G) in FGF21 KO animals with a slight reduction in blood glucose (H) (n = 7 per group). Scale: 1 mm.

Article Snippet: Antibodies were purchased from commercial vendors: FGF21 (AF3057; R&D systems, Minneapolis, MN); Insulin (ab7842; Abcam, Cambridge, MA); β-actin (ab8226; Abcam); α -tubulin (ab7291; Abcam); pERK1/2 (4370; Cell Signaling Technology, Denver, MA); ERK1/2 (9107; Cell Signaling Technology); glucagon (2654; Sigma Aldrich, St. Louis, MO); somatostatin (sc7819; Santa Cruz, Dallas, TX); CD3 (MA1-90582; Thermo Scientific, Waltham, MA).

Techniques:

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor 21 (FGF21) Protects against High Fat Diet Induced Inflammation and Islet Hyperplasia in Pancreas

doi: 10.1371/journal.pone.0148252

Figure Lengend Snippet: HFD consumption caused severe pancreatic inflammation in FGF21 KO mice (A). H&E staining showing, that compared to WT animals (i), FGF21 KO mice developed severe pancreatic periductal inflammation (outlined area in ii-iii) when consuming a HFD diet for 16 weeks. Lymphocytic nature of inflammatory cells is shown in a higher magnification image (iii). (B) Immunohistochemical analysis for the lymphocytic marker CD3 on the pancreas of WT (i) and FGF21 KO animals (ii-iii) is represented. FGF21 KO showed higher number of CD3, T cell receptor antigen. n = 7 per group. Scale: 1 mm, Ai-ii; 2 mm, Bi-ii; 200 μm, iii.

Article Snippet: Antibodies were purchased from commercial vendors: FGF21 (AF3057; R&D systems, Minneapolis, MN); Insulin (ab7842; Abcam, Cambridge, MA); β-actin (ab8226; Abcam); α -tubulin (ab7291; Abcam); pERK1/2 (4370; Cell Signaling Technology, Denver, MA); ERK1/2 (9107; Cell Signaling Technology); glucagon (2654; Sigma Aldrich, St. Louis, MO); somatostatin (sc7819; Santa Cruz, Dallas, TX); CD3 (MA1-90582; Thermo Scientific, Waltham, MA).

Techniques: Staining, Immunohistochemical staining, Marker

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor 21 (FGF21) Protects against High Fat Diet Induced Inflammation and Islet Hyperplasia in Pancreas

doi: 10.1371/journal.pone.0148252

Figure Lengend Snippet: (A) Representative cytofluorometric dot plots of isolated lymphocytes from WT and FGF21 KO animals consuming high fat diet for 16 weeks. FGF21 KO mice show elevated CD45+ (i), TCRb+ and Thy1+ T lymphocytes (ii) and Foxp3+ Treg cells. CD19+ B lymphocytes were not significantly altered (iii). Corresponding summary data is shown in the right panel. The experiment was repeated twice (n = 4 per group). (B) Gene expression analysis of cytokines is presented. n = 7 per group.

Article Snippet: Antibodies were purchased from commercial vendors: FGF21 (AF3057; R&D systems, Minneapolis, MN); Insulin (ab7842; Abcam, Cambridge, MA); β-actin (ab8226; Abcam); α -tubulin (ab7291; Abcam); pERK1/2 (4370; Cell Signaling Technology, Denver, MA); ERK1/2 (9107; Cell Signaling Technology); glucagon (2654; Sigma Aldrich, St. Louis, MO); somatostatin (sc7819; Santa Cruz, Dallas, TX); CD3 (MA1-90582; Thermo Scientific, Waltham, MA).

Techniques: Isolation, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Pyruvate Upregulates Hepatic FGF21 Expression by Activating PDE and Inhibiting cAMP–Epac–CREB Signaling Pathway

doi: 10.3390/ijms23105490

Figure Lengend Snippet: Pyruvate upregulated FGF21 expression and secretion in HepG2 cells. ( A ): pyruvate dose-dependently stimulated FGF21 gene expression after 12 h treatment in HepG2 cells (** p < 0.01 vs. control, n = 3). ( B ): FGF21 protein levels in cell medium significantly increased after pyruvate treatment (** p < 0.01 vs. control, n = 10). ( C ): The cell viability was not influenced by pyruvate treatment, as shown by MTT assay ( n = 10). ( D ): D-LDH levels in cell medium were not changed by pyruvate treatment ( n = 10).

Article Snippet: Human and mouse FGF21 ELISA kits, cAMP assay kits, the ALT activity assay kit, the AST activity assay kit and the LDH assay kit were obtained from Elabscience (Wuhan, China).

Techniques: Expressing, Gene Expression, Control, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Pyruvate Upregulates Hepatic FGF21 Expression by Activating PDE and Inhibiting cAMP–Epac–CREB Signaling Pathway

doi: 10.3390/ijms23105490

Figure Lengend Snippet: cAMP reduction caused pyruvate-stimulated FGF21 expression in HepG2 cells. ( A ): The activation of PPAR-α and AMPK was not involved in pyruvate-stimulated FGF21 expression (** p < 0.01 vs. control, n = 3). ( B ): AC activator forskolin, PDE inhibitor IBMX and 8-Bromo-cAMP administration significantly inhibited FGF21 expression and suppressed pyruvate-stimulated FGF21 expression (* p < 0.05 vs. control, # p < 0.05 vs. pyruvate group, n = 3). ( C ): Forskolin, IBMX and 8-Bromo-cAMP inhibited pyruvate-stimulated increase in FGF21 protein levels in cell medium (* p < 0.05 vs. control, # p < 0.05 vs. pyruvate group, n = 10). ( D ): Pyruvate decreased intracellular cAMP levels in HepG2 cells (** p < 0.01 vs. control, n = 12).

Article Snippet: Human and mouse FGF21 ELISA kits, cAMP assay kits, the ALT activity assay kit, the AST activity assay kit and the LDH assay kit were obtained from Elabscience (Wuhan, China).

Techniques: Expressing, Activation Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Pyruvate Upregulates Hepatic FGF21 Expression by Activating PDE and Inhibiting cAMP–Epac–CREB Signaling Pathway

doi: 10.3390/ijms23105490

Figure Lengend Snippet: Epac and CREB were involved in pyruvate-stimulated FGF21 expression in HepG2 cells. ( A ): Epac inhibitor ESI-09 but not PKA inhibitor H89 upregulated FGF21 expression and eliminated the stimulatory effect of pyruvate on FGF21 expression (** p < 0.01 vs. control, n = 3) ( B ): CREB inhibitor 666-15 upregulated FGF21 expression and eliminated the stimulatory effect of pyruvate on FGF21 expression (** p < 0.01 vs. control, n = 3). ( C , D ): Pyruvate reduced CREB phosphorylation without influencing the total CREB protein levels (** p < 0.01 vs. control, n = 3).

Article Snippet: Human and mouse FGF21 ELISA kits, cAMP assay kits, the ALT activity assay kit, the AST activity assay kit and the LDH assay kit were obtained from Elabscience (Wuhan, China).

Techniques: Expressing, Control, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Pyruvate Upregulates Hepatic FGF21 Expression by Activating PDE and Inhibiting cAMP–Epac–CREB Signaling Pathway

doi: 10.3390/ijms23105490

Figure Lengend Snippet: Pyruvate upregulated FGF21 expression and secretion in mouse hepatic AML-12 cells. ( A , B ): Pyruvate stimulated FGF21 expression and secretion in AML-12 cells (* p < 0.05 and ** p < 0.01 vs. control, n = 6). ( C ): Pyruvate decreased intracellular cAMP levels in AML12 cells (* p < 0.05, n = 5). ( D ): Pyruvate increased PDE activities in AML-12 cells (* p < 0.05, n = 5).

Article Snippet: Human and mouse FGF21 ELISA kits, cAMP assay kits, the ALT activity assay kit, the AST activity assay kit and the LDH assay kit were obtained from Elabscience (Wuhan, China).

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Pyruvate Upregulates Hepatic FGF21 Expression by Activating PDE and Inhibiting cAMP–Epac–CREB Signaling Pathway

doi: 10.3390/ijms23105490

Figure Lengend Snippet: Pyruvate upregulated FGF21 expression in mice in vivo. ( A ): Intraperitoneal injection of pyruvate significantly increased serum pyruvate levels in C57BL/6J mice compared with the control (** p < 0.01, n = 10). ( B ): The pyruvate-treated mice had significantly higher FGF21 gene expression in liver than the control (** p < 0.01, n = 10). ( C ): Serum FGF21 levels were not changed by pyruvate treatment in mice compared with the control ( n = 10). ( D ): cAMP levels in mouse liver were significantly decreased by pyruvate treatment in mice (* p < 0.05, n = 10). ( E ): PDE activity in mouse liver was significantly activated by pyruvate injection in mice (* p < 0.05, n = 10). ( F ): CREB phosphorylation was inhibited in liver after pyruvate injection compared with the control. ( G , H ): ALT and AST activities in mouse serum were not significantly different between pyruvate-treated group and the control group. ( I ): H&E staining showed that the liver tissues had normal morphology in mice of pyruvate-treated group without obvious difference to the control (bar is 20 μm).

Article Snippet: Human and mouse FGF21 ELISA kits, cAMP assay kits, the ALT activity assay kit, the AST activity assay kit and the LDH assay kit were obtained from Elabscience (Wuhan, China).

Techniques: Expressing, In Vivo, Injection, Control, Gene Expression, Activity Assay, Phospho-proteomics, Staining

Journal: International Journal of Molecular Sciences

Article Title: Pyruvate Upregulates Hepatic FGF21 Expression by Activating PDE and Inhibiting cAMP–Epac–CREB Signaling Pathway

doi: 10.3390/ijms23105490

Figure Lengend Snippet: The diagram of pyruvate-stimulated FGF21 expression in hepatocytes. cAMP–Epac–CREB signaling inhibits FGF21 expression in human and mouse hepatocytes. Pyruvate activates PDEs to reduce cAMP levels and then inhibits cAMP–Epac–CREB signaling to upregulate FGF21 expression in hepatocytes.

Article Snippet: Human and mouse FGF21 ELISA kits, cAMP assay kits, the ALT activity assay kit, the AST activity assay kit and the LDH assay kit were obtained from Elabscience (Wuhan, China).

Techniques: Expressing

Journal: Molecular metabolism

Article Title: Fibroblast activation protein (FAP) as a novel metabolic target.

doi: 10.1016/j.molmet.2016.07.003

Figure Lengend Snippet: Figure 2: Chronic FAP inhibition by TB induces metabolic and glycemic benefits and enhances plasma half-life of FGF21 in DIO mice. (AeK) Effects on (A) body weight change, (B) food intake, (C) body composition change, (DeE) intraperitoneal glucose tolerance, (F) plasma insulin, (G) insulin tolerance, (H) plasma cholesterol, (I) total and intact plasma FGF21, (J) body weight-corrected energy expenditure and (K) real-time respiratory quotient of male DIO mice treated daily with vehicle or TB (0.03, 0.1, 0.3 and 1 mg/kg) for 16 days. In D, E, G, J and K, only mice treated with vehicle, 0.3 and 1 mg/kg/day were analyzed. The glucose (D) and insulin (G) tolerance tests were performed in different cohorts of animals at day 7 of treatment. In K, shaded regions represent time during the dark cycle of light. Data are presented as mean SEM; n ¼ 8; *P < 0.05, **P < 0.01, and ***P < 0.001, determined by ANOVA comparing effects following compound administration to vehicle treatment. #P < 0.05, ##P < 0.01, and ###P < 0.001, determined by ANOVA comparing effect following compound administration to vehicle (intact FGF21 value) treatment. In both comparisons, ANOVA was followed by Tukey post hoc multiple comparison analysis to determine statistical significance. Energy expenditure data were analyzed using ANCOVA, with body weight, fat mass and lean mass as covariates. P ¼ 0.002 when compared the highest TB dose (1 mg/kg) to vehicle group. P ¼ 0.003 when comparing vehicle and both TB doses.

Article Snippet: Liquid chromatographyemass spectrometry (LCeMS) In order to check whether mouse FGF21 is susceptible to FAP cleavage, 25 mM mouse FGF21 protein was incubated with or without 125 nM recombinant human FAP (R&D systems) at 37 C. The protein samples were analyzed by LCeMS (Agilent 1260 Infinity coupled with Agilent 6120 Quadrupole mass spectrometer).

Techniques: Inhibition, Clinical Proteomics, Comparison

Journal: Molecular metabolism

Article Title: Fibroblast activation protein (FAP) as a novel metabolic target.

doi: 10.1016/j.molmet.2016.07.003

Figure Lengend Snippet: Figure 3: The metabolic and glycemic benefits of FAP inhibition are blunted in FGF21del mice. (AeE) Effects on (A) body weight change, (B) body composition, (C) fasting blood glucose and (DeE) oral glucose tolerance of male FGF21del mice treated daily with vehicle or TB (0.3 mg/kg) for 7 days. Data are presented as mean SEM; n ¼ 8; *P < 0.05, **P < 0.01, and ***P < 0.001, determined by ANOVA comparing effects following compound administration to vehicle treatment. #P < 0.05, ##P < 0.01, and ###P < 0.001, determined by ANOVA comparing effect of FGF21 ablation to wild type mice. In both comparisons, ANOVA was followed by Tukey post hoc multiple comparison analysis to determine statistical significance.

Article Snippet: Liquid chromatographyemass spectrometry (LCeMS) In order to check whether mouse FGF21 is susceptible to FAP cleavage, 25 mM mouse FGF21 protein was incubated with or without 125 nM recombinant human FAP (R&D systems) at 37 C. The protein samples were analyzed by LCeMS (Agilent 1260 Infinity coupled with Agilent 6120 Quadrupole mass spectrometer).

Techniques: Inhibition, Comparison

Journal: Molecular metabolism

Article Title: Fibroblast activation protein (FAP) as a novel metabolic target.

doi: 10.1016/j.molmet.2016.07.003

Figure Lengend Snippet: Figure 4: Chronic FAP inhibition by TB does not have any metabolic influence and does not alter plasma half-life of FGF21 in lean mice. (AeD) Effects on (A) body weight, (B) food intake, (C) body composition change and (D) total and intact plasma FGF21 of male lean mice treated daily with vehicle or TB (0.3, 1 and 3 mg/kg) for 12 days. (E) Effect of recombinant FAP on FGF21 cleavage in vitro. hFGF21 was incubated with recombinant human FAP in the presence or absence of TB for the indicated times in PBS buffer. The products of the enzymatic reaction were resolved on SDS-PAGE and detected with total, N- and C-terminal specific FGF21 antibodies in Western blots. Data are presented as mean SEM; n ¼ 8; *P < 0.05, **P < 0.01, and ***P < 0.001, determined by ANOVA comparing effects following compound administration to vehicle treatment. ANOVA was followed by Tukey post hoc multiple comparison analysis to determine statistical significance.

Article Snippet: Liquid chromatographyemass spectrometry (LCeMS) In order to check whether mouse FGF21 is susceptible to FAP cleavage, 25 mM mouse FGF21 protein was incubated with or without 125 nM recombinant human FAP (R&D systems) at 37 C. The protein samples were analyzed by LCeMS (Agilent 1260 Infinity coupled with Agilent 6120 Quadrupole mass spectrometer).

Techniques: Inhibition, Clinical Proteomics, Recombinant, In Vitro, Incubation, SDS Page, Western Blot, Comparison

Journal: Endocrinology

Article Title: Dietary Intake Regulates White Adipose Tissues Angiogenesis via Liver Fibroblast Growth Factor 21 in Male Mice.

doi: 10.1210/endocr/bqaa244

Figure Lengend Snippet: Figure 1. Fasting induced adipose-specific VEGF expression and liver Fgf21 expression. Twelve-week-old male C57BL/6J mice were either fed with chow diets ad libitum or fasting for various time periods (6 h, 12 h, and 24 h) as indicated. (A) Serum VEGF levels and (B) the mRNA expression of Vegfa in epididymal WAT (eWAT) (B), subcutaneous iWAT (C), interscapular BAT (D), liver (E), and muscle (F) as determined by real-time PCR ana- lysis. The protein levels of VEGF in eWAT (G) and iWAT (H) as determined by western blot; (I) serum FGF21 levels as determined by enzyme-linked immunosorbent assay; and real-time transcription PCR analysis for Fgf21 mRNA expression levels of liver (J), eWAT (K), iWAT (L), BAT (M), and muscle (N). Serum FFA (O) and ketone bodies (P) levels. Data are mean ± SEM; n = 6/group. Statistical significance was evaluated by 1-way ANOVA with Tukey’s test for multiple comparisons to determine differences between each group. Labeled means without a common letter differ, P < 0.05. Abbreviations: BAT, brown adipose tissue; eWAT, epididymal white adipose tissue; FFA, free fatty acid; FGF21, fibroblast growth factor 21; iWAT, in- guinal white adipose tissue; VEGF, vascular endothelial growth factor.

Article Snippet: Recombinant mouse FGF21 (8409; R&D Systems; Bio-Techne, Minneapolis, MN, USA) or equal volume of phosphate-buffered saline was injected from tail vein at the dose of 1 mg/kg bodyweight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Lysis, Western Blot, Enzyme-linked Immunosorbent Assay, Labeling

Journal: Endocrinology

Article Title: Dietary Intake Regulates White Adipose Tissues Angiogenesis via Liver Fibroblast Growth Factor 21 in Male Mice.

doi: 10.1210/endocr/bqaa244

Figure Lengend Snippet: Figure 2. FGF21 promoted expression and accumulation of VEGF in WAT. The relative mRNA abundance of Vegfa in tissue, adipocytes and stromal vascular fraction isolated from eWAT (A) and iWAT (B) at fed or 24 h of fasting. Twelve-week-old male WT (FGF21fl/fl) and FGF21 LKO mice were fasted for 24 h; the Vegfa mRNA expression in eWAT and iWAT (C). Quantitative reverse transcription PCR analysis for Vegfa mRNA expression in eWAT (D), iWAT (E), and BAT (F) at the indicated time points after tail vein injection of rmFGF21 (1 mg/kg). The protein levels of VEGF at various time points after mice receiving a delivery of rmFGF21 with tail vein injection in eWAT (G) and iWAT (H). n = 6/group. Statistical significance was evaluated by unpaired Student’s t test. *P < 0.05, **P < 0.01, versus control; labeled means without a common letter differ, P < 0.05. Abbreviation: SCV, stromal vascular fraction.

Article Snippet: Recombinant mouse FGF21 (8409; R&D Systems; Bio-Techne, Minneapolis, MN, USA) or equal volume of phosphate-buffered saline was injected from tail vein at the dose of 1 mg/kg bodyweight.

Techniques: Expressing, Isolation, Reverse Transcription, Injection, Control, Labeling

Journal: Endocrinology

Article Title: Dietary Intake Regulates White Adipose Tissues Angiogenesis via Liver Fibroblast Growth Factor 21 in Male Mice.

doi: 10.1210/endocr/bqaa244

Figure Lengend Snippet: Figure 3. Intermittent fasting induced adipose-VEGF expression and angiogenesis depend on liver FGF21 signaling. Mice were fed with HFD ad lib- itum or time-restricted access to food for 16 weeks. HA means WT mice eating a high-fat diet with ad libitum, HT means WT mice eating a high-fat diet with time-restricted access to food, KOHA means FGF21 LKO mice eating a high-fat diet with ad libitum, KOHT means FGF21 LKO mice eating a HFD with time-restricted access to food. (A) Schematic illustration of the experimental design. (B) Body weight. (C) Representative HT mice were remarkably leaner than the HA mice. (D) Body composition was evaluated by EchoMRI. (E) Serum FGF21 levels as determined by enzyme-linked im- munosorbent assay. (F) A representative macroscopic image illustrating increased vascularization in iWAT of HT mice, compared to HA mice but not in FGF21 LKO mice. (G) Real-time quantitative PCR analysis for mRNA expression levels of Vegfa in eWAT and iWAT. Representative protein levels for VEGF in eWAT (G) and iWAT (I). (J) Immunofluorescence staining of CD31 (scale bar, 100 mm) in eWAT and iWAT, illustrating IF increased vascular- ization in WT mice but not in FGF21 LKO mice. Data are mean ± SEM; n = 6–8/group. Statistical significance was evaluated by the 2-way ANOVA test and the Tukey’s test for multiple comparisons to determine differences between each group. HA vs HT, *P < 0.05, **P < 0.01; labeled means without a common letter differ, P < 0.05.

Article Snippet: Recombinant mouse FGF21 (8409; R&D Systems; Bio-Techne, Minneapolis, MN, USA) or equal volume of phosphate-buffered saline was injected from tail vein at the dose of 1 mg/kg bodyweight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Labeling

Journal: Endocrinology

Article Title: Dietary Intake Regulates White Adipose Tissues Angiogenesis via Liver Fibroblast Growth Factor 21 in Male Mice.

doi: 10.1210/endocr/bqaa244

Figure Lengend Snippet: Figure 4. Liver-FGF21 is required for intermittent fasting-induced metabolic benefits. Mice were fed with HFD ad libitum or time-restricted access to food for 16 weeks. (A) O2 consumption (VO2), (B) CO2 production (VCO2). (C and D) GTT shows normal glucose tolerance in HT mice but not for FGF21 LKO mice. (E) Serum insulin levels. real-time quantitative PCR analysis for mRNA expression levels of browning related genes (F), UCP-1 protein level (G) and immunohistochemical staining (H) of UCP-1 in iWAT. (I) The expression of pro-inflammatory cytokines (Tnfα and IL1-β) in eWAT. (J) A working model of dietary intake regulating iWAT browning via FGF21 signaling. Data are mean ± SEM; n = 6–8/group. Statistical significance was evaluated by the 2-way ANOVA test and the Tukey’s test for multiple comparisons to determine differences between each group. *P < 0.05, **P < 0.01; labeled means without a common letter differ, P < 0.05.

Article Snippet: Recombinant mouse FGF21 (8409; R&D Systems; Bio-Techne, Minneapolis, MN, USA) or equal volume of phosphate-buffered saline was injected from tail vein at the dose of 1 mg/kg bodyweight.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunohistochemical staining, Staining, Labeling