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: 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