Journal: International Journal of Molecular Sciences

Article Title: Glucosamine Enhancement of Learning and Memory Functions by Promoting Fibroblast Growth Factor 21 Production

doi: 10.3390/ijms25084211

Figure Lengend Snippet: GLN’s induction of FGF21 expression in the hippocampus, cortex, and striatum. After two weeks of GLN injection, the protein levels of FGF21 were measured in the hippocampus, cortex, and striatum of ND ( A ) or HFD mice ( B ) using Western blotting with β-actin as an internal control. The results represent the means ± S.E.M. ( n = 8). * p < 0.05 and *** p < 0.001 compared with the GLN 0 mg/mouse group.

Article Snippet: The concentration of FGF21 in the culture medium was determined using an ELISA kit for mouse FGF21 (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Injection, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Glucosamine Enhancement of Learning and Memory Functions by Promoting Fibroblast Growth Factor 21 Production

doi: 10.3390/ijms25084211

Figure Lengend Snippet: GLN-induced expression of FGF21 in hippocampal and striatal cells. HT22 hippocampal ( A ) and STHdh Q7/Q7 striatal cells ( B ) were treated with GLN (0, 1.25, 2.5, 5, 10, 20 mM) for 6 h for mRNA determination or 24 h for protein measurement. FGF21 mRNA was quantified using real-time PCR with GAPDH as an internal control, while FGF21 protein was examined through Western blotting with β-actin as an internal control. FGF21 production in the cultured medium was assessed with ELISA. The results represent the means ± S.E.M. ( n = 4). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the GLN 0 mM group.

Article Snippet: The concentration of FGF21 in the culture medium was determined using an ELISA kit for mouse FGF21 (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Glucosamine Enhancement of Learning and Memory Functions by Promoting Fibroblast Growth Factor 21 Production

doi: 10.3390/ijms25084211

Figure Lengend Snippet: Attenuation of GLN-enhanced learning and memory functions and FGF21 production through FGFR1 inhibition in the hippocampus, hippocampal cells, and striatal cells. The ND-fed mice were administrated GLN (0, 30 mg/mouse) alone or in combination with PD173074 (2 mg/kg) through IP injection for 2 weeks, followed by the NORT ( A ). The FGF21 protein levels in the hippocampus ( B ) and in HT22 or STHdh Q7/Q7 cells treated with GLN (20 mM) alone or in combination with PD173074 (0.025, 0.1, 1 μM) for 24 h ( C ) were determined through Western blotting or ELISA. The results represent the means ± S.E.M. from 6 animals ( A ) or 4 animals ( B ) in each treatment group or from 4 independent experiments ( C ). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the GLN 0 mg/mouse group or the GLN 0 mM group. # p < 0.05 and ### p < 0.001 compared with the GLN 30 mg/mouse or the GLN 20 mM only group.

Article Snippet: The concentration of FGF21 in the culture medium was determined using an ELISA kit for mouse FGF21 (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Inhibition, Injection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Glucosamine Enhancement of Learning and Memory Functions by Promoting Fibroblast Growth Factor 21 Production

doi: 10.3390/ijms25084211

Figure Lengend Snippet: Illustration of the potential involvement of signaling molecules in GLN-mediated FGF21 production in hippocampal and striatal cells. ( A ) HT22 cells or ( B ) STHdh Q7/Q7 cells were pretreated with specific inhibitors targeting NF-κB (Parthenolide, 5 µM), MAPK p38 (SB203580, 20 µM), JNK (SP600125, 20 µM), ERK (PD98059, 20 µM), Akt (LY294002, 10 μM), PKA (H89, 5 µM), PKC (Bisindolylmaleimide, BIMI, 5 µM), PPARα (GW6471, 10 µM), or PPARγ (GW9662, 10 µM) for 1 h before the addition of GLN (20 mM) for an additional 24 h. The FGF21 concentration in the cultured medium was quantified using ELISA. The results represent the means ± S.E.M. ( n = 4). * p < 0.05 and *** p < 0.001 compared with the 0 mM group. # p < 0.05 and ### p < 0.001 compared with the GLN 20 mM group.

Article Snippet: The concentration of FGF21 in the culture medium was determined using an ELISA kit for mouse FGF21 (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Glucosamine Enhancement of Learning and Memory Functions by Promoting Fibroblast Growth Factor 21 Production

doi: 10.3390/ijms25084211

Figure Lengend Snippet: Demonstration of the further induction of FGF21 through overexpression of NF-κB p65 in HT22 cells. HT22 cells were transfected with EGFP-p65 or EGFP plasmid and treated with GLN (20 mM) for 24 h. Western blotting was performed to detect the proteins for FGF21, GFP, EGFP-RelA, and p65 using β-actin as an internal control. The results represent the means ± S.E.M. ( n = 3). * p < 0.05 and *** p < 0.001 compared with the 0 mM group. && p < 0.01 between the EGFP and EGFP-RelA groups.

Article Snippet: The concentration of FGF21 in the culture medium was determined using an ELISA kit for mouse FGF21 (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, 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: 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: Journal of Clinical Investigation

Article Title: Peripherally derived FGF21 promotes remyelination in the central nervous system

doi: 10.1172/jci94337

Figure Lengend Snippet: Figure 3. FGF21 secreted from pancreas induces OPC proliferation. (A) Quantitations of Fgf21 mRNA (left) and FGF21 protein (right) in individual organs of intact mice (n = 9); **P < 0.01. (B) Representative images of FGF21-immunolabeled pancreas of intact mice (n = 3). (C) Double IHC staining for FGF21 with the indicated cell markers in the pancreas of adult mice (n = 3). (D) BrdU incorporation in mouse OPCs after stimulation with serum from mice with FGF21 knockdown in the pancreas (n = 4); *P < 0.05, **P < 0.01, as determined by ANOVA with Tukey’s post hoc test. Error bars represent SEM. Scale bars: 50 μm (B); 10 μm (C).

Article Snippet: To assess the role of FGFs, we used recombinant mouse FGF15 (Abcam), recombinant mouse FGF21 (R&D Systems), and recombinant mouse FGF23 (R&D Systems).

Techniques: Immunolabeling, Immunohistochemistry, BrdU Incorporation Assay, Knockdown

Journal: Journal of Clinical Investigation

Article Title: Peripherally derived FGF21 promotes remyelination in the central nervous system

doi: 10.1172/jci94337

Figure Lengend Snippet: Figure 6. FGF21 promotes human OPC proliferation. (A) Representative image of β-klotho expression in an autopsied sample from healthy patient and a patient with multiple sclerosis. Graphs show quantitations as indicated in the images (n = 4 for healthy patients, 3 for multiple sclerosis patients); **P < 0.01. (B) BrdU incorporation in human OPCs after stimulation with recombinant FGF21 (n = 6 for control, 4 for FGF21); *P < 0.05 as determined by Student’s t test. Error bars represent SEM. Scale bar: 20 μm.

Article Snippet: To assess the role of FGFs, we used recombinant mouse FGF15 (Abcam), recombinant mouse FGF21 (R&D Systems), and recombinant mouse FGF23 (R&D Systems).

Techniques: Expressing, BrdU Incorporation Assay, Recombinant, 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 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: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) Cold-induced increase in serum FGF21 levels was blunted by injection of miR-32-ASO as measured by ELISA (n = 7). (B) qRT-PCR showed that cold stress decreased FGF21 mRNA expression in liver but increased it in BAT. Injection of miR-32-ASO significantly repressed the increase in FGF21 expression in BAT during cold exposure when normalized to PPIA . (C) FGF21 immunostaining showed that cold stress decreased FGF21 level in liver but increased it in BAT. Injection of miR-32-ASO significantly repressed the increase in FGF21 expression in BAT during cold exposure. (D) Quantification of FGF21 immunostaining in BAT showed that FGF21 levels within BAT increased greatly after cold exposure but were significantly lower in miR-32-ASO-treated mice compared to control-ASO-treated mice. (E) Quantification of FGF21 immunostaining in liver showed that FGF21 levels within liver decreased greatly after cold exposure but were similar between miR-32-ASO-treated mice and control-ASO-treated mice. (F) Western blot showing cold-induced increases in BAT FGF21 protein levels were blunted by injection of miR-32-ASO. Calnexin served as a loading control. (G) Western blot showing cold-induced decreases in liver FGF21 protein levels were unaffected by injection of miR-32-ASO. Calnexin served as a loading control. (H) Quantification of the western blot results in (F) and (G) using ImageJ. Average intensities were normalized to that of Calnexin. (I) FGF21 mRNA expression in WT-1 cells increased or decreased when transfected with miR-32 mimic or miR-32-ASO, respectively. (J) Western blotting showed that FGF21 protein levels in WT-1 cells increased or decreased when transfected with miR-32 mimic or miR-32-ASO, respectively. Data represent mean ± SEM. *p < 0.05 and ** p < 0.01. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Immunostaining, Control, Western Blot, Transfection

Journal: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) High mirSVR score and favorable binding energy from bioinformatic analysis predicted that miR-32 directly targets the 3′ UTR of Tob1 . (B) The complementary sequence in the Tob1 3′ UTR and the seed region of miR-32 (red letters) are conserved among mammalian species. (C) Tob1 mRNA level in WT-1 cells decreased or increased 24 hr after transfection of miR-32 mimic or miR-32-ASO, respectively. (D) Tob1 protein level decreased or increased in WT-1 cells when transfected with miR-32 mimic or miR-32-ASO, respectively. (E and F) Activities of a luciferase reporter gene linked to the Tob1 3′ UTR decreased or increased 24 hr after transfection of miR-32 mimic (E) or miR-32-ASO (F), respectively. (G and H) Site-directed mutagenesis of the Tob1 3′ UTR sequence complimentary to the miR-32 seed sequence abolished the effects of miR-32 mimic (G) and miR-32-ASO (H) transfection on the luciferase activity. (I) Knockdown of Tob1 by siTob1 suppressed the increase in Tob1 expression and rescued the decrease in FGF21 expression caused by miR-32-ASO transfection. (J) Western blots showed that siTob1 suppressed the increase in Tob1 expression and rescued the decreases of phospho-p38, phospho-ATF2, and FGF21 expression caused by miR-32-ASO transfection. Numbers indicate protein levels quantified using ImageJ relative to control-ASO. (K) qRT-PCR showed miR-32-ASO-treated mice failed to fully downregulate Tob1 level in response to cold stimulation. (L) Western blots showed miR-32-ASO-treated mice fail to downregulate Tob1 levels and activate p38 and ATF2 phosphorylation in BAT in response to cold stimulation. (M) Quantification of relative protein expression in BAT from the western blots showed in (L) using ImageJ. Average intensities were normalized to that of Calnexin. (N) Cold-exposure time course showing miR-32 and Tob1 expression in BAT at various time points after cold exposure. (O) Cold-exposure time course showing FGF21 expression in BAT at various time points after cold exposure. (P) Cold-exposure time course showing Tob1, UCP1, and FGF21 protein expression in BAT at various time points after cold exposure. (Q) Cold-exposure time course showing UCP1 protein expression in iWAT at various time points after cold exposure. Data represent mean ± SEM. *p < 0.05 and ** p < 0.01. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Binding Assay, Sequencing, Transfection, Luciferase, Mutagenesis, Activity Assay, Knockdown, Expressing, Western Blot, Control, Quantitative RT-PCR, Phospho-proteomics

Journal: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) miR-32 expression in BAT but not in liver was blunted in BAT-specific miR-32-ASO-injected mice (n = 8) compared to control-ASO-injected mice (n = 7). (B) miR-32-ASO-BS mice (n = 8) showed lower core body temperatures during cold exposure when compared to control-ASO-BS mice (n = 7). (C) Total energy expenditure was significantly reduced after 7 days’ cold stress in miR-32-ASO-BS mice (n = 6) as compared with control-ASO-BS mice (n = 6). Energy expenditure was normalized to lean body mass. (D) Average total energy expenditure was significantly lower in miR-32-ASO-BS mice (n = 6). (E) Percentage BAT mass was significantly lower in miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). (F) In BAT, mRNA levels of Tob1 were higher in miR-32-ASO-BS mice (n = 8), whereas expression of thermogenic genes was significantly lower in miR-32-ASO-BS mice. (G) Protein levels of Tob1 were higher in miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). However, p-p38, p-ATF2, FGF21, UCP1, and PGC1α were lower in miR-32-ASO-BS mice (n = 8). Total p38 and total ATF2 expression were not significantly different between the two groups. (H) Quantification of relative protein expression using ImageJ showed that protein level of Tob1 was upregulated, but p-p38, p-ATF2, FGF21, UCP1, and PGC1α were significantly lower in BAT of miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). Total p38 and total ATF2 expression were comparable between the two groups. Average intensities were normalized to that of Calnexin. (I) Serum FGF21 levels were decreased in miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). (J) mRNA levels of thermogenic genes in iWAT were significantly reduced in miR-32-ASO-BS mice. Data were normalized to PPIA . (K) Immunoblots showed that miR-32-ASO-BS mice (n = 8) had significantly reduced UCP1 and PGC1α protein levels in iWAT compared to control-ASO-BS mice (n = 7). (L) Quantification of relative UCP1 and PGC1α protein levels using ImageJ. Average intensities were normalized to that of Calnexin. Data represent mean ± SEM. *p < 0.05, **p < 0.01, and *** p < 0.001. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Expressing, Injection, Control, Western Blot

Journal: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) miR-32 expression in BAT but not in liver or iWAT was increased in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). (B) miR-32-AAV-BS mice (n = 8) showed higher core body temperatures during cold exposure when compared to control-AAV-BS mice (n = 7). (C) Total energy expenditure was significantly increased after 7 days’ cold stress in miR-32-AAV-BS mice (n = 6) as compared with control-AAV-BS mice (n = 6). Energy expenditure was normalized to lean body mass. (D) Average total energy expenditure was significantly higher in miR-32-AAV-BS mice (n = 6). (E) Percentage BAT mass was significantly higher in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). (F) In BAT, mRNA levels of Tob1 were lower in miR-32-AAV-BS mice, whereas expression of thermogenic genes was significantly higher in miR-32-AAV-BS mice. (G) Protein levels of Tob1 were lower in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). However, p-p38, p-ATF2, FGF21, UCP1, and PGC1α were higher in miR-32-AAV-BS mice (n = 8). Total p38 and total ATF2 expression were not significantly different between the two groups. (H) Quantification of relative protein expression using ImageJ showed that protein level of Tob1 was reduced, but p-p38, p-ATF2, FGF21, UCP1, and PGC1α were significantly higher in BAT of miR-32-AAV-BS mice compared to control. Total p38 and total ATF2 expression were comparable between the two groups. Average intensities were normalized to that of Calnexin. (I) Serum FGF21 levels were increased in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). (J) mRNA levels of thermogenic genes in iWAT were significantly increased in miR-32-AAV-BS mice. Data were normalized to PPIA . (K) Immunoblots showed that miR-32-AAV-BS mice (n = 8) had significantly increased UCP1 and PGC1α protein levels in iWAT compared to control-AAV-BS mice (n = 7). (L) Quantification of relative UCP1 and PGC1α protein levels using ImageJ. Average intensities were normalized to that of Calnexin. Data represent mean ± SEM. *p < 0.05, **p < 0.01, and *** p < 0.001. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Expressing, Control, Western Blot

Journal: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) FGF21 mRNA expression in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6). (B) FGF21 protein expression in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6). (C) Quantification of relative protein expression using ImageJ showed that protein level of FGF21 in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6). (D) Serum FGF21 levels were decreased in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6) compared to wild-type mice (n = 4). (E) miR-32-AAV-BS+Cre mice (n = 6) showed higher core body temperatures only during first 48 hr of cold exposure when compared to control-AAV-BS+Cre mice (n = 6). (F) Total energy expenditure was similar after 7 days’ cold stress in miR-32-AAV-BS+Cre mice (n = 6) as compared with control-AAV-BS+Cre mice (n = 6). Energy expenditure was normalized to lean body mass. (G) Average total energy expenditure was slightly higher in miR-32-AAV-BS+Cre mice (n = 6) than control-AAV-BS+Cre mice (n = 6) but not statistically significant. (H) Percentage BAT mass was slightly higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6). (I) In BAT, mRNA levels of Tob1 were lower in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6), whereas expression of several thermogenic genes including UCP1 was higher in miR-32-AAV-BS+Cre mice. (J) Protein levels of PGC1α and UCP1 were higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6). (K) Quantification of relative protein expression using ImageJ showed that protein levels of PGC1 and UCP1 were higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6). Average intensities were normalized to that of Calnexin. (L) mRNA levels of thermogenic genes in iWAT were similar in both groups of mice (both n = 6). Data were normalized to PPIA . (M) Immunoblots showed that miR-32-AAV-BS+Cre mice (n = 6) had similar UCP1 and PGC1α protein levels in iWAT compared to control-AAV-BS+Cre mice (n = 6). (N) Quantification of relative UCP1 and PGC1α protein levels using ImageJ. Average intensities were normalized to that of Calnexin. (O) Proposed mechanism by which miR-32 promotes BAT thermogenesis by inhibiting Tob1, activating p38/MAPK signaling and driving UCP1, PGC1α, and FGF21 expression in BAT. The BAT secreted FGF21 functions in a paracrine fashion to promote further thermogenic gene expression in BAT as well as in an endocrine fashion to promote iWAT browning. Data represent mean ± SEM. *p < 0.05, **p < 0.01, and *** p < 0.001. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Expressing, Control, Western Blot, Gene Expression