Journal: The Journal of Clinical Investigation

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

doi: 10.1172/JCI94337

Figure Lengend Snippet: (A) Concentration-dependence of BrdU incorporation in A2B5+ OPCs cultured with adult mice serum (n = 6). (B) BrdU incorporation in A2B5+ OPCs 1 day after stimulation with adult mouse serum heated or pretreated with the indicated reagents (n = 6). (C) BrdU incorporation in OPCs after serum stimulation with PD173074 (10 nM), an inhibitor of FGFR (n = 4). (D) BrdU incorporation in OPCs after serum stimulation with NF449 (10 μM), an inhibitor of FGFR3 (n = 4). (E) BrdU incorporation in mouse OPCs with FGFR and β-klotho knockdown after serum stimulation (n = 7). (F) BrdU incorporation in OPCs after stimulation with recombinant FGF15, FGF21, and FGF23 (n = 4). (G) BrdU incorporation in OPCs after serum stimulation with neutralizing antibody against FGF21 (n = 5), determined by Student’s t test or by ANOVA with Tukey’s post hoc test or Dunnett’s test. Error bars represent SEM. *P < 0.05, **P < 0.01.

Article Snippet: To assess cell proliferation, cells were cultured in DMEM supplemented with or without recombinant human FGF21 (R&D Systems) at a final concentration of 6 μg/ml.

Techniques: Concentration Assay, BrdU Incorporation Assay, Cell Culture, Recombinant

Journal: The Journal of Clinical Investigation

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

doi: 10.1172/JCI94337

Figure Lengend Snippet: (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 cell proliferation, cells were cultured in DMEM supplemented with or without recombinant human FGF21 (R&D Systems) at a final concentration of 6 μg/ml.

Techniques: Immunolabeling, Immunohistochemistry, BrdU Incorporation Assay

Journal: The Journal of Clinical Investigation

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

doi: 10.1172/JCI94337

Figure Lengend Snippet: (A) Quantitation of FGF21 protein in the spinal cord 1 day and 3 days after LPC injection (n = 5 for control, 5 for d1, 4 for d3). (B) Representative images of spinal cord sections double-labeled for PDGFRα and Ki67. Sections were obtained from FGF21-KO mice and control littermates 7 days after LPC injection. Graph shows quantitations as indicated in the images (n = 5). Arrows indicate Ki67+ cells colabeled with PDGFRα; arrowheads indicate Ki67+ cells not labeled with PDGFRα. (C) Representative images of spinal cord sections labeled for MBP. Sections were obtained from mouse spinal cord 14 days after LPC injection. Graph shows quantitations as indicated in the images (n = 5). (D) Representative immunoelectron microscopy images of myelin in the spinal cord. Sections were obtained from FGF21-KO mice and control littermates 14 days after LPC injection. Graphs show quantitations of g-ratio indicated in the images (n = 3). (E) Motor function was assessed by ladder-walk test (n = 11 for control littermates, 9 for FGF21-KO mice). (F) Representative images of spinal cord sections labeled for MBP. Graph shows quantitations as indicated in the images (n = 5 for control littermates + vehicle, 5 for FGF21-KO mice + vehicle, 4 for FGF21-KO mice + FGF21). (G) Representative images of spinal cord sections labeled for MBP. Graph shows quantitations as indicated in the images (n = 4); **P < 0.01 as determined by Student’s t test or by ANOVA with Tukey’s post hoc test or Dunnett’s test. *P < 0.05, **P < 0.01. Error bars represent SEM. Scale bars: 50 μm (B); 200 μm (C, F, and G); 2 μm (D).

Article Snippet: To assess cell proliferation, cells were cultured in DMEM supplemented with or without recombinant human FGF21 (R&D Systems) at a final concentration of 6 μg/ml.

Techniques: Quantitation Assay, Injection, Labeling, Immuno-Electron Microscopy

Journal: The Journal of Clinical Investigation

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

doi: 10.1172/JCI94337

Figure Lengend Snippet: (A) Representative images of β-klotho expression in the mouse spinal cord 3 days after LPC injection. (B) Representative images of spinal cord sections double-labeled for PDGFRα and Ki67. Graph shows quantitations as indicated in images (n = 3 for control, 3 for CKO); *P < 0.05. Arrows indicate Ki67+ cells colabeled with PDGFRα. (C) Representative images of spinal cord sections labeled for MBP. Graph shows quantitations as indicated in the images (n = 4 each); **P < 0.01. (D) Representative images of brain sections double-labeled for PDGFRα and Ki67 in the mouse cortex, 7 days after traumatic brain injury. FGF21 was administered i.c.v. 24 hours after LPC injection (n = 4); *P < 0.05. Arrows indicate Ki67+ cells colabeled with PDGFRα; arrowheads indicate Ki67+ cells not labeled with PDGFRα. (E) Representative images of brain sections labeled for MBP in the mouse cortex, 14 days after traumatic brain injury. Graph shows quantitations as indicated in the images (n = 6 for vehicle, n = 4 for FGF21); *P < 0.05. (F) Representative immunoelectron microscopy images of myelin in the mouse cortex, 14 days after traumatic brain injury. Graphs show quantitations of g-ratio indicated in the images (n = 4); **P < 0.01 as determined by Student’s t test. Error bars represent SEM. Scale bars: 20 μm (A); 50 μm (B and D); 200 μm (C and E); 2 μm (F).

Article Snippet: To assess cell proliferation, cells were cultured in DMEM supplemented with or without recombinant human FGF21 (R&D Systems) at a final concentration of 6 μg/ml.

Techniques: Expressing, Injection, Labeling, Immuno-Electron Microscopy

Journal: The Journal of Clinical Investigation

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

doi: 10.1172/JCI94337

Figure Lengend Snippet: (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 cell proliferation, cells were cultured in DMEM supplemented with or without recombinant human FGF21 (R&D Systems) at a final concentration of 6 μg/ml.

Techniques: Expressing, BrdU Incorporation Assay, Recombinant

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: Novel Insights into the Cardio-Protective Effects of FGF21 in Lean and Obese Rat Hearts

doi: 10.1371/journal.pone.0087102

Figure Lengend Snippet: FGF21 mRNA [ Fig. 2A1 ] and protein expression [ Fig. 2A2 ]; βKlotho mRNA [ Fig. 2A3 ] and protein expression [ Fig. 2A4 ] in isolated rat cardiomyocytes [A] and rat heart [B]. Fig . 1B:Immunocyto/histochemistry and confocal analysis of FGF21 protein in isolated adult rat cardiomyocytes [ B1 ] and rat heart [ B2 ]. MW.- Molecular Weight Marker for PCR products; BP.- Base Pairs; kDa.-kilo daltons. Fig. 2C -( a ): Trace recordings of left ventricular developed pressure [LVDP (mmHg)] and left ventricular contractility (dp/dt) in control (saline treated) groups; and Fig. 2C -( b ):[LVDP-mmHg] and dp/dt ratio in FGF21 treated groups - following 30 mins of global ischemia and 120 mins reperfusion. Fig. 2D: Rate pressure product [RPP (mmHg/min)] during global ischemia and reperfusion with or without FGF21 treatment [** P <0.01 vs. control]. Fig. 2E : Graphical representation of infarct area (%) in rat hearts treated with or without FGF21. Data shown are means ± SEM (n = 6, in triplicates). ***P<0.001; **P<0.01 vs. control.

Article Snippet: The drugs used for the study were: rat recombinant FGF21 [R&D systems, UK], wortmannin (PI3K inhibitor), U1026 (MAPK inhibitor), Compound C (AMPK inhibitor), TO-901317 [ROR-α (Retinoic acid receptor-related receptor alpha) inhibitor]; TO-901317 (was initially intended as a therapeutic Liver X receptor (LXR) agonist in dementia , however recently, work by Uebanso T et al, have demonstrated its role in the regulation of FGF21 expression ) and collagenase (type I+protease type XIV).

Techniques: Expressing, Isolation, Molecular Weight, Marker, Control, Saline

Journal: PLoS ONE

Article Title: Novel Insights into the Cardio-Protective Effects of FGF21 in Lean and Obese Rat Hearts

doi: 10.1371/journal.pone.0087102

Figure Lengend Snippet: Fig. 3A1: Cardiomyocytes treated with FGF21 (100 nM) for 5–30 minutes. Phosphorylated ERK 1/2 [Fig. 3A1]; Akt [Fig. 3A2] and AMPK [Fig. 3A3] proteins are represented in relation to total proteins, expressed as fold increase over basal. ***P<0.001, **P<0.01, *P<0.05 vs. basal, n = 6 per group. Fig. 3B1: RPP with ischemia/reperfusion and FGF21 treatment following pre-incubation with inhibitors (TO-901317; wortmanin; Compound C or U0126) a P<0.05, b P<0.01 vs. FGF21 only treatment, n = 6 per group. Fig. 3B2: Infarcted area (%) following ischemia/reperfusion and FGF21 treatment following pre-incubation with inhibitors (TO-901317; wortmanin; Compound C or U0126) **P<0.01, *P<0.05 vs. FGF21 treated, n = 6 per group.

Article Snippet: The drugs used for the study were: rat recombinant FGF21 [R&D systems, UK], wortmannin (PI3K inhibitor), U1026 (MAPK inhibitor), Compound C (AMPK inhibitor), TO-901317 [ROR-α (Retinoic acid receptor-related receptor alpha) inhibitor]; TO-901317 (was initially intended as a therapeutic Liver X receptor (LXR) agonist in dementia , however recently, work by Uebanso T et al, have demonstrated its role in the regulation of FGF21 expression ) and collagenase (type I+protease type XIV).

Techniques: Incubation

Journal: PLoS ONE

Article Title: Novel Insights into the Cardio-Protective Effects of FGF21 in Lean and Obese Rat Hearts

doi: 10.1371/journal.pone.0087102

Figure Lengend Snippet: Fig. 4A :FGF21 mRNA expression levels in cardiomyocytes following FGF21 (100 nM) treatment with or without pathway inhibitors [(U0126; wort (wortmanin); Comp C (Compound C) or TO (TO-901317)] (normalised to GAPDH and expressed as fold changes over basal). Fig. 4B : Graphical analysis of FGF21 protein levels following FGF21 treatment. Fig. 4C : Graphical representation of FGF21 ELISA measurements in the conditioned media following FGF21 treatment. Fig. 4D : Graphical representation of FGF21 ELISA measurements of rat heart Langendorff exudates following global ischemia for 5–30 mins and 120 mins of reperfusion; with or without prior FGF21 (100 nM) infusion. Data shown are means ± SEM of triplicates. The values represented are relative to basal. *** P <0.001; ** P <0.01; * P <0.05 vs. FGF21 only treated, a P <0.001 vs. basal, n = 6 per group.

Article Snippet: The drugs used for the study were: rat recombinant FGF21 [R&D systems, UK], wortmannin (PI3K inhibitor), U1026 (MAPK inhibitor), Compound C (AMPK inhibitor), TO-901317 [ROR-α (Retinoic acid receptor-related receptor alpha) inhibitor]; TO-901317 (was initially intended as a therapeutic Liver X receptor (LXR) agonist in dementia , however recently, work by Uebanso T et al, have demonstrated its role in the regulation of FGF21 expression ) and collagenase (type I+protease type XIV).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Novel Insights into the Cardio-Protective Effects of FGF21 in Lean and Obese Rat Hearts

doi: 10.1371/journal.pone.0087102

Figure Lengend Snippet: Using rat heart Langendorff model and inducing global ischemia for 30[ Fig. 5A1 ], protein [ Fig. 5A2 ], and secretion of FGF21 in Langendorff coronary exudates [ Fig. 5A3 ] were measured. Similarly, changes in FGFR1 mRNA expressions [ Fig. 5A4 ] were measured. Data shown are means ± SEM of triplicates. The values represented are relative to basal. *** P <0.001; ** P <0.01vs. control. n = 6 per group. FGF21 mRNA [ Fig. 5B1 ], protein [ Fig. 5B2 ], FGF21 secretion (in Langendorff coronary exudates) [ Fig. 5B3 ] and FGFR1 mRNA [ Fig. 5B4 ] expressions were measured in obese and lean rat hearts. Graphical representation of a key signalling component of FGF21/FGFR1; βKlotho protein level in obese and lean hearts [ P <0.05 vs.lean; Figure 5C 1 ]. Graphical representation of ERK 1/2 [ Fig. 5C2 ], Akt [ Fig. 5C3 ] and AMPK [ Fig. 5C4 ] phosphorylation levels in lean and obese hearts with FGF21 (100 nM) pre-infusion. Data shown are means ± SEM of triplicates. The values represented are relative to basal. * P <0.05, ** P <0.01 vs. lean control, NS -non-significant; n = 6 per group.

Article Snippet: The drugs used for the study were: rat recombinant FGF21 [R&D systems, UK], wortmannin (PI3K inhibitor), U1026 (MAPK inhibitor), Compound C (AMPK inhibitor), TO-901317 [ROR-α (Retinoic acid receptor-related receptor alpha) inhibitor]; TO-901317 (was initially intended as a therapeutic Liver X receptor (LXR) agonist in dementia , however recently, work by Uebanso T et al, have demonstrated its role in the regulation of FGF21 expression ) and collagenase (type I+protease type XIV).

Techniques: Control, Phospho-proteomics

Journal: PLoS ONE

Article Title: Novel Insights into the Cardio-Protective Effects of FGF21 in Lean and Obese Rat Hearts

doi: 10.1371/journal.pone.0087102

Figure Lengend Snippet: LVDP (mmHg) and left ventricular contractility (dp/dt) in obese control (saline treated) groups [ Fig. 6A1 ] and lean control (saline treated) groups [ Fig. 6A2 ]; following global ischemia and reperfusion. Graphical representation of RPP (mmHg/min)] during global ischemia and reperfusion in obese and lean control rat hearts [ Fig. 6B ]. Graphical representation of the infarcted area (%) in obese and lean control rat hearts following global ischemia and reperfusion [ Fig. 6C ]. Graphical representation of FGF21 levels in obese and lean rat heart coronary effluents following global ischemia and reperfusion [ Fig. 6D ]. Data shown are means ± SEM of triplicates. The values represented are relative to basal. *** P <0.001 vs. t [0] time point, n = 6 per group.

Article Snippet: The drugs used for the study were: rat recombinant FGF21 [R&D systems, UK], wortmannin (PI3K inhibitor), U1026 (MAPK inhibitor), Compound C (AMPK inhibitor), TO-901317 [ROR-α (Retinoic acid receptor-related receptor alpha) inhibitor]; TO-901317 (was initially intended as a therapeutic Liver X receptor (LXR) agonist in dementia , however recently, work by Uebanso T et al, have demonstrated its role in the regulation of FGF21 expression ) and collagenase (type I+protease type XIV).

Techniques: Control, Saline

Journal: PLoS ONE

Article Title: Novel Insights into the Cardio-Protective Effects of FGF21 in Lean and Obese Rat Hearts

doi: 10.1371/journal.pone.0087102

Figure Lengend Snippet: LVDP (mmHg) and left ventricular contractility (dp/dt) in obese control (saline treated) groups [ Fig. 7A1 ], obese (FGF21 treated) groups [ Fig. 7A2 ] and lean (FGF21 treated) groups [ Fig. 7A3 ]; following global ischemia and reperfusion. Graphical representation of the infarcted area (%) in obese control, obese and lean FGF21 (100 nM) treated rat hearts following global ischemia and reperfusion [ Fig. 7B ]. Graphical representation of FGF21 levels in obese and lean FGF21 (100nM) pre-treated rat heart coronary effluents following global ischemia and reperfusion [ Fig 6C ] . Data shown are means ± SEM of triplicates. The values represented are relative to basal. *** P < 0.001 vs. t [0] time point, # P < 0.05 vs. lean at t(5); n = 6 per group.

Article Snippet: The drugs used for the study were: rat recombinant FGF21 [R&D systems, UK], wortmannin (PI3K inhibitor), U1026 (MAPK inhibitor), Compound C (AMPK inhibitor), TO-901317 [ROR-α (Retinoic acid receptor-related receptor alpha) inhibitor]; TO-901317 (was initially intended as a therapeutic Liver X receptor (LXR) agonist in dementia , however recently, work by Uebanso T et al, have demonstrated its role in the regulation of FGF21 expression ) and collagenase (type I+protease type XIV).

Techniques: Control, Saline

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

Journal: PLoS ONE

Article Title: FGF19 Regulates Cell Proliferation, Glucose and Bile Acid Metabolism via FGFR4-Dependent and Independent Pathways

doi: 10.1371/journal.pone.0017868

Figure Lengend Snippet: (A) GAL-Elk1 luciferase assay in rat L6 cells. L6 cells were cotransfected with expression vectors for KLB and the indicated FGFR together with GAL-Elk1, SV40-renilla Luciferase, and Gal-responsive firefly luciferase reporter. Transfected cells were incubated with media containing increasing concentrations of FGF19 (○) or FGF21(▴) for 6 hours before luciferase assays. Transcriptional activation was assessed by the relative firefly luciferase activity normalized by renilla luciferase activity and expressed as relative luciferase unit (RLU). (B) Drawings (to scale) of FGF19 (top), FGF21 (bottom), and various chimeric proteins with amino acid composition at left. Based on the results of repeated GAL-Elk1 assays such as shown in (C), each chimera was classified into class (I), (II) or (III) as indicated at right (see text). Chimeras which did not exhibit an equivalent FGFR1c activity to FGF21 or FGF19 when conditioned medium was used were not shown here. (C) Representative results of GAL-Elk-1 assay for chimeras shown in (B). L6 cells were cotransfected with expression vectors for KLB and/or FGFR as indicated at right. Each FGF construct was expressed in transiently transfected 293 cells and the conditioned medium was used in the assay. The results are shown as a fold induction over control media conditioned with mock transfected cells. (D) Similar to (A). Purified FGF19 (○) and FGF19v (▾), (the construct #4 in (B) and (C)), were tested for FGFR activation in the presence or absence of KLB coexpression as indicated. (E) Solid phase binding assay of FGF19 and FGF19v to FGFR4 fused to Fc fragment was tested as described in the method section. Schematic diagram for the experiments is shown at right. Bold Y indicates antibody against FGF19 (black) or Fc fragment (gray). HRP: horseradish peroxidase. (F) A control ELISA experiment to show that anti-FGF19 antibody used in (E) recognize FGF19 and FGF19v at indistinguishable affinity. Schematic diagram for the experiments is shown at right.

Article Snippet: For some experiments, E. coli derived FGF21 (2539-FG/CF, R&D systems) was used.

Techniques: Luciferase, Expressing, Transfection, Incubation, Activation Assay, Activity Assay, Construct, Control, Purification, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: FGF19 Regulates Cell Proliferation, Glucose and Bile Acid Metabolism via FGFR4-Dependent and Independent Pathways

doi: 10.1371/journal.pone.0017868

Figure Lengend Snippet: 11-week-old ob/ob mice were subcutaneously implanted with an osmotic pump to infuse 1 ng/hr FGF protein (0.4 mg/kg/day) or PBS control (N = 7). (A) Changes in body weight and random fed blood glucose level. The osmotic pump was implanted on day 0. (B) Blood glucose levels at random fed condition and after overnight fast. (C) Serum non-esterified fatty acids (NEFA) levels on day 8. (D) Glucose tolerance test conducted on day 6. Mice were overnight fasted and i.p. injected with 1 g/kg glucose at t = 0. (E) Organ/body weight ratio on day 8. (F) qPCR gene expression profiles on indicated organs. p values: *<0.05, **<0.005, ***<0.0005 (vs PBS control), ##p<0.005 (FGF21 vs FGF19v).

Article Snippet: For some experiments, E. coli derived FGF21 (2539-FG/CF, R&D systems) was used.

Techniques: Control, Injection, Gene Expression

Journal: PLoS ONE

Article Title: FGF19 Regulates Cell Proliferation, Glucose and Bile Acid Metabolism via FGFR4-Dependent and Independent Pathways

doi: 10.1371/journal.pone.0017868

Figure Lengend Snippet: (A) Schematic diagram showing hepatic biosynthetic pathways that convert cholesterol into bile acids. The classical (neutral) pathway (center) is initiated by Cyp7A1, whereas the alternate (acidic) pathway (right) is initiated by Cyp27a1 and Cyp7b1. According to our model, FGF19 suppresses the classical pathway through transcriptional regulation of Cyp7a1 and Cyp8b1, shifting BA synthesis towards production of CDCA or its derivatives. (B) Distinct FGFR/KLB receptor complexes mediate various biological activities of FGF19, FGF19v, and FGF21. In addition to the model depicted, FGF21 and FGF19 can also suppress Cyp7a1 expression acutely in FGFR4-independent manner .

Article Snippet: For some experiments, E. coli derived FGF21 (2539-FG/CF, R&D systems) was used.

Techniques: Expressing

Journal: Journal of Diabetes

Article Title: Renoprotective effects of brown adipose tissue activation in diabetic mice

doi: 10.1111/1753-0407.12938

Figure Lengend Snippet: Effects of CL316,243 on serum adipokines (adiponectin and fibroblast growth factor [FGF] 21) and renal expression of Fgf21 components in diabetic mice. A, Serum adiponectin and B, serum Fgf21 concentrations; C, Fgf21 protein and D, relative expression of β‐klotho ( Klb ) and FGF receptor 1c ( Fgfr1c ) mRNA in renal cortical tissues. Data are the mean ± SEM (n = 6 per group). * P < 0.05, ** P < 0.01, *** P < 0.001 compared with control (non‐diabetic) mice; † P < 0.05, †† P < 0.01 compared with the diabetic control (DM‐Con) group. DM + CL, HFD combined with STZ‐induced diabetic mice treated with CL316,243

Article Snippet: Serum creatinine (Cr) was measured using a commercially available kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and FGF21 protein in the renal cortex was determined by ELISA (Elabscience Biotechnology, Wuhan, China).

Techniques: Expressing, Control, Mouse Assay

Journal: Physiological Reports

Article Title: Identification of FGF21 ‐inducing rare sugars that reduces sugar appetite in male BL /6 mice

doi: 10.14814/phy2.70618

Figure Lengend Snippet: Identification of FGF21‐inducing rare sugars and their effects on blood glucose. (a–d) FGF21 level of mouse primary hepatocytes treated for 24 h with vehicle (distilled water‐negative control) or 25 mM D‐glucose, D‐fructose (positive control), or rare sugars (46 samples). n = 3 mice. (e) Plasma FGF21 levels over 24 h after gastric gavage of vehicle (distilled water), D‐glucose, D‐tagatose, D‐allulose, or D‐sorbitol at 5 g/kg in nine‐week‐old male WT mice with ad libitum access to normal chow (NC) diet. n = 4 mice per group. (f) Area under the curve (AUC) over 24 h of data in (e). n = 4 mice per group. (g) Blood glucose levels of the mice receiving the same dose in (e) of either vehicle (distilled water), D‐glucose, D‐tagatose, D‐allulose, or D‐sorbitol after 16 h fasting. n = 4 mice per group. Statistical analyses were done by one‐way ANOVA for (a–d, f) and repeated measures ANOVA for (e, g), followed by Tukey's HSD test. ** p < 0.01; **** p < 0.0001.

Article Snippet: Mouse FGF21 protein levels in each plasma sample were determined using a Mouse/Rat FGF‐21 Quantikine ELISA Kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Negative Control, Positive Control, Clinical Proteomics

Journal: Physiological Reports

Article Title: Identification of FGF21 ‐inducing rare sugars that reduces sugar appetite in male BL /6 mice

doi: 10.14814/phy2.70618

Figure Lengend Snippet: FGF21‐inducing rare sugars activated oxytocin neurons of paraventricular nucleus of hypothalamus in BL/6 mice. (a–e) Immunostaining of mice brain after gastric gavage with either (a) vehicle (distilled water) or 5 g/kg body weight of D‐glucose (b), D‐tagatose (c), D‐allulose (d), or D‐sorbitol (e). Blue arrow ( ) indicates c‐Fos‐positive oxytocin neurons, shown by dark brown spot on the light brown neurons ( ). Magenta arrow ( ) indicates c‐Fos‐negative oxytocin neurons, shown by light brown anti‐oxytocin staining ( ). n = 4 mice per group. Scale bar represents 100 μ m. (f) Percentage of activated oxytocin neurons (c‐Fos positive oxytocin neurons) from the total of PVH oxytocin neurons in the mice receiving either vehicle (distilled water), D‐glucose, or FGF21‐inducing rare sugars in (a–e). Data are presented as box‐whiskers in (f), where the middle line represents median, bottom/ top edges represent 25th/75th percentile of the data, and whiskers represent maximum and minimum value of the data. Statistical analyses were done by one‐way ANOVA, followed by Dunnett's test.

Article Snippet: Mouse FGF21 protein levels in each plasma sample were determined using a Mouse/Rat FGF‐21 Quantikine ELISA Kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Immunostaining, Staining

Journal: Physiological Reports

Article Title: Identification of FGF21 ‐inducing rare sugars that reduces sugar appetite in male BL /6 mice

doi: 10.14814/phy2.70618

Figure Lengend Snippet: Intragastric administration of FGF21‐inducing rare sugars reduced sucrose preference in BL/6 mice. (a–c) 50 mM sucrose intake in mice receiving 5 g/kg body weight of D‐allulose (a), D‐tagatose (b), or D‐sorbitol (c). (d–f) sucrose preference of the same mice in (a–c). n = 10 mice per group for (a, d) and n = 5 mice per group for (b, c, e, f). Data are presented as box‐whiskers, where the middle line represents the median, bottom/top edges represent the 25th/75th percentile of the data, and whiskers represent the maximum and minimum value of the data. Statistical analyses were done by Student's paired t ‐test between “pre” and “post” of each treatment.

Article Snippet: Mouse FGF21 protein levels in each plasma sample were determined using a Mouse/Rat FGF‐21 Quantikine ELISA Kit (R&D Systems) according to the manufacturer's instructions.

Techniques:

Journal: Physiological Reports

Article Title: Identification of FGF21 ‐inducing rare sugars that reduces sugar appetite in male BL /6 mice

doi: 10.14814/phy2.70618

Figure Lengend Snippet: Mixing FGF21‐inducing rare sugars into sucrose solution reduced solution intake and preference in BL/6 mice. (a–c) Intake of 100 mM (final concentration) sucrose solution that was mixed with either vehicle (distilled water), 600 mM (final concentration) of D‐glucose, or 600 mM (final concentration) of D‐allulose (a), D‐tagatose (b), or D‐sorbitol (c). (d–f) Sucrose preference of the same mice in (a–c). n = 6 mice per group. Data are presented as box‐whiskers, where the middle line represents the median, bottom/top edges represent the 25th/75th percentile of the data, and whiskers represent the maximum and minimum value of the data. Statistical analyses were done by repeated measures ANOVA, followed by Tukey's HSD test.

Article Snippet: Mouse FGF21 protein levels in each plasma sample were determined using a Mouse/Rat FGF‐21 Quantikine ELISA Kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Hepatic FGF21 preserves thermoregulation and cardiovascular function during bacterial inflammation

doi: 10.1084/jem.20202151

Figure Lengend Snippet: Increased circulating FGF21 during bacterial inflammation is hepatic in origin and required for survival. (A) Kaplan–Meier survival curve after 12.5 mg/kg i.p. LPS for WT and Fgf21-Tg mice. n = 10–11/group; pooled data from two independent experiments. (B) WT mice were challenged with 12.5 mg/kg i.p. LPS. Plasma FGF21 levels were measured by ELISA, n = 4–5/time point. Data are from one of two independent experiments with similar results. (C) Relative abundance (RA) of mRNA expression in whole-liver tissue 4 h after PBS vehicle (VEH) or 12.5. mg/kg i.p. LPS challenge in WT mice, shown relative to Rpl13a . n = 5–6/group; pooled data are from two of four independent experiments with similar results. (D) mRNA expression in whole-liver tissue, shown relative to Rpl13a , 18 h after PBS vehicle (Fed), 15 mg/kg i.p. LPS, or fasting in WT mice, n = 4/group; data are from one of six independent experiments with similar results. (E) mRNA expression of whole-liver tissue, shown relative to Rpl13a , 20 h after PBS vehicle or 15 mg/kg i.p. LPS in WT and Ppara −/− mice. n = 3–7/group; data are from one of three independent experiments with similar results. (F) mRNA expression of whole-liver tissue, shown relative to Rpl13a , from ad libitum–fed or 24 h fasted WT and Ppara −/− mice. n = 5–6/group; data are from one of three independent experiments with similar results. (G) Plasma FGF21 levels by ELISA 18 h after PBS vehicle or 15 mg/kg i.p. LPS in WT and Ppara −/− mice. n = 4–5/group; data are from one of two independent experiments. (H) mRNA expression in whole tissue from Fgf21 fl/fl and Fgf21 ΔLiv mice, shown relative to Rpl13a . n = 3–5/group; data are from one of two independent experiments. BAT, brown adipose tissue; eWAT, epididymal white adipose tissue. (I) Plasma FGF21 levels measured by ELISA 20 h after 5 mg/kg i.p. LPS or PBS vehicle in Fgf21 fl/fl and Fgf21 ΔLiv mice. Vehicle n = 4–6/group; LPS n = 8–12/group; pooled data are from three independent experiments. (J) Kaplan–Meier survival curve after 10 mg/kg i.p. LPS for Fgf21 fl/fl and Fgf21 ΔLiv mice. n = 5–6/group; data are from one of three independent experiments with similar results. (K) Kaplan–Meier survival curve after CLP for Fgf21 fl/fl and Fgf21 ΔLiv mice. n = 9–13/group; pooled data are from two independent experiments. (L) CFUs cultured from mouse peritoneal lavage fluid and blood 24 h after CLP, n = 7–9/group; pooled data are from two independent experiments. **, P < 0.01; ***, P < 0.001; ****, P < 0.000, log-rank (Mantel–Cox) test (A, J, and K); two-sided, unpaired t test (C and L); one-way ANOVA with Dunnett's multiple comparisons test (D); two-way ANOVA with Sidak’s multiple comparisons test (E–I). Data are expressed as mean ± SEM.

Article Snippet: Recombinant FGF21 (R&D Systems) was used for FGF21 rescue and signaling experiments.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Hepatic FGF21 preserves thermoregulation and cardiovascular function during bacterial inflammation

doi: 10.1084/jem.20202151

Figure Lengend Snippet: FGF21 deficiency results in decreased body temperature after LPS challenge. (A) Rectal temperatures of Fgf21 fl/fl and Fgf21 ΔLiv mice 24 h after CLP, LPS high dose (10 mg/kg i.p.), and LPS low dose (5 mg/kg i.p.). n = 4–11/group; data are from one of two independent experiments (CLP, LPS high dose), and pooled data are from three independent experiments (LPS low). (B and C) Energy expenditure (EE) and oxygen consumption (VO 2 ) before and after 2.5 mg/kg i.p. LPS challenge in Fgf21 fl/fl and Fgf21 ΔLiv mice. n = 4/group; data are from one of two independent experiments with similar results. (C) Area under the curve (AUC) from B. (D–H) WT and Fgf21 −/− mice were challenged with 12.5 mg/kg i.p. LPS. (D) Blood glucose. n = 5/group; data are from one of four independent experiments with similar results. (E) Plasma free fatty acids (FFAs) by enzymatic assay. n = 5/group; data are from one of two independent experiments with similar results. (F) Plasma adiponectin measured by ELISA. n = 5/group; data are from one of two independent experiments with similar results. (G) Plasma corticosterone measured by ELISA. n = 3–4/group; data are from one of two independent experiments with similar results. (H) Alanine aminotransaminase (ALT) activity measured by enzymatic activity assay and troponin measured by ELISA 24 h after LPS. n = 8–9/group; pooled data are from two independent experiments. (I) Plasma creatinine measured by HPLC 24 h after PBS vehicle (VEH) or 10 mg/kg i.p. LPS in WT and Fgf21 −/− mice, n = 6–9/group; pooled data are from two independent experiments. (J) Plasma lipase and amylase activity measured by enzymatic assay before and after LPS 5 mg/kg i.p. challenge; data are from one of two independent experiments with similar results. (K) mRNA expression in whole-liver tissue 20 h after vehicle or 5 mg/kg i.p. challenge, shown relative to Rpl13a . n = 7–8/group; pooled data are from two independent experiments. Data are expressed as mean ± SEM. *, P < 0.05, **; P < 0.01; ***, P < 0.001; ****, P < 0.0001; two-way ANOVA with Sidak’s multiple comparisons test (A, C–G, and I–K) or unpaired two-sided t test (H).

Article Snippet: Recombinant FGF21 (R&D Systems) was used for FGF21 rescue and signaling experiments.

Techniques: Clinical Proteomics, Enzymatic Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Enzyme Activity Assay, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Hepatic FGF21 preserves thermoregulation and cardiovascular function during bacterial inflammation

doi: 10.1084/jem.20202151

Figure Lengend Snippet: FGF21-deficient mice do not have defects in thyroid hormone axis or differences in lactate levels or blood gases after LPS challenge. (A and B) C57BL/6J (WT) and Fgf21 −/− mice were challenged with LPS 10 mg/kg i.p., n = 9–10/group; pooled data are from two independent experiments. (A) Thyroid-stimulating hormone (TSH) measured by ELISA. (B) Free thyroxine (T4) measured by ELISA. (C) Venous blood lactate measured by the i-STAT1 Handheld Analyzer CG4+ cartridge 24 h after 12.5 mg/kg i.p. LPS in WT and Fgf21 −/− mice; n = 4–5/group. Data are from one of two independent experiments. (D) Respiratory rate and O 2 saturation measured before and after LPS challenge in WT and Fgf21 −/− mice; n = 5/group. Data are from one of two independent experiments with similar results. (E) Venous blood gas measured by the i-STAT 1 Handheld Analyzer CG8+ cartridge 24 h after PBS vehicle (VEH) or 12.5 mg/kg i.p. LPS in WT and Fgf21 −/− mice; n = 3–9/group. Pooled data are from two independent experiments. Unpaired two-sided t test (A–C) or two-way ANOVA with Sidak’s multiple comparisons test (D and E). Data are expressed as mean ± SEM.

Article Snippet: Recombinant FGF21 (R&D Systems) was used for FGF21 rescue and signaling experiments.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Hepatic FGF21 preserves thermoregulation and cardiovascular function during bacterial inflammation

doi: 10.1084/jem.20202151

Figure Lengend Snippet: FGF21 deficiency causes bradycardia during LPS endotoxemia. (A and B) Echocardiography performed on Fgf21 fl/fl and Fgf21 ΔLiv mice before (baseline) and 18 h after 5 mg/kg i.p. LPS. n = 10/group; pooled data are from three independent experiments. (B) Representative echocardiogram windows. (C) Plasma troponin in Fgf21 fl/fl and Fgf21 ΔLiv mice 20 h after PBS vehicle or 5 mg/kg i.p. LPS. Vehicle n = 3/group; LPS n = 10–12/group; pooled data are from three independent experiments. (D) Ambulatory blood pressure and heart rate of Fgf21 fl/fl and Fgf21 ΔLiv mice measured by in vivo telemetry before and after 2 mg/kg i.p. LPS. n = 4/group; pooled data are from three independent experiments. Mean arterial pressure and heart rate measured every minute and shown as mean ± SEM within each group. (E–G) Fgf21 ΔLiv mice were challenged with LPS 5 mg/kg i.p. Recombinant mouse FGF21 10 ng i.v. or PBS vehicle was administered starting at 6 h after LPS and then every 6 h for three doses. n = 9–10/group; pooled data are from two independent experiments. Experimental workflow shown in E. (F) Rectal temperatures and areas under the curve (AUC). (G) Heart rate by echocardiography was performed at 20 h after LPS injection. (H) Klb mRNA expression in whole tissue from WT mice, shown relative to Rpl13a ; inset shows RA of heart Klb after PBS vehicle or LPS treatment. n = 3–4/group; results of one of two independent experiments with similar results are shown. qPCR Ct values are as follows: brown adipose tissue (BAT), 22; white adipose tissue (WAT), 22; liver, 23; aorta, 25; heart, 30 at baseline, 32 after LPS. FB, forebrain; MB, midbrain; HB, hindbrain; Sm Int, small intestine; DRG, dorsal root ganglia. (I) Whole-tissue protein lysates from Klb TdTm mice immunoblotted for RFP (to detect KLB-TdTomato), p-ERK, and total ERK 10 min after 1 mg/kg i.p. recombinant human FGF21 (rhFGF21). The hindbrain region, including AP and NTS, was grossly dissected. Pancreas tissue was included as a positive control. Representative blots from one of three independent experiments are shown. (J) Brains from Klb TdTm mice were harvested 10 min after 1 mg/kg i.p. rhFGF21 treatment. 50-micron fixed brain vibratomed sections were immunostained for RFP and p-ERK. Representative images of the AP from one of two independent experiments are shown. Scale bars represent 20 µm. (K) Brains from Klb TdTm mice were harvested 18 h after 15 mg/kg i.p. LPS or PBS vehicle. 50-micron fixed brain vibratomed sections were immunostained for RFP and cFos. Representative images from one of three independent experiments are shown. PVN, paraventricular nucleus. Scale bars represent 200 µm. *, P < 0.05; ***, P < 0.001; two-way ANOVA with Sidak’s multiple comparisons test (A and C), two-way ANOVA (D), or unpaired two-sided t test (F and G). Data are expressed as mean ± SEM.

Article Snippet: Recombinant FGF21 (R&D Systems) was used for FGF21 rescue and signaling experiments.

Techniques: Clinical Proteomics, In Vivo, Recombinant, Injection, Expressing, Positive Control

Journal: The Journal of Experimental Medicine

Article Title: Hepatic FGF21 preserves thermoregulation and cardiovascular function during bacterial inflammation

doi: 10.1084/jem.20202151

Figure Lengend Snippet: FGF21 deficiency results in depressed heart rate and increased heart rate variability during endotoxemia. Ambulatory blood pressure and heart rate measured by in vivo telemetry before and after 2 mg/kg i.p. LPS, related to . (A) Hourly averages of heart rate and mean arterial pressure after LPS injection in Fgf21 fl/fl vs Fgf21 ΔLiv . Floating bars represent minimum and maximum hourly averages per genotype group. (B and C) Telemetry measurements were recorded every minute. Heart rate differences between each recording are shown. (B) Minute-to-minute HRV before and after LPS injection. (C) Minute-to-minute HRV after LPS injection, with Fgf21 ΔLiv (red curve) graphed in front to show the difference. n = 4/group; pooled data are from three independent experiments. ****, P < 0.0001, two-way ANOVA. Data are expressed as mean ± SEM.

Article Snippet: Recombinant FGF21 (R&D Systems) was used for FGF21 rescue and signaling experiments.

Techniques: In Vivo, Injection

Journal: The Journal of Experimental Medicine

Article Title: Hepatic FGF21 preserves thermoregulation and cardiovascular function during bacterial inflammation

doi: 10.1084/jem.20202151

Figure Lengend Snippet: Effects of FGF21 deficiency on cardiac markers of inflammation, fatty acid oxidation, acute stress response, and cell apoptosis during endotoxemia. Fgf21 fl/fl and Alb-Cre;Fgf21 fl/fl ( Fgf21 ΔLiv ) mice given vehicle or 5 mg/kg i.p. LPS treatment, whole heart tissue harvested 20 h after vehicle or LPS treatment. (A) RA of mRNA expression, shown relative to Rpl13a . Vehicle (Veh), n = 7/group; LPS, n = 14–15/group. mRNA data are pooled from four independent experiments. (B) Whole heart tissue protein lysates immunoblotted for caspase-3. Positive control (+ctrl) generated from Hepa1-6 cells treated with 5 μM staurosporine for 24 h. Representative blot from one of two independent experiments. (C) Quantification of TUNEL-positive cells per 400×-power field. n = 5/vehicle group, n = 6/LPS group; data are pooled from two independent experiments. Data are expressed as mean percentage of TUNEL-positive cells per total cells per high-power field (hpf). (D) Representative images of heart sections from the left ventricle using TACS Blue Labeling to stain TUNEL-positive cells and Nuclear Fast Red heart to stain nuclei. Terminal deoxynucleotidyl transferase enzyme was omitted for the unlabeled negative control. TACS-nuclease treatment was used for the positive control. Scale bars represent 100 µm. Two-way ANOVA with Sidak’s multiple comparisons tests were not statistically significant (A and C). Data are expressed as mean ± SEM.

Article Snippet: Recombinant FGF21 (R&D Systems) was used for FGF21 rescue and signaling experiments.

Techniques: Expressing, Positive Control, Generated, TUNEL Assay, Labeling, Staining, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: Hepatic FGF21 preserves thermoregulation and cardiovascular function during bacterial inflammation

doi: 10.1084/jem.20202151

Figure Lengend Snippet: Primers used for qRT-PCR

Article Snippet: Recombinant FGF21 (R&D Systems) was used for FGF21 rescue and signaling experiments.

Techniques: