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: Journal of Biological Chemistry

Article Title: Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

doi: 10.1074/jbc.m112.375907

Figure Lengend Snippet: FIGURE 1. Nonesterified unsaturated fatty acids and chylomicron remnants increase hepatic FGF21 mRNA abundance. A, nutritional regulation of hepatic FGF21 expression in rats. Sprague-Dawley rats were fed a chow diet, a standard purified diet, or an HF-LC ketogenic diet for 7 days. The composition of the diets is described under “Experimental Procedures.” A fourth group of animals was fed the standard purified diet for 6 days and then starved for 30 h. Animals were killed 6 h after the start of the dark cycle, total RNA was isolated from liver, and the abundance of FGF21 mRNA was measured by quantitative real time PCR. Hepatic FGF21 mRNA abundance was also measured in a fifth group of animals fed a standard purified diet for 6 days followed by starvation for 30 h and refeeding for 5 h. The level of FGF21 mRNA in animals fed the chow diet was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of six animals. An asterisk indicates that the mean is significantly (p 0.05) higher compared with that of animals fed the standard (Std) purified diet. B–F, effect of nutrients and hormones on FGF21 expression in primary cultures of rat hepatocytes. Rat hepatocytes were prepared as described under “Experimental Procedures” and incubated in serum-free Medium 199. B, time course of the effect of nonesterified fatty acids (250 M) on FGF21 mRNA abundance. Hepatocytes were incubated with the indicated fatty acids complexed to BSA. Hepatocytes receiving no additions (NA) were incubated in medium containing an equivalent concentration of BSA. The level of FGF21 mRNA in cells incubated with fatty acids for 0 h was set at 1, and the other values were adjustedproportionately.ValuesaremeansS.E.(errorbars)offourexperiments.C,effectofdifferentoleatetreatmentprotocolsonFGF21mRNAabundance. Hepatocytes were incubated with or without oleate for 2 and 6 h without a medium change. A third treatment group (designated 4 2) was incubated with oleate for 6 h with a medium change 2 h prior to the end of the treatment period. The level of FGF21 mRNA in hepatocytes incubated without oleate for 2 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. Different superscript letters indicate that means are significantly (p 0.05) different. D, time course of the effect of GW7647 on FGF21 mRNA abundance. Hepatocytes were incubated with or without GW7647 (1 M) for the indicated time periods. The level of FGF21 mRNA in cells incubated with GW7647 for 0 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated in the absence of GW7647 for the same time period. E, effect of chylomicrons and chylomicron remnants on FGF21 mRNA abundance. Hepatocytes were incubated with chylomicrons and chylomicron remnants prepared from rats fed safflower oil. FGF21 mRNA was measured after 2 h of treatment. The level of FGF21 mRNA in cells incubated with no additions was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of five experiments. The asterisk indicates that the mean is significantly (p 0.05) different. F, effects of glucagon, corticosterone, GW0742, TCPOBOP, glucose, insulin, T3, and leptin on FGF21 mRNA abundance. Rat hepatocytes were incubated in serum-free Medium 199 containing low glucose (5.5 mM) or high glucose (25 mM) with or without glucagon (100 nM), corticosterone (1 M), GW0742 (1 M), TCPOBOP (1 M), insulin (50 nM), T3 (150 nM), or leptin (125 nM). FGF21 mRNA abundance was measured after 6 h of treatment. The level of FGF21 mRNA incubated with 5.5 mM glucose and vehicle was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of three experiments. Different superscript letters indicate that means are significantly (p 0.05) different.

Article Snippet: Goat polyclonal antibodies against mouse FGF21 (AF3057), human FGF21 (TA303289), and albumin (sc-46293) were obtained from R&D Systems, Origene, and Santa Cruz Biotechnology, respectively.

Techniques: Expressing, Purification, Isolation, Real-time Polymerase Chain Reaction, Incubation, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

doi: 10.1074/jbc.m112.375907

Figure Lengend Snippet: FIGURE 3. Bile acids increase hepatic FGF21 secretion and plasma FGF21 concentration. The effect of bile acids on FGF21 accumulation in the culture medium was investigated using primary rat hepatocyte cultures (A) and human HepG2cells(B).Cellswereincubatedwithorwithouttheindicatedbileacids(100 M) in serum-free medium. After 6 and 12 h of treatment, the level of FGF21 and albumin in the culture medium was measured by Western analysis. Top panels, Western analysis of FGF21 from a representative experiment. Bottom panels, sig- nals for FGF21 protein were quantitated. The level of FGF21 protein in the medium of cells incubated with no additions (NA) for 6 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. C, effect of consuming a diet containing cholate on plasma FGF21 concentration. Rats were starved for 24 h and then fed a standard (Std) purifieddietsupplementedwithorwithout1%cholatefor5h.TheplasmaFGF21 concentrationwasmeasuredusinganELISA.Valuesarethemeansofsixanimals. An asterisk indicates that the mean is significantly (p 0.05) different compared withthatofcellsoranimalstreatedwithoutofbileacidforthesametimeperiod. T-MCA, tauro--muricholic acid.

Article Snippet: Goat polyclonal antibodies against mouse FGF21 (AF3057), human FGF21 (TA303289), and albumin (sc-46293) were obtained from R&D Systems, Origene, and Santa Cruz Biotechnology, respectively.

Techniques: Clinical Proteomics, Concentration Assay, Western Blot, Incubation

Journal: Journal of Biological Chemistry

Article Title: Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

doi: 10.1074/jbc.m112.375907

Figure Lengend Snippet: FIGURE 4. FGF19 increases hepatic FGF21 secretion and plasma FGF21 concentration. A and B, effect of FGF19 and GLP-1 on FGF21 mRNA abundance and FGF21 secretion. Primary rat hepatocyte cultures (A) and human HepG2 cells (B) were incubated with or without FGF19 (100 ng/ml) or GLP-1 (100 nM) in serum-free medium for the indicated treatment times. Cells and culture medium were harvested, total RNA was isolated, and FGF21 mRNA abundance and FGF21 protein levels were measured as described under “Experimental Procedures.” Left panels, effect of FGF19 and GLP-1 on FGF21 mRNA abundance. The level of FGF21 mRNA in cells incubated with vehicle for 2 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) offourexperiments.Rightpanels,effectofFGF19andGLP-1onFGF21secretionintotheculturemedium.TheFGF21proteinlevelintheculturemediumofcells incubated with vehicle for 1 (rat hepatocytes) or 2 h (HepG2) was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated with vehicle for the same timeperiod.C,effectofFGF19onplasmaFGF21concentration.MicewereinjectedintravenouslywithvehicleorrecombinantFGF19(0.4mg/kg).Animalswere killed 4 h after injection. Hepatic FGF21 mRNA abundance (left panel) and plasma FGF21 concentration (right panel) were measured. D, interaction between FGF19andCDCAintheregulationofFGF21mRNAabundanceandFGF21secretion.PrimaryrathepatocytecultureswereincubatedwithorwithoutCDCA(100 M), FGF19 (100 ng/ml), or CDCA plus FGF19 for 6 h. The level of FGF21 mRNA (left panel) and the level of FGF21 protein in the culture medium (right panel) of cells incubated with vehicle was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of three experiments. *, mean is significantly (p 0.05) different from that of cells or mice treated with vehicle for the same time period. **, mean is significantly (p 0.05) higher than that of any other treatment of the same time period.

Article Snippet: Goat polyclonal antibodies against mouse FGF21 (AF3057), human FGF21 (TA303289), and albumin (sc-46293) were obtained from R&D Systems, Origene, and Santa Cruz Biotechnology, respectively.

Techniques: Clinical Proteomics, Concentration Assay, Incubation, Isolation, Injection

Journal: Journal of Biological Chemistry

Article Title: Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

doi: 10.1074/jbc.m112.375907

Figure Lengend Snippet: FIGURE 5. Activation of FXR increases hepatic FGF21 mRNA abundance and FGF21 secretion. A and B, effect of the FXR-selective agonist GW4064 on FGF21 mRNA abundance and FGF21 secretion in primary rat hepatocyte cultures (A) and human HepG2 cells (B). Cells were incubated with or without GW4064 (3 M) for the indicated time periods. Cells and culture medium were harvested, total RNA was isolated, and FGF21 mRNA abundance and FGF21 protein levels were measured as described under “Experimental Procedures.” Left panels, FGF21 mRNA abundance in cells incubated with GW4064 for 0 h was set at 1, and the other values were adjusted proportionately. Right panels, the FGF21 protein level in the culture medium of cells incubated with vehicle (DMSO) for 6 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated with vehicle for the same time period. C, effect of GW4064 administration on FGF21 and BSEP mRNA levels in intact mice. Wild-type mice (Fxr/) and FXR knock-out mice (Fxr/) were orally administered GW4064 (30 mg/kg twice a day) or vehicle (2-hydroxypropyl--cyclodextrin) for 7 days, and FGF21 and BSEP mRNA levels were measured in liver. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of animals of the same genotype treated with vehicle.

Article Snippet: Goat polyclonal antibodies against mouse FGF21 (AF3057), human FGF21 (TA303289), and albumin (sc-46293) were obtained from R&D Systems, Origene, and Santa Cruz Biotechnology, respectively.

Techniques: Activation Assay, Incubation, Isolation, Knock-Out

Journal: Journal of Biological Chemistry

Article Title: Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

doi: 10.1074/jbc.m112.375907

Figure Lengend Snippet: FIGURE 6. Identification of an FXRE in the FGF21 gene. A, hepatocytes were transiently transfected with a series of plasmids containing fragments of the rat FGF21 gene linked to the luciferase (Luc) gene as described under “Experimental Procedures.” After transfection, cells were treated with or without GW4064 for 24 h. Cells were harvested, extracts were prepared, and luciferase assays were performed. Left, the constructs used in these experiments. The number at the left of each construct is the 5-end of FGF21 DNA in nucleotides relative to the transcription initiation site. The 3-end of each construct is 68 bp. The location of the FXRE (1222 to 1210 bp) is indicated by a vertical line. A mutation of the FXRE (FXRE Mut) is indicated by an X through the vertical line. Right, luciferase activity of cells transfected with the 2949 to 68 bp FGF21 construct and treated with vehicle was set at 1, and all other activities were adjusted proportion- ately. The -fold stimulation by GW4064 was calculated by dividing the luciferase activity for cells treated with GW4064 by that for cells treated with vehicle. The -fold responses were calculated for individual experiments and then averaged. The results are the means S.E. (error bars) of three experiments. B, the sequence of the rat FGF21 gene between 1231 and 1196 bp. The hexameric half-sites comprising the FXRE are indicated by arrows. The sequence of a mutation of the FXRE (FXRE Mut) is shown underneath. Mutated sequences are boxed. C, gel mobility shift assays were performed using recombinant FXR and/or RXR and an oligonucleotide probe containing the FGF21 FXRE (1231 to 1196 bp). In lanes 4–10, competition analyses were performed with a 2.5-, 5-, and 10-fold molar excess of unlabeled competitor DNA. The sequences of the probe and competitor DNAs are shown in B. In lanes 12 and 14, the receptor preparations were incubated with antibodies against FXR or nuclear factor 1 (NF1) prior to the addition of the probe. Positions of FXR/RXR and supershifted complexes are indicated by arrows.

Article Snippet: Goat polyclonal antibodies against mouse FGF21 (AF3057), human FGF21 (TA303289), and albumin (sc-46293) were obtained from R&D Systems, Origene, and Santa Cruz Biotechnology, respectively.

Techniques: Transfection, Luciferase, Construct, Mutagenesis, Activity Assay, Sequencing, Mobility Shift, Recombinant, Incubation

Journal: Journal of Biological Chemistry

Article Title: Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

doi: 10.1074/jbc.m112.375907

Figure Lengend Snippet: FIGURE7.DeletionofFXRsuppressesthestimulatoryeffectofHF-LCcon- sumption on hepatic FGF21 mRNA abundance and plasma FGF21 con- centration. FXR knock-out mice (Fxr/) and wild-type mice (Fxr/) were fed a standard (Std) purified diet or an HF-LC ketogenic diet for 7 days. A third group was fed a standard purified diet for 6 days and then starved for 24 h. Animals were killed 6 h after the start of the dark cycle, and the FGF21 mRNA abundance (A) and plasma FGF21 concentration (B) were measured. The level of FGF21 mRNA in Fxr/ mice fed the standard purified diet was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of seven animals. The asterisk indicates that the mean is signifi- cantly (p 0.05) lower compared with that of Fxr/ mice fed the HF-LC ketogenic diet. Carb, carbohydrate.

Article Snippet: Goat polyclonal antibodies against mouse FGF21 (AF3057), human FGF21 (TA303289), and albumin (sc-46293) were obtained from R&D Systems, Origene, and Santa Cruz Biotechnology, respectively.

Techniques: Clinical Proteomics, Knock-Out, Purification, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

doi: 10.1074/jbc.m112.375907

Figure Lengend Snippet: FIGURE 8. Proposed model for how consumption of an HF-LC ketogenic diet increases hepatic FGF21 gene expression and secretion. Consumption of an HF-LC ketogenic diet enhances the hepatic delivery of multiple signaling molecules that stimulate FGF21 secretion. These signaling factors include dietary unsaturated fatty acids derived from the hepatic hydrolysis of triacylglycerols in chylomicron remnants and the extrahepatic hydrolysis of triacylglycerols in chylomicrons (i.e. nonesterified fatty acid spillover). HF-LC consumption also increases the enterohepatic circulation of bile acids and intestinal secretion of FGF19. Unsaturated fatty acids and bile acids increase hepatic FGF21 gene transcription and secretion by activating FXR and PPAR, respectively. Unsaturated fatty acids and bile acids may also act through FXR- and PPAR-independent pathways to increase FGF21 gene expression. FGF19 activates fibroblast growth factor receptor 4 (FGFR4)/-Klotho causing an increase in FGF21 secretion via a translational and/or posttranslational mechanism. NEFA, non-esterified fatty acid; LPL, lipoprotein lipase.

Article Snippet: Goat polyclonal antibodies against mouse FGF21 (AF3057), human FGF21 (TA303289), and albumin (sc-46293) were obtained from R&D Systems, Origene, and Santa Cruz Biotechnology, respectively.

Techniques: Gene Expression, Derivative 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: 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