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: Scientific Reports

Article Title: Fgf21 regulates T-cell development in the neonatal and juvenile thymus

doi: 10.1038/s41598-017-00349-8

Figure Lengend Snippet: Fgf21 mRNA is enriched in mature mTECs. ( A and B ) Expression levels of Fgf21 mRNA in the various tissues from 4-week-old C57BL/6 mice ( A ) and in the thymus of E15.5 and 0-, 4-, 8- and 12-week-old ( B ). Relative Fgf21 mRNA expression was normalised against 18S rRNA expression. ( C and D ) Thymic cell suspensions from 4-week-old C57BL/6 mice were assessed by flow cytometric analysis using anti-CD45 mAb, anti-EpCAM mAb, anti-MHC II (I-A/I-E) mAb, and UEA-1 lectin, and expression levels of Fgf21 mRNA were determined by RT-realtime PCR in the subpopulations of thymic cells from 4-week-old C57BL/6 mice. ( C ) Anti-CD45 and anti-EpCAM staining discriminated CD45 + , TEC (CD45 − EpCAM + ), and non-TEC stromal cell (CD45 − EpCAM − ) subpopulations. ( D ) Gated TECs were subdivided into cTEC hi (UEA-1 − MHC high ), cTEC lo (UEA-1 − MHC low ), mTEC hi (UEA-1 + MHC high ), and mTEC lo (UEA-1 + MHC low ) subpopulations. ( E ) Thymocytes were removed from embryonic thymi by 1.35 mM dGuo treatment for 6 days, and remaining stromal cells were stimulated with 1 μg/ml RANKL to induce the maturation of TECs. Fgf21 and Aire mRNA were increased along with TEC maturation. Graphs represent the mean ± SD; n ≧5 replicates per group from at least 2 independent experiments.

Article Snippet: Medium containing 500 ng/ml of recombinant human FGF21 (R&D Systems) was replaced at 3-day intervals.

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: Fgf21 regulates T-cell development in the neonatal and juvenile thymus

doi: 10.1038/s41598-017-00349-8

Figure Lengend Snippet: CD4SP and CD8SP populations are decreased in Fgf21 KO mice. ( A and B ) Thymi and spleens were isolated from 4-week-old WT and Fgf21 KO mice, and their weights were measured. ( C ) Cell numbers of enzymatically digested suspensions from thymi and spleens were counted. ( D ) Thymocytes and splenocytes from WT and Fgf21 KO mice were stained with anti-CD4 and anti-CD8 mAb. Representative flow cytometry plots show CD4/CD8 analysis of thymocytes and splenocytes from 1-week-old WT and Fgf21 KO mice. ( E ) Charts show the percentage of DN, DP, CD8SP, and CD4SP cells from 1- and 4-week-old WT and Fgf21 KO mice. ( F ) Charts show the percent of CD4SP and CD8SP cells from 1- and 4-week-old WT and Fgf21 KO mice. ( G ) Thymocytes from 1-week-old WT and Fgf21 KO mice were defined by TCRβ and CD69 levels and subdivided into 5 subsets (T1; TCRβ − CD69 − , T2; TCRβ int CD69 − , T3; TCRβ int CD69 + , T4; TCRβ hi CD69 + , and T5; TCRβ hi CD69 − ). ( H ) Charts show the percentage of the 5 subsets from 1-week-old WT and Fgf21 KO mice. ( I ) Gated CD4SP and CD8SP cells from 1-week-old WT and Fgf21 KO mice were defined by CD62L and CD24 levels and subdivided into CD24 + CD62L − immature and CD24 − CD62L + mature SP cells. ( J ) Charts show the percentage of CD24 + CD62L − immature and CD24 − CD62L + mature SP subsets from 1-week-old WT and Fgf21 KO mice. All data shown are the mean ± SD from ≧6 mice per genotype from 2 independent experiments. *P < 0.05, *P < 0.01 versus WT mice.

Article Snippet: Medium containing 500 ng/ml of recombinant human FGF21 (R&D Systems) was replaced at 3-day intervals.

Techniques: Isolation, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Fgf21 regulates T-cell development in the neonatal and juvenile thymus

doi: 10.1038/s41598-017-00349-8

Figure Lengend Snippet: Thymic stromal cells are not obviously altered in Fgf21 KO mice. ( A ) Enzymatically digested thymic cell suspensions were stained with anti-CD45 mAb and anti-EpCAM mAb, and the TEC (CD45 − EpCAM + ) number was counted from the thymi of E14.5, E15.5, E18.5, and 4-week-old WT and Fgf21 KO mice. ( B ) Immunohistochemistry of WT and Fgf21 KO thymi with anti-Keratin-5 antibody for a medullary TEC marker (left panels) and anti-Ly-51 antibody for a cortical TEC marker (right panels). ( C ) The ratio of Keratin-5-positive thymic medullary regions was measured from immunostained sections. ( D ) Gated TECs of 1-weeks old were subdivided into mTEC hi (Ly51 − MHC high ), mTEC lo (Ly51 − MHC low ), cTEC hi (Ly51 + MHC high ), and cTEC lo (Ly + MHC low ) subpopulations. Graphs represent the percentage of subpopulations. ( E ) The mean fluorescence intensity (MFI) of surface MHCII on the TEC was measured by flow cytometry with anti-MHCII antibody. ( F ) Gated CD45 − thymic stromal cells of 1-weeks old were stained with anti-CD140a and anti-CD31 antibodies. Graphs represent the cellularity of CD45 − CD140a + fibroblasts and CD45 − CD31a + endothelial cells in the thymus of WT and Fgf21 KO mice. ( G ) The expression levels of thymic factors were measured in the thymus of 1-week-old WT and Fgf21 KO mice. Graphs represent the mean ± SD; n ≧ 5 replicates per group from at least 3 independent experiments.

Article Snippet: Medium containing 500 ng/ml of recombinant human FGF21 (R&D Systems) was replaced at 3-day intervals.

Techniques: Staining, Immunohistochemistry, Marker, Fluorescence, Flow Cytometry, Expressing

Journal: Scientific Reports

Article Title: Fgf21 regulates T-cell development in the neonatal and juvenile thymus

doi: 10.1038/s41598-017-00349-8

Figure Lengend Snippet: FGF21 treatment alters T-cell development in FTOC. WT and Fgf21 KO foetal thymi were cultured with or without recombinant human FGF21 protein (500 ng/ml) for 14 days. ( A ) Total cell numbers of thymocytes in FTOCs are presented. Flow cytometry analysis of CD4/8 ( B ) and TCRβ/CD69 ( C ) distribution and thymocyte subset number ( D and E ). ( F ) Representative flow cytometry data for each FTOC. ( G ) Numbers of mTEC (CD45 − EpCAM + UEA-1 + ), cTEC (CD45 − EpCAM + UEA-1 − ), and non-TEC stromal cells (CD45 − EpCAM − ) are shown. Data represents the mean ± SD of two separate experiments. *,# P < 0.05, **,## P < 0.01, and *** P < 0.001 versus WT without rhFGF21, $ P < 0.05, and $$ P < 0.01 versus Fgf21 KO without rhFGF21.

Article Snippet: Medium containing 500 ng/ml of recombinant human FGF21 (R&D Systems) was replaced at 3-day intervals.

Techniques: Cell Culture, Recombinant, Flow Cytometry

Journal: Scientific Reports

Article Title: Fgf21 regulates T-cell development in the neonatal and juvenile thymus

doi: 10.1038/s41598-017-00349-8

Figure Lengend Snippet: FGF21 treatment results in increased apoptosis of immature thymocytes. FTOC was performed in the presence or absence of recombinant human FGF21 (500 ng/ml) for 14 days. Annexin V staining was performed to detect apoptotic T cells by flow cytometry. ( A ) Representative histograms show the percentage of Annexin V + cells in gated thymocyte subsets. ( B ) Graph showing the analysis of the percentage of Annexin V + cells in thymocytes from FTOCs. All data shown are the mean ± SD. **P < 0.01 versus control.

Article Snippet: Medium containing 500 ng/ml of recombinant human FGF21 (R&D Systems) was replaced at 3-day intervals.

Techniques: Recombinant, Staining, Flow Cytometry, Control

Journal: Scientific Reports

Article Title: Fgf21 regulates T-cell development in the neonatal and juvenile thymus

doi: 10.1038/s41598-017-00349-8

Figure Lengend Snippet: Fgf21 induced by protein-free diet does not affect thymic change by protein malnutrition. ( A ) Serum Fgf21 levels of 4-week-old C57BL/6 mice fed for 1 week with normal chow diet (NC) or protein-free diet (PF) were determined with ELISA assays. Data shown are the mean ± SD from ≧6 mice. ** P < 0.001 versus NC. ( B ) Relative expression levels of Fgf21 mRNA in the liver, thymus, subcutaneous white adipose tissue (sWAT), and skeletal muscle from mice fed with NC or PF. Data shown are the mean ± SD from ≧6 mice. ** P < 0.01, ** P < 0.001 versus NC. ( C–F ) Body weight ( C ), blood glucose ( D ), thymic weight ( E ), and thymic cell number ( F ) of WT and Fgf21 KO mice fed with NC or PF. Data shown are the mean ± SD from ≧5 mice. * P < 0.05, # P < 0.05 versus WT and Fgf21 KO mice fed with NC, respectively. $ P < 0.05 versus WT mice fed with PF. ( G and H ) Thymocytes from WT and Fgf21 KO mice fed with NC or PF were stained with anti-CD4 and anti-CD8 mAb ( G ), or anti-TCRβ and anti-CD69 mAb ( H ). All data shown are the mean ± SD from ≧5 mice. * P < 0.05, # P < 0.05 versus WT and Fgf21 KO mice fed with NC, respectively. $ P < 0.05 versus WT mice fed with PF.

Article Snippet: Medium containing 500 ng/ml of recombinant human FGF21 (R&D Systems) was replaced at 3-day intervals.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Staining

Journal: Cerebral Cortex (New York, NY)

Article Title: Fibroblast growth factor 21 alleviates diabetes-induced cognitive decline

doi: 10.1093/cercor/bhad502

Figure Lengend Snippet: FGF21 mitigates cognitive deficits in DICD mice. (A) After 1 wk of acclimation, diabetes was induced in the mice by intraperitoneally injecting STZ consecutively for 5 days, and the MWM was conducted to determine the DM mice’s cognitive function at week 12 after STZ treatment. When the DM mice exhibited impaired cognitive functions, 3 μg of FGF21 was administered to the DICD mice through i.c.v administration. After a 2-wk restoration period, [1- 13 C]-glucose was continually injected into the mice through the left jugular veins, and MWM was performed to test the learning/memory abilities of the DICD mice after FGF21 treatment ( n = 6). (B) Escape latency during the 4-day training period of MWM test. (C) A swimming path was used for the probe test on the fifth day. (D) Crossing number at the initial platform position, (E) time, (F) distance in the goal area, and (G) swimming length percentage within the target quadrant. Data are expressed as the mean ± SD. The significance of difference among three groups was analyzed by using ANOVA with a Bonferroni-adjusted test for pairwise comparisons. * P < 0.05, * * P < 0.01, * * * P < 0.001 vs. normal mice; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. DICD mice.

Article Snippet: Recombinant FGF21 (3 μg, Cloud-Clone Corp, Wuhan, China) was dissolved in saline at a dose of 1 μg/μL and i.c.v-injected into the DICD mice by using a microinfusion pump at the rate of 0.2 μL/20 s. Thereafter, the needle was removed with no blood reflux following 2-min dwelling, the burr hole was filled with bone wax, and the skin was sutured.

Techniques: Injection

Journal: Cerebral Cortex (New York, NY)

Article Title: Fibroblast growth factor 21 alleviates diabetes-induced cognitive decline

doi: 10.1093/cercor/bhad502

Figure Lengend Snippet: FGF21 attenuated the neuronal loss in the hippocampi and cortices of the DICD mice. Representative Nissl staining images of (A) hippocampus and (B) cortex tissues of mice and corresponding quantitative results ( n = 3). Bar = 500 μm. Data are expressed as the mean ± SD. The significance of difference among three groups was analyzed by using ANOVA with a Bonferroni-adjusted test for pairwise comparisons. Cornu ammonis area 1, CA1; Cornu ammonis area 3, CA3; dentate gyrus, DG.

Article Snippet: Recombinant FGF21 (3 μg, Cloud-Clone Corp, Wuhan, China) was dissolved in saline at a dose of 1 μg/μL and i.c.v-injected into the DICD mice by using a microinfusion pump at the rate of 0.2 μL/20 s. Thereafter, the needle was removed with no blood reflux following 2-min dwelling, the burr hole was filled with bone wax, and the skin was sutured.

Techniques: Staining

Journal: Cerebral Cortex (New York, NY)

Article Title: Fibroblast growth factor 21 alleviates diabetes-induced cognitive decline

doi: 10.1093/cercor/bhad502

Figure Lengend Snippet: FGF21 alleviated HG-induced SH-SY5Y cell apoptosis. SH-SY5Y cells were treated with 50-mM glucose for indicated time points (1, 2, 3, 4, 5, 6, and 7 days) in the absence or presence of the treatment with 100 ng/mL FGF21 or 5.5 mM glucose as control, and day 4 was selected as further time points. (A) The CCK-8 assay was conducted to measure cell viability ( n = 3). (B, C) Hoechst 33342 nuclear staining was performed to assess cell apoptosis ( n = 3). (D) The western blot assay was conducted to determine the Bax, bcl-2, caspase-3, and cleaved caspase-3 protein levels in brain tissues of the normal, DICD, and FGF21-treated mice ( n = 3). (E) Bax expression was quantified based on bcl-2 expression. (F) Cleaved caspase-3 expression normalized to caspase-3 expression. Bar = 200 μm. Data are expressed as the mean ± SD. The significance of difference among three groups was analyzed by using ANOVA with a Bonferroni-adjusted test for pairwise comparisons. * P < 0.05, * * P < 0.01, * * * P < 0.001 vs. NG group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. HG group.

Article Snippet: Recombinant FGF21 (3 μg, Cloud-Clone Corp, Wuhan, China) was dissolved in saline at a dose of 1 μg/μL and i.c.v-injected into the DICD mice by using a microinfusion pump at the rate of 0.2 μL/20 s. Thereafter, the needle was removed with no blood reflux following 2-min dwelling, the burr hole was filled with bone wax, and the skin was sutured.

Techniques: CCK-8 Assay, Staining, Western Blot, Expressing

Journal: Cerebral Cortex (New York, NY)

Article Title: Fibroblast growth factor 21 alleviates diabetes-induced cognitive decline

doi: 10.1093/cercor/bhad502

Figure Lengend Snippet: Metabolic alterations of brain tissues in the DICD mice following FGF21 treatment. (A) Representative 600 MHz 1 H-NMR spectra from all three groups of mice. (B) The PLS-DA score plot based on brain metabolomics among the Con, DICD, and FGF21-treated mice. (C) OPLS-DA score plot, (D) corresponding loading plot, and (E) −log 10 ( P ) values between the Con and DICD mice. (F) OPLS-DA score plot, (G) corresponding loading plot, and (H) −log 10 ( P ) values between the DICD and FGF21-treated mice. Changes of (I) glucose, (J) lactate, (K) GABA, (L) glutamate, (M) NAA, and (N) aspartate levels in brain tissues among Con, DICD, and FGF21-treated mice. Data are expressed as the mean ± SD. The significance of difference among three groups was analyzed by using ANOVA with a Bonferroni-adjusted test for pairwise comparisons. * P < 0.05, * * P < 0.01, * * * P < 0.001 vs. normal mice; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. DICD mice.

Article Snippet: Recombinant FGF21 (3 μg, Cloud-Clone Corp, Wuhan, China) was dissolved in saline at a dose of 1 μg/μL and i.c.v-injected into the DICD mice by using a microinfusion pump at the rate of 0.2 μL/20 s. Thereafter, the needle was removed with no blood reflux following 2-min dwelling, the burr hole was filled with bone wax, and the skin was sutured.

Techniques:

Journal: Cerebral Cortex (New York, NY)

Article Title: Fibroblast growth factor 21 alleviates diabetes-induced cognitive decline

doi: 10.1093/cercor/bhad502

Figure Lengend Snippet: [1- 13 C]-glucose metabolic fate of brain tissues among Con, DICD, and FGF21-treated mice. Analysis of metabolic pathways by using the KEGG and SMPDB databases. Data are expressed as the mean ± SD. The significance of difference among three groups was analyzed by using ANOVA with a Bonferroni-adjusted test for pairwise comparisons. * P < 0.05, * * P < 0.01, * * * P < 0.001 vs. normal mice; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. DICD mice.

Article Snippet: Recombinant FGF21 (3 μg, Cloud-Clone Corp, Wuhan, China) was dissolved in saline at a dose of 1 μg/μL and i.c.v-injected into the DICD mice by using a microinfusion pump at the rate of 0.2 μL/20 s. Thereafter, the needle was removed with no blood reflux following 2-min dwelling, the burr hole was filled with bone wax, and the skin was sutured.

Techniques:

Journal: Cerebral Cortex (New York, NY)

Article Title: Fibroblast growth factor 21 alleviates diabetes-induced cognitive decline

doi: 10.1093/cercor/bhad502

Figure Lengend Snippet: FGF21 treatment significantly activated DICD-mediated PI3K/AKT/GSK-3β pathway suppression. (A) Western blotting and (B) quantitative analysis of p-GSK-3β, GSK-3β, p-AKT, AKT, and p-PI3K, PI3K expressions in the hippocampus of mice. (C) Western blot and (D) quantitative analysis of p-GSK-3β, GSK-3β, p-AKT, and AKT expressions in the cortex of mice. Data are expressed as the mean ± SD. The significance of difference among three groups was analyzed by using ANOVA with a Bonferroni-adjusted test for pairwise comparisons. * P < 0.05, * * P < 0.01, * * * P < 0.001 vs. normal mice; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. DICD mice.

Article Snippet: Recombinant FGF21 (3 μg, Cloud-Clone Corp, Wuhan, China) was dissolved in saline at a dose of 1 μg/μL and i.c.v-injected into the DICD mice by using a microinfusion pump at the rate of 0.2 μL/20 s. Thereafter, the needle was removed with no blood reflux following 2-min dwelling, the burr hole was filled with bone wax, and the skin was sutured.

Techniques: Western Blot

Journal: Cerebral Cortex (New York, NY)

Article Title: Fibroblast growth factor 21 alleviates diabetes-induced cognitive decline

doi: 10.1093/cercor/bhad502

Figure Lengend Snippet: Possible metabolic mechanism of FGF21 in preventing DICD: FGF21 effectively suppressed diabetes-induced neuronal loss and apoptosis, improved brain glucose homeostasis and neurotransmitter metabolism, and consequently ameliorated cognitive deficits of DICD mice, possibly by activating the PI3K/AKT/GSK-3β signaling pathway. Red arrow indicates changes in DICD, and blue arrow indicates changes after i.c.v FGF21 infusion.

Article Snippet: Recombinant FGF21 (3 μg, Cloud-Clone Corp, Wuhan, China) was dissolved in saline at a dose of 1 μg/μL and i.c.v-injected into the DICD mice by using a microinfusion pump at the rate of 0.2 μL/20 s. Thereafter, the needle was removed with no blood reflux following 2-min dwelling, the burr hole was filled with bone wax, and the skin was sutured.

Techniques:

Journal: Molecular metabolism

Article Title: Fibroblast growth factor 21 has no direct role in regulating fertility in female mice.

doi: 10.1016/j.molmet.2016.05.010

Figure Lengend Snippet: Figure 3: High fat diet reverses the infertility in FGF21-Tg mice. A) Body weight of WT and FGF21-Tg mice fed either with chow or HFD (n ¼ 4e6). B) Body composition as represented as absolute fat and lean mass (n ¼ 6e7). C) Assessment of estrus cycle in FGF21-Tg mice fed either chow or HFD. (1: leukocytes [diestrus/metestrus], 2: cornified cells [estrus], 3: nucleated cells [proestrus]) D) Representative vaginal cytology from FGF21 Tg mice fed with HFD as (i) diestrus, (ii) proestrus, (iii) estrus, (iv) metestrus. Scale: 200 mm (ieiv). (n ¼ 8). E) Ovarian histology from FGF21-Tg mice; (i) chow, (ii) HFD, (iiieiv) HFD fed pregnant females. Corpus lutea (CL) are encircled with dotted lines. Scale: 1000 mm. Statistical significance was evaluated by Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Mouse FGF21 and human FGF21 proteins were measured by ELISA (R & D Systems, MN).

Techniques:

Journal: Molecular metabolism

Article Title: Fibroblast growth factor 21 has no direct role in regulating fertility in female mice.

doi: 10.1016/j.molmet.2016.05.010

Figure Lengend Snippet: Figure 5: Infertility of FGF21-Tg mice is independent of FGF21. Serum levels of total FGF21 (A) and active FGF21 (B) in FGF21-Tg mice fed with chow or HFD (n ¼ 9e10). AVP and Kiss-1 gene expression in SCN (C) and AVPV (D), respectively of WT and FGF21-Tg mice fed chow or HFD (n ¼ 8e10). All mice were in the diestrus phase. Statistical significance was evaluated by Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Mouse FGF21 and human FGF21 proteins were measured by ELISA (R & D Systems, MN).

Techniques: Gene Expression