Journal: EBioMedicine

Article Title: Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

doi: 10.1016/j.ebiom.2015.05.028

Figure Lengend Snippet: Selective activation of FGFR1c/KLB complex by bFKB1 (A) A proposed model for FGFR1c–KLB–bispecific Ab (bFKB1) complex formation for signal activation. D1, D2 and D3 denote each of the Immunoglobulin-like domains in FGFR1 extra cellular domain (ECD). KD: kinase domain. (B) A predicted model for FGFR1c–KLB–FGF21 complex formation for signal activation. (C) GAL–ELK1 luciferase assay of human FGF21 and bFKB1 activity using FGFR1 –KO HEK293T cells generated by CRISPR/Cas9-mediated genome editing. Cells were transfected to express indicated receptors. h: human, c: cynomolgus monkey, and m: murine. Mean relative luciferase unit (RLU) ± SEM is shown. (D) GAL–ELK1 luciferase assay in L6 cells. Cells were co-transfected to express indicated receptors. Transfected cells were incubated with various concentrations of bFKB1, hFGF21, hFGF19 or R1MAb1. Mean fold change ± SEM in RLU is shown. (E) Western blot analysis of primary human adipocytes treated with indicated protein (hFGF21 (100 nM) or IgG (33 nM)) for 1 h. Each treatment was performed in duplicate. (F) Expression of UCP1 mRNA in primary human adipocytes treated with indicated protein at 30 nM for 48 h. N = 3. p < 0.005 (**). Data are presented as mean fold over vehicle control ± SEM.

Article Snippet: Antibodies used for western blot analysis were from Cell Signaling Technology: pFRS2a (T196) (#3864), pMEK1/2 (S217/221) (#9154), MEK (#9126), pERK1/2 (T202/204) (#4370), ERK1/2 (#4695), HSP90 (#4874), β-Actin (#5125), from abcam: UCP1 (ab10983), or from R&D Systems: KLB (AF2619).

Techniques: Activation Assay, Luciferase, Activity Assay, Generated, CRISPR, Transfection, Incubation, Western Blot, Expressing, Control

Journal: Molecular Metabolism

Article Title: FGF21 resistance is not mediated by downregulation of beta-klotho expression in white adipose tissue

doi: 10.1016/j.molmet.2017.03.009

Figure Lengend Snippet: Decreased expression of β-klotho in white adipose tissue correlates with elevated levels of circulating FGF21 . (A) Plasma FGF21 protein levels in male WT C57Bl/6 mice ad libitum fed chow or 60% high fat diet (HFD) for 16 weeks (n = 6–7/group). (B–G) Quantitative real-time PCR (QPCR) analysis of Klb and Fgfr1c mRNA expression in epididymal white adipose tissue (eWAT; B, C), liver (D–E), and hypothalamus (F–G) (n = 6–7/group). (H) Western blot analysis of KLB protein expression in eWAT of male WT C57Bl/6 mice ad libitum fed chow or 60% high fat diet (HFD) for 16 weeks. (I) Representative western blots of KLB protein expression in eWAT, brown adipose tissue (BAT), and liver of WT C57Bl/6 mice ad libitum fed 60% high fat diet (HFD) for the indicated time (5–6 mice/group). (J) Plasma FGF21 and insulin levels from the mice in (I). (K) Liver Fgf21 mRNA levels from mice in (I). Values are mean +/− SEM. (*, P < 0.05; **, P < 0.01; ***, P < 0.005; and #, P < 0.001 compared to WT; for (J) significance is relative to time 0).

Article Snippet: Antibody information: β-klotho (R&D Systems, #AF2619), β-actin (Sigma, #A5316), IR-β (Santa Cruz, #sc-711), phospho-FRS2α (Cell Signaling, #3864), phospho-ERK1/2 (Cell Signaling, #9101), total ERK1/2 (Cell Signaling, #9102), phospho-MEK (Cell Signaling, #9121), and total MEK (Cell Signaling, #9122).

Techniques: Expressing, Clinical Proteomics, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Metabolism

Article Title: FGF21 resistance is not mediated by downregulation of beta-klotho expression in white adipose tissue

doi: 10.1016/j.molmet.2017.03.009

Figure Lengend Snippet: Generation of adipose-specific β-klotho transgenic mice . (A) Schematic of inducible β-klotho transgenic construct (inKLB). (B) Western blot analysis of KLB protein expression in epididymal white adipose tissue (eWAT) of 16 week chow-fed and high fat diet (HFD)-fed wild-type (WT) mice compared to HFD fed WT and adipose-specific β-klotho transgenic (KLB AdipoTG) mice. QPCR analysis of Klb and Tdtomato mRNA expression in eWAT (C, D), inguinal white adipose tissue (iWAT; E, F), brown adipose tissue (BAT; G, H), and liver (I, J) of WT and KLB AdipoTG mice fed chow or 60% HFD for 12 weeks (n = 7–11/group). (K) Western blot analysis of cytoplasmic and membrane KLB protein expression in epididymal white adipose tissue of wild-type (WT) and KLB adipose-specific transgenic mice (KLB AdipoTG). Values are mean +/− SEM. (*, P < 0.05; ***, P < 0.005; and #, P < 0.001 compared to WT; nd = not detected).

Article Snippet: Antibody information: β-klotho (R&D Systems, #AF2619), β-actin (Sigma, #A5316), IR-β (Santa Cruz, #sc-711), phospho-FRS2α (Cell Signaling, #3864), phospho-ERK1/2 (Cell Signaling, #9101), total ERK1/2 (Cell Signaling, #9102), phospho-MEK (Cell Signaling, #9121), and total MEK (Cell Signaling, #9122).

Techniques: Transgenic Assay, Construct, Western Blot, Expressing, Membrane

Journal: Molecular Metabolism

Article Title: FGF21 resistance is not mediated by downregulation of beta-klotho expression in white adipose tissue

doi: 10.1016/j.molmet.2017.03.009

Figure Lengend Snippet: Maintenance of β-klotho expression in white adipose tissue does not improve metabolic profiles . (A) Body weight curves and (B) body composition of male WT and KLB AdipoTG mice on HFD for 12 weeks (n = 8–10/group). (C) Glucose tolerance (GTTs) and (D) insulin tolerance tests (ITTs) of lean, male WT and KLB AdipoTG mice (n = 6/group). (E) GTTs and (F) ITTs of diet-induced obese (DIO), male WT and KLB AdipoTG mice on HFD for 12 and 14 weeks, respectively (n = 6/group). (G) Hepatic triglyceride levels in DIO WT and KLB AdipoTG mice. Values are mean +/− SEM. (*, P < 0.05; ***, P < 0.005; and #, P < 0.001 compared to WT).

Article Snippet: Antibody information: β-klotho (R&D Systems, #AF2619), β-actin (Sigma, #A5316), IR-β (Santa Cruz, #sc-711), phospho-FRS2α (Cell Signaling, #3864), phospho-ERK1/2 (Cell Signaling, #9101), total ERK1/2 (Cell Signaling, #9102), phospho-MEK (Cell Signaling, #9121), and total MEK (Cell Signaling, #9122).

Techniques: Expressing

Journal: Molecular Metabolism

Article Title: FGF21 resistance is not mediated by downregulation of beta-klotho expression in white adipose tissue

doi: 10.1016/j.molmet.2017.03.009

Figure Lengend Snippet: White adipose tissue and systemic FGF21 sensitivity is not improved by preservation of β-klotho expression . (A) Plasma FGF21 and (B) hepatic Fgf21 mRNA levels in WT and KLB AdipoTG mice fed chow or HFD for 14 weeks (n = 8–9/group). (C) Western blot analysis of phospho-ERK1/2, total ERK1/2, phospho-FRS2α, phospho-MEK, and total MEK from eWAT of chow and HFD fed WT and KLB AdipoTG mice administered either vehicle or FGF21 (0.1 mg/kg) for 15 min. Values are mean +/− SEM. (#, P < 0.001 compared to WT).

Article Snippet: Antibody information: β-klotho (R&D Systems, #AF2619), β-actin (Sigma, #A5316), IR-β (Santa Cruz, #sc-711), phospho-FRS2α (Cell Signaling, #3864), phospho-ERK1/2 (Cell Signaling, #9101), total ERK1/2 (Cell Signaling, #9102), phospho-MEK (Cell Signaling, #9121), and total MEK (Cell Signaling, #9122).

Techniques: Preserving, Expressing, Clinical Proteomics, Western Blot