FGF-21 stimulates glucose uptake and modulates GLUT1 expression. The values (± SE) shown are the average of at least 3 independent measurements. *P < 0.02, **P < 0.001 compared with no stimulation or vehicle control. FGF-21 dose response on 3T3-L1 (A) and human primary adipocytes (B) in glucose uptake assay. (C) FGF-21 augments insulin activity. Cells were pretreated with or without FGF-21 and then stimulated with insulin as indicated. (D) Cycloheximide diminishes FGF-21 bioactivity in glucose uptake assay. 3T3-L1 adipocytes were stimulated with FGF-21 for 24 hours in the presence or absence of cycloheximide. P < 0.001 at all doses for FGF-21 versus FGF-21 plus cycloheximide stimulations. FGF-21 affects GLUT1 mRNA (E) and protein (F) levels and does not upregulate GLUT4 protein (F) in 3T3-L1 adipocytes (immunoblot). Cells were starved and then stimulated with FGF-21 or vehicle as indicated. Quantitative PCR and immunoblotting analyses were used to measure mRNA and protein levels, respectively. (G) GLUT1 mRNA is upregulated in adipose tissue of FGF-21–injected ob/ob mice. Two groups (5 animals each) of 8-week-old mice were injected s.c. with FGF-21 or vehicle. Quantitative PCR analysis was used to measure mRNA.

FGF-21 stimulates phosphorylation in 3T3-L1 adipocytes. (A) FGF-21 induces phosphorylation of MAPK and FRS-2 in 3T3-L1 adipocytes. Upon stimulation, cells were lysed, and phospho-specific antibodies were used to determine phosphorylation of MAPK and FRS-2 in immunoblots. After immunoblots were stripped, anti-MAPK and anti–FRS-2 antibodies were used to confirm that protein loads were equal. For MAPK experiment, cells were stimulated with FGF-21 for the indicated times. For FRS-2 experiment, cells were stimulated with FGF-21 or FGF-1 (positive control). (B) FGF-21 stimulates tyrosine phosphorylation of FGFR-1 and FGFR-2 in 3T3-L1 adipocytes. Cells were stimulated with FGF-21 and lysed. FGFR-1 and FGFR-2 immunoprecipitates were analyzed in immunoblots with anti-phosphotyrosine antibodies. After stripping, anti–FGFR-1 and anti-FGFR-2 antibodies were used to confirm that protein loads were equal. pErk, phospho-Erk; PY, phosphotyrosine.

FGF-21 does not induce proliferation and does not block FGF-7–dependent mitogenicity on 4MBr5 cells. Cells were stimulated as indicated with different concentrations of FGF-7, FGF-21, and FGF-21 in the presence of a constant concentration of heparin and FGF-21 in the presence of a constant concentration of FGF-7.

FGF-21 injection studies in rodents. The values (± SE) shown are the average of the measurements of at least 5 animals in a group. *P < 0.05, **P < 0.02, and ^#P < 0.001 compared with vehicle control. Fed blood glucose (A) and triglyceride levels (B) in ob/ob mice treated with FGF-21. FGF-21 was administered once daily, and blood glucose and triglyceride levels were measured 1 hour after the last injection. (C) Fed blood glucose levels in db/db mice at days 18 and 46 during 8-week constant-infusion study. Mice were infused s.c. with 11 μg/kg/h FGF-21 using ALZET minipumps. (D and E) FGF-21 lowers glucose in obese ZDF rats and does not induce hypoglycemia in lean ZDF rats. Fed blood glucose levels were measured in obese (D) and lean (E) ZDF rats that were administered s.c. twice daily with FGF-21, Humulin, or vehicle at indicated total daily doses for 1 week. (F) FGF-21 induces extended lowering of fed blood glucose in ob/ob mice. FGF-21 was administered once daily for 7 days, and blood glucose levels were measured after the last injection at indicated time points. (G and H) FGF-21 affects insulin levels (G) and glucose disposal (H) during OGTT in ob/ob mice. At indicated time points, blood samples were obtained for glucose and insulin measurements.

Histological analysis of FGF-21–transgenic and FGF-21–infused animals. (A) H&GE staining of brown fat. Notice an increase in intensity of brown fat in the FGF-21–transgenic mouse compared with the wild-type mouse. (B) H&GE staining of subcutaneous white fat. Notice the smaller adipocytes in the FGF-21 mouse compared with the wild type. (C) H&GE staining of livers from FGF-21–transgenic and wild-type mice. There are no differences between the 2. Magnification, ×200 (A–C). (D) PCNA staining of the livers from FGF-21–infused and saline-treated db/db mice. PNCA immunostaining shows very low proliferation (less than 5%) of hepatocytes (brown staining, arrows) in both the control and treated groups. Magnification, ×400.

FGF-21–transgenic mice are resistant to diet-induced weight gain and fat accumulation. (A) Average cumulative weight gain of FGF-21–transgenic and wild-type mice fed an HFHC diet for 15 weeks. The values (± SE) shown are the average of the measurements of at least 5 animals in a group. P < 0.05 for all time points for male (M) wild-type versus FGF-21–transgenic mice; and for weeks 6–15 for female (F) wild-type versus FGF-21–transgenic mice. (B) Body composition of male FGF-21–transgenic and wild-type mice before and after feeding on an HFHC diet for 15 weeks as determined by nuclear magnetic resonance. P < 0.002 for lean and fat mass of FGF-21–transgenic versus wild-type mice.

FGF-21 inhibits glucagon secretion in isolated rat pancreatic islets. The values (± SE) shown are the average of 6 measurements. *P < 0.05 and **P < 0.02 compared with vehicle controls. Islets were stimulated as indicated with FGF-21 in the presence of different glucose concentrations.