An FGF21-adiponectin-ceramide axis controls energy expenditure and insulin action in mice.

FGF21 promotes adiponectin secretion and diminishes ceramide accumulation in serum. Results are mean ± SEM Asterisks indicate p< 0.05 for FGF21 effects. Dagger denotes p<0.05 for TNFα effect. (a) Adiponectin from media conditioned for 2 h after L1 adipocytes were cultured for 16 hours with TNFα (10 ng/mL) or BSA control, in the presence of FGF21 (100 ng/mL, grey bars) or PBS (black bars). n=8 from separate experiments (b) Primary murine adipocytes were isolated and equal aliquots were cultured for 4 hours with the TZD rosiglitazone (1 nM) or FGF21 (100 ng/mL). Conditioned media was collected and subjected to SDS-PAGE and western blots were performed with antibodies to Adiponectin (αAdn). Data are representative of 5 independent experiments from separate animals. (c) Serum adiponectin was measured from sera collected at indicated time-points after injection of FGF21 (1 mg/kg, IP) into WT mice. (d) WT mice were injected with indicated doses of human FGF21. Adiponectin was measured by ELISA on serum samples obtained before or after FGF21 treatment, and the change in adiponectin was calculated (n=5–6 per group). (e) HMW (black bars), LMW (grey bars) and trimeric (hatched bars) adiponectin from WT mice, WT mice 2-hours post-FGF21 treatment (1mg/kg, IP), or transgenic (Tg) FGF21 over-expressing mice (n=5/group). (f) Plasma ceramides from WT, FGF21 overexpressing mice, and FGF21 knockout mice (n=5/group). (g) Plasma ceramides from lean (white bars) or DIO mice after treatment with vehicle (black bars), FGF21 (1mg/kg/day, grey bars), or rosiglitazone (10 mg/kg/day, hatched bars) (n=5/group).

Adiponectin critically regulates improvements in glucose homeostasis, but not body weight regulation in DIO mice. Results are mean ± SEM. Asterisks indicate p< 0.05 for FGF21 effects. Dagger denotes p<0.05 for Adn−/− vs. WT of same treatment. Body weight (a) and blood glucose (b) from WT (squares) or adiponectin knockout (triangles) treated with FGF21 (0.3 mg/kg/d, open shapes/dashed lines) or PBS (filled shapes/solid lines). (n=8/group) (c) Average RER during the night from WT and Adn−/− mice treated with PBS (black) or FGF21 (grey). Oxygen consumption (d), and carbon dioxide production (e) from WT (squares) or Adn−/− mice (triangles) treated with FGF21 (0.3 mg/kg/d, open shapes/dashed lines) or PBS (filled shapes/solid lines). (n=8/group) (f) Serum lipid concentrations were determined and expressed as a percentage of the PBS/WT controls [control values: 80.67±8.88 mg/dL Triglyceride (TG); 160.16± 5.49 mg/dL Cholesterol (CHO); 4.20±0.17 mg/dL β-hydroxybutyrate (β-HB); 1.64±0.15 µg/mL ceramide] following treatments with FGF21 (0.3 mg/kg/d, grey) or PBS (black) by mini-pump (n=6 per group).

Adiponectin is essential for enhanced insulin action after acute administration of FGF21. After 6-weeks of high fat diet, WT and Adn−/− mice received chronic indwelling jugular catheters and were allowed to recover to presurgical weight. FGF21 (1µg/kg/min, iv, grey bars) or PBS (black bars) was infused for 90 minutes prior to initiating hyperinsulinemia and throughout the duration of the experiment (n=4–5 per group). (a-b) Hyperinsulinemic euglycemic clamps were performed on conscious unrestrained mice a) The glucose infusion rate required to maintain euglycemia was determined and (b) the rate of glucose efflux from the liver was calculated from kinetic measurements of 3H-glucose turnover. (c) Lipids were extracted from livers obtained from mice following experiments and triglyceride (TG), diacylglycerol (DAG), and ceramide concentrations were quantified and expressed as a percentage of the PBS/WT controls (control values: 108.4±25.3 mg/g TG; 867.7±168.0 nmol/g DAG; 172.0±7.8 nmol/g Ceramide). (d-f) Hyperinsulinemic clamps were initiated for 15 minutes and tissues were snap frozen for analysis of insulin signaling intermediates. (d) Representative samples of 32P-labeled 3,4,5-trisphosphoinositol [PI(3)P] from IRS2-associated PI3 kinase assays (Top), and immunoblots against phosphorylated (serine 473) Akt and total Akt1. (e) Insulin-stimulated activation of IRS2-associated PI3 kinase activity was quantified and graphed as fold stimulation over non-insulin treated samples. (f) Insulin-stimulated phosphorylation of Akt kinase was quantified and normalized to total Akt. Data are graphed as fold stimulation over non-insulin treated samples. Results are mean ± SEM. Asterisks indicate p< 0.05 for FGF21 effects. Dagger denotes p<0.05 for Adn−/− vs. WT of same treatment.

Adiponectin critically regulates improvements in glucose homeostasis in Lepob/ob mice. Results are mean ± SEM. Asterisks indicate p< 0.05 for FGF21 effects. (a) Changes in body weight and (b) blood glucose during infusion of FGF21 (0.3 mg/kg/d, dashed lines, open shapes) or PBS (solid lines, filled shapes) by subcutaneous mini-pumps into Lepob/ob mice (squares) or Lepob/ob -Adn−/− mice (DKO, triangles). (n=4/group) (c-d) Hyperinsulinemic euglycemic clamps were performed during acute infusion of FGF21 (1µg/kg/min, iv, grey bars) or PBS (black bars). (c) The glucose infusion rate required to maintain euglycemia and (d) the rate of glucose efflux from the liver was determined. (n=4/group) (e) Liver ceramide content after 10-day treatment with FGF21 (grey bars) or PBS (black bars).sphingosine-2 (f) Lipids were extracted from livers obtained from mice following acute administration of FGF21 (clamp experiments) and triglyceride (TG), diacylglycerol (DAG), and ceramide concentrations were quantified and expressed as a percentage of the PBS/ Lepob/ob controls (control values: 100.8±2.8 mg/g TG; 1347.9±192.8 nmol/g DAG; 174.6±10.7 nmol/g Ceramide) (n=4/group).