Fig 2: Macrophage Spry4 deficiency activates AMPK pathway through CaMKK2. (A) Spry4FL/FL mice and Spry4MKO mice were subjected to CLP surgery for 24 h, and CaMKK2 phosphorylation was detected using western blot (n = 6). (B) Spry4FL/FL or Spry4MKO BMDMs were stimulated with LPS (100 ng/mL) for 12 h in the presence or absence of STO (5 mmol/L), and AMPK phosphorylation was detected using western blot (n = 6). (C) Quantification of IL-1ß and IL-6 in cell medium (n = 6). (D) Relative ROS level in macrophages as assessed by DCF intensity (n = 6). (E) Quantification of H2O2 and O2- in macrophages (n = 6). Data are expressed as the mean ± SD. *P < 0.05. NS indicates no significance.

Fig 3: Schematic diagram of FGF21-mediated enhancement of endothelium-dependent relaxation.The normal function of vascular endothelial cell is impaired by oxidative stress that is initiated by metabolic disorders. FGF21 binds to FGFRs (i.e., FGFR1) to potentiate CaMKK2 and AMPKα activities. On the one hand, AMPKα upregulates the expressions of CAT, Nrf-2 and HO-1 to abrogate oxidative stress. On the other hand, AMPKα directly enhances the activity of eNOS. The strengthened eNOS activity leads to an increased production of nitric oxide and restoration of endothelium-dependent vasodilatation

Fig 4: RFGF21 induces relaxation of aorta via CaMKK2/ AMPKa mediated activation of eNOS.a Dose-dependent relaxation of aorta from C57BL/6J mice to rFGF21 pretreated with FIIN-4 (10 µM) or Compound C (10 µM) for 30 mins (n = 7–12). b, c Phosphorylation level of eNOS (b) and AMPKa (c) in aortas from C57BL/6 J mice pretreated with FIIN-4 (10 µM) or Compound C (10 µM) for 30 mins and exposed to rFGF21 (0.01 mg/ml) for 3 mins as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 3). d Dose-dependent relaxation of aorta from C57BL/6J mice to rFGF21 pretreated with STO-609 (5 µg/ml) for 30 mins (n = 9–10). e–g Phosphorylation level of eNOS (e), AMPKa (f) and ACC (g) in aortas from C57BL/6J mice pretreated with STO-609 (5 µg/ml) for 30 mins and exposed to rFGF21 (0.01 mg/ml) for 3 mins as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). All data are presented as mean ± SEM. *p < 0.05 vs Control; #p < 0.05 vs FGF21

Fig 5: Muscle-based non-shivering thermogenesis is activated by apelin-AMPK axis. (a) Myogenic cell isolation from male ß-actinCre/Apjflox/+ weanling mice and treated with apelin. (b) Cropped western blots to show Prkaa1 deficiency in the primary myogenic cells (ß-tubulin was used for normalization; n = 3). (c–f) mRNA expression of Sln, Ucp3, Ppargc1a, and Acaa2 in the primary myogenic cells with/without apelin treatment (n = 3). (g) Cropped western blots of SLN, UCP3 and p-CaMKK2 in the primary myogenic cells with/without apelin treatment (ß-tubulin was used for normalization; n = 3). (h) Working model: ME-induced apelin activates AMPK and CaMKK2 phosphorylation, thereby leading to SLN and UCP3-dependent non-shivering thermogenesis. Data are mean ± SEM, and each dot represents one litter; P values by two-way ANOVA followed by post hoc (Tukey's test) analysis (c–g).