Fig 2: 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 3: RFGF21 activates CaMKK2 to upregulate AMPKa activity, improve endothelium-dependent relaxation and alleviate oxidative stress in aortas challenged with HG.a Aortas isolated form C57BL/6J mice were pretreated with FIIN-4 (10 µM) for 30 mins and exposed to either HG (30 mM) alone or HG plus rFGF21 (0.01 mg/ml) for an additional 2 h. Phosphorylation level of CaMKK2 as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). b–g Aortas isolated form C57BL/6J mice were pretreated with STO-609 (5 µg/ml) for 30 mins and exposed to either HG (30 mM) alone or HG plus rFGF21 (0.01 mg/ml) for an additional 2 h. b Dose-dependent relaxation to ACh (n = 5). c NO2 and NO3 levels stimulated by ACh (6 × 10-8 M) for 3 mins (n = 5). d Phosphorylation level of eNOS stimulated by ACh (6 × 10-8 M) for 3 mins as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). e Immunofluorescent DHE staining (n = 6). The upper panel shows DHE staining and the lower panel shows quantitation using ImageJ software. Scale bars, 100 µm. (f, g) Phosphorylation level of AMPKa (f) and ACC (g) 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 HG; †p < 0.05 vs HG + FGF21

Fig 4: Maternal exercise long-termly activates muscle thermogenic gene expression impaired due to HFD feeding. One female and one male offspring per dam were weaned onto either a control diet (CD) or high fat diet (HFD) for 8 weeks (M-Ctrl_CD, M-Ctrl_HFD and M-Ex_HFD). (a-b) mRNA levels of Sln (a) and Ucp3 (b) genes in female and male offspring challenged with CD or HFD (n = 6). (c) Cropped western blots of SLN, UCP3 and p-CaMKK2 in female and male offspring challenged with CD or HFD (β-tubulin was used for normalization; n = 6). (d-e) mRNA expression of mitochondrial fatty acid oxidation (d) or lipid metabolism (e)-related genes (n = 6). Data are mean ± SEM, and each dot represents one litter; P values by one way ANOVA followed by post hoc (Tukey's test) analysis (a-e).

Fig 5: Apelin induces muscle-based thermogenesis in fetal muscle. (a) Circulating apelin levels in the fetal serum at E18.5 (n = 6). (b) Cropped western blots of apelin in female and male fetuses from maternal exercise (ME) study (ß-tubulin was used for normalization; n = 6). (c-d) mRNA expression of Sln and Ucp3 in fetal muscle of CD/PBS, OB/PBS, and OB/APN (n = 6). (e–h) Cropped western blots of SLN (F), UCP3 (g) and CaMKK2 phosphorylation (h) in fetal muscle of apelin supplementation study (ß-tubulin was used for normalization; n = 6). (i,j) mRNA expression of mitochondrial biogenic genes in fetal muscle of CD/PBS, OB/PBS and OB/APN (n = 6). (k) Gene expression related to mitochondrial fatty acid oxidation by RNA-seq data in fetal muscle of CD/PBS, OB/PBS, and OB/APN (n = 4). Data are mean ± SEM, and each dot represents one litter; P values by one way ANOVA followed by post hoc (Tukey's test) analysis (a-j).