Fig 2: Direct effects of norepinephrine (NE) on the expression of GLP-1R (glucagon-like peptide-1 receptor) and neprilysin (in vitro). A, Representative gel and mean protein expressions of GLP-1R and neprilysin after NE incubation in kidney epithelial LLC-PK1 cells. The cells were incubated with NE (0.32–200 ng/mL) for 24 hours. B, Neprilysin activity of kidney LLC-PK1 cells after incubation with NE for 24 hours. *P<0.05 vs control group without NE treatment.

Fig 3: MS—275 stimulates GLP-1R-mediated GSIS and prevents fatty-acid-induced pancreatic beta-cell death.(A) GSEA Blue–Pink O’ gram of the representative genes involved in insulin secretion in control and MS-275-treated pancreatic beta cells. The graph depicting the profile of the running enrichment score (ES) and the position of the representative Gene Set members in the rank order list. NES = Normalized enrichment score, FDR = False Discovery Rate. (B) Effect of MS-275 on GLP-1R-mediated GSIS in BRIN-BD11 pancreatic beta cells. Insulin secretion is reported as ng/mg protein and expressed as fold over the basal secretion. Data are mean (± S.E) of three independent experiments; ***p<0.001 was determined by analysis of variance (ANOVA) using Tukey’s multiple comparison test comparing the effect of MS-275 on GSIS in the presence and absence of 0.1 nM liraglutide. (C) Effect of MS-275 on GLP-1R-induced GSIS mediated by 1 nM liraglutide in cultured rat islets. Results are the mean (± S.E) of three independent experiments; ***p<0.001 was determined by analysis of variance (ANOVA) using Tukey’s multiple comparison test comparing the effect of MS-275 on GSIS in rat islets in the presence and absence of liraglutide. (D) Effect of Rab5A S34N on GLP-1R-induced GSIS mediated by 1 nM liraglutide in MS-275-treated pancreatic beta cells. GSIS was evaluated in the presence of 16.7 mM glucose. Results are mean (± S.E) of three independent experiments; **p<0.01 was determined by analysis of variance (ANOVA) using Tukey’s multiple comparison test. (E) Effect of Bafilomycin on GLP-1R-induced GSIS mediated by 1 nM liraglutide in control and MS-275-treated pancreatic beta cells. Results are mean (± S.E) of three independent experiments; *p<0.05 was determined by the analysis of variance (ANOVA) using Tukey’s multiple comparison test. GSIS was evaluated in the presence of 16.7 mM glucose. (F) String analysis of DEGs related to the cAMP signaling cascade, SNARE, and insulin secretion pathway that is modulated upon MS-275 treatment. Networks in which there are overlaps between pathways based on the co-occurrence of genes are shown. Enrichment score: 1.0e-16. SNARE (GO) red; cAMP pathway (KEGG) blue; insulin secretion pathway (KEGG) green. The white/gray node indicates the second shell of interactors. (G) The effect of MS-275 on the mRNA expression of Syt-8; the quantification being carried out using the 2-??CT method and the data normalized using 18S rRNA as reference. Results are represented as the mean (± SE) of three independent experiments. ***p<0.001 was determined by Welch’s t-test (unpaired) (H) Impact of MS-275 on SNAP 25 protein expression in cultured pancreatic beta cells. The immunoblot, representative of three independent experiments, is quantified using image J and the intensity (arbitrary units) is expressed as a ratio of SNAP 25 and RPL13a that serves as a loading control. Results are represented as the mean (± SE) of three independent experiments. *p<0.05 was determined by Welch’s t-test (unpaired) (I) The effect of MS-275 on the mRNA expression of Ano1; the quantification being carried out using the 2-??CT method and the data normalized using 18S rRNA as reference. Results are represented as the mean (± SE) of three independent experiments. ***p<0.001 was determined by Welch’s t-test (unpaired) (J) The effect of MS-275 on the mRNA expression of Syt-7; the quantification being carried out using the 2-??CT method and the data normalized using 18S rRNA as reference. Results are represented as the mean (± SE) of three independent experiments. ns: non-significant.Figure 4—source data 1.Source Data Figure 4B: The effect of MS-275 on GLP-1R-mediated GSIS in BRIN-BD11 pancreatic beta cells.Figure 4—source data 2.Source Data Figure 4H: Western blot pictures (uncut) showing the impact of MS-275 on SNAP 25 protein expression in cultured pancreatic beta cells; RPL-13a immunoblot was considered as the loading control.

Fig 4: Ex-4 regulates the expression of MSTN through GLP-1R-mediated PKA and AKT signalling pathways. (A) The protein expression level of GLP-1R in the pancreas and TA muscle from WT (Glp1r +/+ Cre -), heterozygous (Glp1r flox/+ Cre +), and homozygous (Glp1r flox/flox Cre +) mice with a representative blot. (B) C2C12 myotubes were pretreated with 20 nM of Ex-9 (GLP-1R antagonist) for 10 min and then were further treated with 20 nM of Ex-4 for 30 min. cAMP production was measured using cAMP ELISA kit. (C–E) The treated C2C12 myotubes were examined for the activation of GLP-1R downstream mediators including PKA and HSF-1 (C) and AKT and NF-?B (D) signals using western blot. (E) The protein level of MSTN with a representative blot. (F–I) GLP-1R siRNA-transfected C2C12 myotubes were treated with 20 nM Ex-4 for 30 min. (F) Representative blot of GLP-1R protein levels. (G–I) C2C12 myotubes were examined for the activation of downstream mediators of GLP-1R, including PKA and HSF-1 (G) and AKT and NF-?B (H) by western blotting. (I) Representative blot of MSTN protein levels. All values are expressed as the mean ± standard error. Significant differences are indicated as **P < 0.01 compared with Con + vehicle. n = 3. Con, control; Ex-4, exendin-4; Ex-9, exendin-9.

Fig 5: GLP-1 attenuated apoptosis of PC12 cells induced by CML. Results in (A) showed that GLP-1 restored the PC12 cells apoptosis induced by CML. (B) Showed the western blotting results of bcl2, bax, GLP-1R and PPAR-?. “a” in (C) showed the down-regulated bcl2, PPAR-? and GLP-1R as well as up-regulated bax in PC12 cells with 50 µg/ml CML, compared with those without CML (all P<0.05). “b” in (C) showed up-regulated bcl2, PPAR-? and GLP-1R as well as down-regulated bax in PC12 cells with 50 µg/ml CML and 100 nM/ml GLP-1, compared with those with 50 µg/ml CML but without GLP-1 (all P<0.05). Data are represented as mean ± SD; n = 3 per group for results of western blotting.