Fig. 3: Sam68 enhances glucagon signaling by inhibiting CRTC2 degradation.
a WT and Sam68−/− primary hepatocytes were treated with glucagon (100 nM) for 0−30 min and 1−4 h; then, protein levels of phosphorylated CREB (p-CREB), total CREB, and CRTC2 were evaluated via Western blot. b WT and Sam68−/− hepatocytes were treated with glucagon, forskolin (10 μM), or Bt2-cAMP (100 μM) for 30 min; then, CRTC2 occupancy of the promoters for PGC-1α, PEPCK, and G6Pase was evaluated via chromatin immunoprecipitation (ChIP) assay (n = 3). c–i Sam68LKO and Sam68f/f mice were administered adenoviral vectors coding for GFP or a ubiquitination-defective CRTC2K628R mutant (2 × 109 IFU per mouse). Four days later, blood glucose levels were measured (c) under feeding conditions (Sam68f/f; Ad-GFP, n = 9. Sam68LKO; Ad-GFP, n = 9. Sam68f/f; Ad-CRTC2K628R, n = 8; Sam68LKO; Ad-CRTC2K628R, n = 9), (d) after fasting for 16 h (Sam68f/f; Ad-GFP, n = 9. Sam68LKO; Ad-GFP, n = 9. Sam68f/f; Ad-CRTC2K628R, n = 10; Sam68LKO; Ad-CRTC2K628R, n = 9), and in the (e) pyruvate-tolerance test (PTT; Sam68f/f; Ad-GFP, n = 9. Sam68LKO; Ad-GFP, n = 9. Sam68f/f; Ad-CRTC2K628R, n = 10; Sam68LKO; Ad-CRTC2K628R, n = 11), (f) glucagon-tolerance test (GcTT; Sam68f/f; Ad-GFP, n = 9. Sam68LKO; Ad-GFP, n = 10. Sam68f/f; Ad-CRTC2K628R, n = 9. Sam68LKO; Ad-CRTC2K628R, n = 9), and (g) glucose-tolerance test (GTT; Sam68f/f; Ad-GFP, n = 9. Sam68LKO; Ad-GFP, n = 10. Sam68f/f; Ad-CRTC2K628R, n = 9. Sam68LKO; Ad-CRTC2K628R, n = 9); and (h) mRNA and (i) protein levels of gluconeogenic genes in the liver were analyzed under feeding condition or after fasting for 16 h (h; n = 6. i; n = 3). Data are expressed as mean ± s.e.m.; *p < 0.05, **p < 0.01, ***p < 0.001 (two-way ANOVA). “n” denotes biologically independent samples of primary hepatocytes, peripheral blood, or liver tissues. Source data are provided as a Source Data file.
