Fig. 5: mTORC1 regulates α-cell transcriptional factor MafB.

a Relative expression of glucagon, Arx, and MafB in 2-week-old WT and βRapKO^GFP β-cells by qRT-PCR (n = 4). b Quantification of Gcg⁺GFP⁺, Arx⁺GFP⁺, and MafB⁺GFP⁺ cells in GFP⁺ cells in 2-week-old WT and βRapKO^GFP mice (n = 3, Gcg: p = 0.58; Arx: p = 0.86; MafB: p = 9.00122E-07). At least 1783 GFP⁺ cells were used for quantifications. c Representative immunofluorescent staining for MafB (green) and GFP (red) in 2-week-old WT and βRapKO^GFP mice (n = 3). Yellow box showed the specific area of the islet, which was enlarged and represented by arrows to demonstrate MafB⁺GFP⁺ cell. Scale bars, 20 μm. d Relative expression of selected transcripts associated with α-cell-enriched genes in INS-1 cells which were transfected with SiNC or SiRaptor in the presence or absence of SiMafB by qRT-PCR (n = 4 independent cell experiments, p values included in source data). e, f Western blotting showed the expression of C/EBPβ(LAP) in 8-week-old WT and βRapKO^GFP mice (n = 3, p = 0.049). g, h INS-1 cells were treated with Rapamycin (25 nM), C/EBPβ(LAP) protein expression was assayed by immunoblot (n = 3 independent cell experiments). i Luciferase reporter gene assays revealed that Rapamycin treatment for 12 h positively regulated the luciferase activity of MafB (n = 5 independent cell experiments, p = 0.0011). j Immunoblotting to evaluate the LAP and LIP protein levels after LAP and LIP overexpression (n = 2 independent cell experiments). k Luciferase reporter assay using a rat MafB promoter reporter. INS-1 cells were transfected with GFP or LAP overexpression vector (n = 3 independent cell experiments, p = 0.001). l qRT-PCR analysis of MafB expression in GFP and LAP overexpressed INS-1 cells (n = 4 independent cell experiments, p = 0.0023). m Luciferase reporter assay using a rat MafB promoter reporter. INS-1 cells were transfected with GFP or LIP overexpression vector in the absence and presence of Rapamycin (n = 3 independent cell experiments, GFP + Vehicle vs GFP + Rapamycin, p < 0.001; LIP + Vehicle vs LIP + Rapamycin, p = 0.001). n qRT-PCR analysis of MafB expression in GFP and LIP overexpressed INS-1 cells in the absence or presence of Rapamycin (n = 4 independent cell experiments, GFP + Vehicle vs GFP + Rapamycin, p = 0.002; LIP + Vehicle vs LIP + Rapamycin, p = 0.177). Data represent means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by two-sided Student’s t-test and one-way ANOVA.