Fig 2: Regulation of L Cells and Protein O-GlcNAcylation by Gut Microbiota and Diet(A) Representative images of O-GlcNAc immunohistochemistry of the colon from ad-libitum-fed, overnight-fasted, and 4-h-refed mice (n = 3).(B) Immunoblotting of global protein O-GlcNAcylation in the colon of mice fed normal chow (NC; n = 4) or a high-fat diet (HFD; n = 5) for 2 months.(C) Expression of the Gcg and Pyy genes in isolated colonic epithelial cells from 2-month-old SPF and GF mice (n = 5).(D) GLP-1 levels in the serum of 2-month-old SPF and GF mice (n = 6), determined by ELISA.(E and F) Representative images (E) and quantification (F) of O-GlcNAc immunostaining of the colon from 2-month-old SPF and GF mice (n = 5).(G and H) Representative images (G) and quantification (H) of O-GlcNAc immunostaining of the colon from 2-month-old mice gavaged with H2O or antibiotics (ABX) for 2 weeks (n = 6).(I) Four-month-old mice were treated with saline or ABX for 1 week, followed by 2 doses (24 h and 4 h before sacrifice) of sodium butyrate (BUT) or SCFAs (3 g/kg body weight). Colon protein was extracted for immunoblotting with an anti-O-GlcNAc antibody.(J) Caco-2 cells were serum-starved overnight, treated with SCFAs (8 µM each) for 24 h, and subjected to O-GlcNAc immunoblotting.(K and L) Ileal organoids were treated with SCFAs and OSMI-1, as indicated, for 2 days. Gcg gene levels (K, n = 5–7) and GLP-1 secretion (L, n = 4–5) were determined.Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed unpaired Student’s t test (C, D, F, and H) or one-way ANOVA with post hoc Dunnett’s (K and L) test.

Fig 3: O-GlcNAcylation Modulates the Transcriptional Activity of FOXO1(A) Transcriptional repressor of Ngn3-luciferase activity by OGT in Caco-2 cells (n = 6). RLU, relative luminescence unit.(B and C) Transcriptional activation of FOXO1 on the insulin-responsive element (IRE) with OGT coexpression (B) or OGA inhibition (C). Luciferase activity was normalized to co-transfected ß-galactosidase (ß-gal) activity in HepG2 cells (n = 4).(D) O-GlcNAcylation levels of WT- and 4A-FOXO1 expressed in 293 cells in the absence or presence of PUGNAc, an OGA inhibitor.(E) Changes in the transcriptional activation activity of FOXO1 on the IRE-luciferase (luc) reporter after co-expression with OGT in HepG2 cells (n = 4).(F) Transcriptional repression of Ngn3-luciferase activity by WT and 4A-FOXO1 in Caco-2 cells (n = 4–5).(G–J) Ileal organoids were treated with or without OSMI-1 in the presence or absence of AS1842856 for 2 days (n = 4). Expression of the Nng3 (G), Gcg (H), and Pyy (I) genes and GLP-1 secretion into the medium (J) were determined. Log2 values of fold change (FC) after OSMI-1 treatment were calculated and plotted.Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed unpaired Student’s t test (A and G–J), one-way ANOVA with post hoc Turkey’s (B and E) or Dunnett’s (F) test, or two-way ANOVA with post hoc Tukey’s test (C).