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: Loss of OGT in the Intestinal Epithelium Promotes EEC Development(A and B) Expression of EEC marker genes in the ileum (A) and colon (B) of 2-month-old WT and Vil-Ogt KO male mice (n = 4–10).(C–E) Representative images (left) and quantified numbers (right) of GLP-1+ cells in the ileum (C, 133 and 92 crypt/villus units counted from 5 WT and 4 KO) and colon (D, 176 and 138 crypts counted from 5 WT and 5 KO) and PYY+ cells in the colon (E, 1,005 and 867 crypts counted from 5 WT and 4 KO) of 10-week-old WT and Vil-Ogt KO male mice.(F) Serum GLP-1 levels of WT (n = 9) and Vil-Ogt KO (n = 10) male mice at the age of 8–10 weeks.Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed unpaired Student’s t test.

Fig 4: Intestinal SIRT1 deficiency increases the number of EECs in HFD-fed mice. (A and B) ChgA staining images (green, ChgA+ cells; blue, 4′,6-diamidino-2-phenylindole [DAPI]) and the quantification of ChgA-positive cells in the proximal jejunum of HFD-flox and HFD-VilKO mice (n = 4 per group). (C and D) Immunostained L cells (green, L cells; blue, DAPI) and the quantification of L cells in the ileum of HFD-flox and HFD-VilKO mice (n = 3 per group). (E) Serum GLP-1 levels in HFD-flox and HFD-VilKO mice after oral gavage of glucose (n = 8–9 per group). (F and G) Somatostatin (SST) staining images (green, SST+ cells; blue, DAPI) and the quantification of SST-positive cells in the proximal jejunum of HFD-flox and HFD-VilKO mice (n = 3 per group). (H and I) PYY staining images (green, PYY+ cells; blue, DAPI) and the quantification of PYY-positive cells in the ileum of HFD-flox and HFD-VilKO mice (n = 3 per group). (J and K) ChgA staining images (green, ChgA+ cells; blue, DAPI) and the quantification of ChgA-positive cells in the proximal jejunum of CD-flox and CD-VilKO mice (n = 4 per group). (L) Serum GLP-1 levels in CD-flox and CD-VilKO mice after oral gavage of glucose (n = 9–10 per group). (A, C, F, H, and J) Original magnification, 200×. (A, C, F, H, and J) Scale bar: 50 μm. Values represent the means ± SEM. ∗P < .05, ∗∗P < .01. (B, D, E, G, K, and L) Student t test, and (I) Welch t test.

Fig 5: Gastrin‐SiO2 microspheres indirectly increase the secretion of incretins. A) Serum levels of GLP‐1 (glucagon‐like peptide 1), B) PYY (peptide YY), C) GIP (glucose‐dependent insulinotropic polypeptide) in ND‐Villin‐Cckbr+/+ mice (n = 13) ND‐Villin‐Cckbr−/− mice (n = 19) mice, HFD‐Villin‐Cckbr+/+ mice (n = 12), and HFD‐Villin‐Cckbr−/− (n = 12) mice; D) Expression of L cell marker genes in the small intestine (ileum); E,F) GLP‐1 (marker of L cells) immunofluorescence staining in the small intestine (ileum) in ND‐Villin‐Cckbr+/+ and ND‐Villin‐Cckbr−/− mice cells (GLP‐1, Red; DAPI, Blue); G) Serum levels of GLP‐1, H) PYY, and I) GIP in C57BL/6J mice from the ND (normal diet) group (n = 12), ND+Gas group (n = 12), HFD group (n = 12), and HFD+Gas group (n = 12); J–L) Isolated mouse intestines (entire intestinal tract) were treated at different conditions for 15, 30, 45, and 60 min. The levels of gut hormones (GLP‐1, PYY, and GIP) in supernatants were measured by ELISA, and the final concentration was corrected by isolated intestine protein concentration (pmol/L/prot). As a control, 20 mm mannitol was added to 5 mm glucose containing medium, Control (5 mm glucose and 20 mm mannitol) n = 3, Glucose (25 mm) n = 3, Gastrin (10−9 mm) (n = 3), Glucose (25 mm) + Gastrin (10−9 mm) n = 3; M) Immunofluorescence of GIP (marker of K cells, red), CCK (marker of I cells, red), and GLP‐1/PYY (marker of L cells, red) in the small intestine (ileum). CCKBR was stained green; white arrows mark the co‐localization of the marker proteins with CCKBR; N–P) Cell supernatant media were collected after 1 h treatment under the following conditions (Control, 5 mm glucose and 20 mm mannitol) n = 6; Glucose (25 mm), n = 7; Gastrin (10−9 mm), Glucose (25 mm) + Gastrin (10−9 mm), n = 7; 25 mm glucose+ phlorizin (100 µm), n = 7). The levels of gut hormones (GLP‐1, PYY, and GIP) in the supernatant were measured by ELISA, and the final concentration divided by NCL‐H716 overall protein concentration (pmol/L/prot). All data are expressed as mean ± SEM; unpaired Student's t‐test (F), ND‐Villin‐Cckbr+/+ mice versus ND‐Villin‐Cckbr−/− mice; two way ANOVA, post hoc Scheffe test (A–D, and G–I), ND‐Villin‐Cckbr+/+ mice versus ND‐Villin‐Cckbr−/− mice, HFD‐Villin‐Cckbr−/− mice versus HFD‐Villin‐Cckbr +/+ , ND+Gas group versus ND group, HFD group versus ND group, HFD+Gas group versus HFD group, Glucose (25 mm) group versus Control group, Gastrin (10−9 mm) versus Control group, Glucose (25 mm) + Gastrin (10−9 mm) versus Glucose group, 25 mm glucose+ phlorizin (100 µm) versus Glucose (25 mm); three‐way ANOVA, post hoc Scheffe test (J–L), Glucose (25 mm) group versus Control group, Gastrin (10−9 mm) versus Control group, Glucose (25 mm) +Gastrin (10−9 mm) versus Glucose group, 25 mm glucose+ phlorizin (100 µm) versus Glucose (25 mm), *p < 0.05, **p < 0.01, ***p < 0.001, NS: Not significant, Control group: 60 min versus 15 min, Glucose (25 mm) group: 60 min versus 15 min, Gastrin (10−9 mm) group: 60 min versus 15 min, 25 mm glucose+ Gas (10−9 mm) group: 60 min versus 15 min, NS: Not significant.