Volume 121, Issue 6, Pages 1400-1406 (December 2001) D-glucose releases 5-hydroxytryptamine from human BON cells as a model of enterochromaffin cells  Minsoo Kim, Helen J. Cooke, Najma H. Javed, Hannah V. Carey, Fievos Christofi, Helen E. Raybould  Gastroenterology  Volume 121, Issue 6, Pages 1400-1406 (December 2001) DOI: 10.1053/gast.2001.29567 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of 50 μmol/L brefeldin A (BFA) on basal and 10 μmol/L forskolin-stimulated 5-HT release expressed as percent change from baseline. *P < 0.05 vs. zero control; n = 3. Gastroenterology 2001 121, 1400-1406DOI: (10.1053/gast.2001.29567) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of increasing concentrations of D-glucose and mannitol on 5-HT release expressed as percent change from baseline. (A) Noncloned BON cells. Basal 5-HT release was 0.86 ± 0.07 pmol/well per 20 minutes; n = 5. (B) Subcloned BON cells. Basal 5-HT release was 0.56 ± 0.15 pmol/well per 20 minutes; n = 3–4. *P < 0.05 vs. zero control. Gastroenterology 2001 121, 1400-1406DOI: (10.1053/gast.2001.29567) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Comparison of nonmetabolizable αMG with other stimulants of 5-HT release. GLU, glucose; MAN, mannitol; ISO, isoproterenol. 5-HT release is expressed as percent change from baseline. BON0 cells were treated with 75 mmol/L αMG, GLU, or MAN, and 0.1 mmol/L ISO was used as a positive control. Baseline 5-HT release was 0.73 ± 0.06 pmol/well per 20 minutes; n = 7–9. *P < 0.05 vs. zero controls. Gastroenterology 2001 121, 1400-1406DOI: (10.1053/gast.2001.29567) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of 0.1 mmol/L phloridzin on 75 mmol/L glucose (GLU)– and 75 mmol/L αMG–stimulated 5-HT release expressed as percent change from baseline. Basal.5-HT release was 0.41 ± 0.04 pmol/well per 20 minutes; n = 4–11. *P < 0.05 vehicle vs. phloridzin. Gastroenterology 2001 121, 1400-1406DOI: (10.1053/gast.2001.29567) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Comparison of the effects of 50 mmol/L D-glucose (GLU), 50 mmol/L galactose (GAL), and 50 mmol/L fructose (FRU) on 5-HT release from BON7 cells, expressed as percent change from baseline. Basal 5-HT release was 1.2 ± 0.16 pmol/well per 20 minutes; n = 4–10. *P < 0.05. Gastroenterology 2001 121, 1400-1406DOI: (10.1053/gast.2001.29567) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 cDNA amplified by polymerase chain reaction. Reverse-transcribed cDNA from HEK293 cells was a positive control (HEK). For negative control, water was substituted for the cDNA (H2O). The 208-bp polymerase chain reaction product (arrow) from the BON cell was excised, purified, and sequenced. Gastroenterology 2001 121, 1400-1406DOI: (10.1053/gast.2001.29567) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Western blotting of SGLT1, GLUT2, and GLUT5. Protein preparations from human jejunum and BON cells were solubilized in sample buffer and separated by 10%–12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were transferred to the nitrocellulose membrane. (A) Membrane probing with polyclonal anti-SGLT1 antibody (Ab) indicated the presence of a specific bands of 55–60 kilodaltons in both human jejunum and BON cell preparations. Preabsorption of anti-SGLT1 antibody with immunogenic blocking peptide prevented appearance of the band. (B) Membrane probing with polyclonal anti-GLUT2 and anti-GLUT5 antibodies revealed specific bands of 47–50 kilodaltons and 45 kilodaltons, respectively, in human jejunum and BON cells. Gastroenterology 2001 121, 1400-1406DOI: (10.1053/gast.2001.29567) Copyright © 2001 American Gastroenterological Association Terms and Conditions