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1 TITLE The regulation of K- and L-cell activity by GLUT2 and CasR in rat small intestine AUTHORS Oliver J Mace, Marcus Schindler and Sonal Patel ADDRESS Prosidion Limited, Windrush Court, Watlington Road, Oxford, OX4 6LT, UK RUNNING TITLE Control of K- and L-cell activity by GLUT2 and CasR KEYWORDS Calcium sensing receptor, facilitative glucose transporter 2, gluco-insulinotropic peptide, glucagon-like peptide-1, peptide tyrosine tyrosine

2 Non-technical summary In the intestine, anti-diabetic hormones including gluco-insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) are secreted from gut enteroendocrine cells in response to dietary derived stimuli.

The success of GLP-1 mimetics and the reversal of type II diabetes following bariatric surgery has driven interest in targeting the gut endocrine system to stimulate anti-diabetic hormone secretion. Here we show that the secretion of GIP, GLP-1 and PYY is modulated by two proteins, the facilitative glucose transporter, GLUT2, and the calcium sensing receptor, CasR which can detect glucose and amino acids, respectively. Using rat whole small intestine, we were able to inhibit GIP, GLP-1 and PYY secretion stimulated by glucose, artificial sweetener, dipeptide, fatty acids and bile acids with inhibitors of GLUT2 and GIP, GLP-1 and PYY secretion stimulated by L-amino acids with inhibitors of CasR. We show GLUT2 and CasR are important for nutrient and non-nutrient stimulation of gut hormone secretion and may be of potential therapeutic value.

3 Key points ? In intestine, nutrients including glucose &

amino acids and non-nutrients including bile acids increase secretion of anti-diabetic gut peptides such as GIP, GLP-1 and PYY ? Facilitative glucose transporter pathways in addition to active electrogenic transporter pathways contribute to GIP, GLP-1 and PYY secretion, in particular, GLUT2 is involved ? Sucralose, in the presence of glucose, can strongly and acutely upregulate GIP, GLP-1 and PYY secretion in a time scale of minutes ? Amino acid stimulated GIP, GLP-1 and PYY secretion is acutely regulated by CasR ? The results establish new functions for GLUT2 and CasR as regulators of gut peptide secretion that sense nutrients and provide signaling pathways for the release of GIP, GLP-1 and PYY Key points word count

113 4 Abstract Intestinal enteroendocrine cells (IECs) secrete gut peptides in response to both nutrients and non-nutrients. Glucose and amino-acids both stimulate gut peptide secretion. Our hypothesis was that the facilitative glucose transporter, GLUT2, could act as a glucose sensor and the calcium sensing receptor, CasR, could detect amino acids in the intestine to modify gut peptide secretion. We used isolated loops of rat small intestine to study the secretion of gluco-insulinotropic peptide (GIP), glucagon- like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) secretion stimulated by luminal perfusion of nutrients or bile acid. Inhibition of the sodium-dependent glucose cotransporter

1 (SGLT1) with phloridzin partially inhibited GIP, GLP-1 and PYY secretion by

45 %, suggesting another glucose sensor might be involved in modulating peptide secretion. The response was completely abolished in the presence of the GLUT2 inhibitors, phloretin or cytochalasin B. Given that GLUT2 modified gut peptide secretion stimulated by glucose, we investigated whether it was involved in the secretion of gut peptide by other gut peptide secretagogues. Phloretin completely abolished gut peptide secretion stimulated by artificial sweetener (sucralose), dipeptide (glycylsarcosine), lipid (oleoylethanolamine), short chain fatty acid (propionate) and major rat bile acid (taurocholate) indicating a fundamental position for GLUT2 in the gut peptide secretory mechanism. We investigated how GLUT2 was able to influence gut peptide secretion mediated by a diverse range of stimulators and discovered that GLUT2 affected membrane depolarisation through the closure of K+ ATP-sensitive channels. In the absence of SGLT1 activity (or presence of phloridzin), the secretion of GIP, GLP-1 and PYY was sensitive to K+ ATP- sensitive channel modulators, tolbutamide and diazoxide. L-amino acids phenylalanine (Phe), tryptophan (Trp), asparagine (Asn), arginine (Arg) and glutamine (Gln) also stimulated GIP, GLP-1 and PYY secretion, which was completely abolished when extracellular Ca2+ was absent. The gut peptide response stimulated by the amino-acids was also blocked by the CasR inhibitor Calhex