The regulation of K- and L-cell activity by GLUT2 and the calcium-sensing receptor CasR in rat small intestine

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 231 and augmented by the CasR agonist NPS-R568. GLUT2 and CasR regulate K- and L-cell activity in response to nutrient and non-nutrient stimuli.

Figure 1. SGLT1 and GLUT2 regulate GIP, GLP-1 and PYY secretion

A–F, rat small intestine was perfused with 5 mm glucose in KHB. At 30 min, 25, 50 or 100 mm glucose was introduced for a further 30 min. Samples were analysed for GIP, GLP-1 and PYY. AUC_reactive was calculated for 1st (30–40 min) and 2nd (40–60 min) phases for GIP, GLP-1 and PYY. Significance was determined using Student's unpaired t test vs. 5 mm glucose where *P < 0.05, **P < 0.01 and ***P < 0.001. G–L, rat small intestine was perfused with 5 mm glucose in KHB. At 30 min, the perfusate was switched to one containing 25, 50 or 100 mm glucose (25, 50 or 100 mm) + phloridzin (0.5 mm) for a further 30 min. Samples were analysed for GIP, GLP-1 and PYY. AUC_reactive was calculated for the 1st (30–40 min) and 2nd (40–60 min) phases for GIP, GLP-1 and PYY. Significance was determined using Student's unpaired t test vs. 25 mm glucose + phloridzin where *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 2. SGLT1 and GLUT2 inhibitors reduce GIP, GLP-1 and PYY secretion

A–F, rat small intestine was perfused with glucose (100 mm) + phloridzin (0.5 mm) in KHB. Phloretin (0.5 mm) or cytochalasin B (10 μm) was added at 30 min. Control perfusions with 100 mm glucose were also conducted and phloretin (0.5 mm) added from 30 min. Samples were analysed for GIP, GLP-1 and PYY. G–I, rat small intestine was perfused with glucose (100 mm) in KHB in which Na⁺ was replaced with choline. At t= 30 min, phloretin was added. Samples were analysed for GIP, GLP-1 and PYY. J–L, AUC was calculated for t= 0–30 min and t= 30–60 min for GIP, GLP-1 and PYY. Significance was determined using Student's paired t test vs.. t= 0–30 min where ***P < 0.001.

Figure 3. Gut peptide secretion stimulated by sucralose is blocked by GLUT2 inhibition

A–F, rat small intestine was perfused with glucose (5 mm; •) in KHB. At t= 30 min, sucralose (1 mm) was introduced. In a separate series of experiments rat small intestine was perfused with glucose (5 mm) containing sucralose (1 mm; ○) in KHB. At t= 30 min, sucralose was removed. Samples were analysed for GIP, GLP-1 and PYY. AUC was calculated for t= 0–30 min and t= 30–60 min for GIP, GLP-1 and PYY. Significance was determined using Student's paired t test vs. the control period (t= 0–30 min) where **P < 0.01 and ***P < 0.001. G–L, rat small intestine was perfused with glucose (5 mm) + sucralose (1 mm) in KHB. At t= 30 min, phlorein (0.5 mm; ○) or phloridzin (0.5 mm; •) was added. Samples were analysed for GIP, GLP-1 and PYY. AUC was calculated for t= 0–30 min and t= 30–60 min for GIP, GLP-1 and PYY. Significance was determined using Student's paired t test vs. the control period (t= 0–30 min) where ***P < 0.001.

Figure 4. Gut peptide secretion stimulated by OEA, glycylsarcosine or taurocholate is blocked by inhibition of GLUT2

Rat small intestine was perfused with glucose (5 mm) and OEA (10 μm) + URB597 (10 μm; •), or glycylsarcosine (5 mm) at pH 6.8 (▪) or taurocholate (50 mm; ▴) in KHB. Phloretin (0.5 mm) was added at 30 min. Samples were analysed for GIP, GLP-1 and PYY. AUC was calculated for t= 0–30 min and t= 30–60 min for GIP, GLP-1 and PYY. Significance was determined using Student's paired t test vs. the control period (t= 0–30 min) where ***P < 0.001.

Figure 5. K⁺_ATP-sensitive channels regulate GIP, GLP-1 and PYY secretion

Rat small intestine was perfused with glucose (5 mm) + phloridzin (0.5 mm) in KHB. At t= 30 min, tobutamide (500 μm; •) or diazoxide (340 μm; ▪) was added. Samples were analysed for GIP, GLP-1 and PYY. AUC was calculated for t= 0–30 min and t= 30–60 min for GIP, GLP-1 and PYY. Significance was determined using Student's paired t test vs. t= 0–30 min where *P < 0.05 and ***P < 0.001.

Figure 6. Calhex 231 antagonises gut peptide secretion stimulated by l-amino acids

Rat small intestine was perfused with KHB ± 1.25 mm Ca²⁺. At 30 min, 10 mm Gln (•), Phe (□), Trp (▴), Asn (▿) or Arg (◆) was introduced. At 60 min, Calhex 231 (10 μm) was introduced. Samples were analysed for GIP, GLP-1 and PYY content. The AUC_reactive was calculated for t= 30–60 and 60–90 min periods. Student's paired t tests were used to determine significance between the experimental periods where **P < 0.01 and ***P < 0.001.

Figure 7. NPS R-568 agonism of gut peptide secretion stimulated by l-amino acids

Rat small intestine was perfused with KHB containing KHB ± 1.25 mm Ca²⁺_.At 30 min, 10 mm Gln (•), Phe (▪) or Trp (▴) was introduced. At 60 min, NPS R-568 was added into the perfusate. Samples were analysed for GIP, GLP-1 and PYY content. The AUC for t= 0–30, 30–60 and 60–90 min periods were calculated. Student's paired t tests were used to determine significance between the t= 0–30 and t= 30–60 min where **P < 0.01 and ***P < 0.001, and the t= 30–60 and t= 60–90 min periods where ^§P < 0.05, ^§§P < 0.01 and ^§§§P < 0.001

Figure 8. l-Phe increases the potency of extracellular Ca²⁺-stimulated gut peptide secretion

A, rat small intestine was perfused with KHB deplete of Ca²⁺± 10 mm Phe (+Phe, ○, and –Phe, •). At 20 min, Ca²⁺ was re-introduced cumulatively into the perfusate every 15 min. Samples were analysed for GIP, GLP-1 and PYY content. A–C depict the time courses for the perfusions; D–F show extracellular Ca²⁺–gut peptide response relationships using the calculated AUC following each Ca²⁺ addition. Student's paired t tests were used to determine significance between additions where *P < 0.05, **P < 0.01 and ***P < 0.001. Student's unpaired t tests were used to determine significance between control and l-Phe where ^§P < 0.05, ^§§P < 0.01 and ^§§§P < 0.001

Figure 9. Proposed working model

Absorptive epithelial cells and EECs share common signalling elements initiating gut peptide secretion, namely SGLT1, GLUT2, CasR, GPCRs, voltage-gated Ca²⁺ and K⁺_ATP-sensitive channels. Glucose (glu) can stimulate gut peptide secretion by (1) Na⁺-coupled glucose transport via SGLT1 to directly depolarise the membrane (ΔΨ), opening voltage-gated Ca²⁺ channels or (2) transport of glucose into cells via GLUT2 leads to increased metabolism and closure of K⁺_ATP-sensitive channels located close to the tight junction (TJ). This generates depolarisation of the membrane. A rise in intracellular Ca²⁺ results with gut peptide secretion from EECs.