International Journal of Obesity (2014) 38, 1365–1373 © 2014 Macmillan Publishers Limited All rights reserved 0307-0565/14 www.nature.com/ijo
ORIGINAL ARTICLE
Gastrointestinal hormonal responses on GPR119 activation in lean and diseased rodent models of type 2 diabetes S Patel1,3,9, OJ Mace1,4,9, IR Tough2,9, J White1,5, T-A Cock1,6, U Warpman Berglund1,7, M Schindler1,8 and HM Cox2 BACKGROUND: G protein-coupled receptor 119 (GPR119) has emerged as a potential target for the treatment of type 2 diabetes (T2D) and tool compounds have been critical in the evaluation of GPR119 functions. METHODS: We synthesised a novel small-molecule GPR119 agonist, PSN-GPR119, to study GPR119 signalling activities in cells overexpressing GPR119. We measured GPR119-stimulated peptide hormone release from intestinal loops and oral glucose tolerance in vivo from lean (C57BL/6J mouse or Sprague-Dawley (SD) rat) and diabetic (ob/ob mouse or ZDF rat) models. To evaluate the direct effects of GPR119 agonism on gastrointestinal (GI) tissue, we measured vectorial ion transport (measured as ISC; short-circuit current) across rodent GI mucosae and from normal human colon specimens. RESULTS: GPR119 activation by PSN-GPR119 increased cAMP accumulation in hGPR119-overexpressing HEK293 cells (EC50, 5.5 nM), stimulated glucagon-like peptide 1 (GLP-1) release from GLUTag cells (EC50, 75 nM) and insulin release from HIT-15 cells (EC50, 90 nM). In vivo, PSN-GPR119 improved glucose tolerance by ~50% in lean mice or rats and ~60% in the diabetic ob/ob mouse or ZDF rat models. Luminal addition of PSN-GPR119 to isolated loops of lean rat small intestine stimulated GLP-1, glucose insulinotropic peptide (GIP) and peptide YY (PYY) release under basal (5 mM) and high glucose (25 mM) conditions. Activation of GPR119 also reduced intestinal ion transport. Apical or basolateral PSN-GPR119 addition (1 μM) to lean or T2D rodent colon mucosae reduced ISC levels via PYY-mediated Y1 receptor agonism. The GPR119 response was glucose sensitive and was abolished by Y1 receptor antagonism. Similarly, in human colon, mucosa PSN-GPR119 acted via a Y1-specific mechanism. CONCLUSIONS: Our results show that functional GPR119 responses are similar in lean and diabetic rodent, and human colon; that GPR119 stimulation can result in glucose lowering through release of intestinal peptide hormones and that PSN-GPR119 is a useful tool compound for future studies. International Journal of Obesity (2014) 38, 1365–1373; doi:10.1038/ijo.2014.10 Keywords: GPR119; intestinal responses; incretins; peptide YY and Y1 receptors; glucagon-like peptide 1; type 2 diabetes rodent models
INTRODUCTION New improved anti-diabetic therapies would be advantageous to address the increasing epidemic of type 2 diabetes (T2D) and obesity. G protein-coupled receptor 119 (GPR119) is primarily expressed in gastrointestinal (GI) enteroendocrine cells and pancreatic β cells1–3 in rodents and humans, attracting significant interest as a therapeutic target for T2D. GPR119 agonists in vivo have shown impressive lowering of postprandial glucose in rodent models of T2D3–5 and GPR119 agonists have also demonstrated beneficial effects on body weight,2 offering potential advantages to obese T2D patients over therapeutics already in the market. As such, new GPR119 molecules have advanced into the clinic for the treatment of T2D.6,7 The ability of GPR119 agonism to improve glucose tolerance and reduce body weight shows that GPR119 biology warrants further investigation. GPR119 agonists have direct effects on
insulin-secreting cell lines and on rodent islets, which have been characterised extensively.5,8–10 Importantly, insulin secretion mediated by GPR119 is glucose dependent and agonism will not cause the hypoglycaemia associated with other diabetic agents.11,12 Furthermore, GPR119 expression is significantly increased in the islets of obese diabetic db/db mice when compared with islets from lean models13 adding credence to the potential therapeutic value of this receptor. In addition, GPR119 agonists have greater oral efficacy as compared with intravenous efficacy, suggesting that GPR119 may control glycaemia via additional incretin-based mechanisms.12 The secretion of endogenous anti-diabetic hormones, including glucose insulinotropic peptide (GIP) and glucagon-like peptide 1 (GLP-1), is a consequence of GPR119 agonism in vivo.9,14 In purified enteroendocrine cells and isolated murine intestinal L cells, GLP-1 secretion is mediated by signalling pathways
1 Prosidion Ltd, Oxford, UK and 2King's College London, Wolfson Centre for Age-Related Diseases, London, UK. Correspondence: Professor HM Cox, King’s College London, Wolfson Centre for Age-Related Diseases, Hodgkin Building, Guy’s Campus, Newcomen Street, London SE1 1UL, UK. E-mail: [email protected] 3 Current address: Bioelectronics R&D, GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY, UK. 4 Current address: Heptares Therapeutics, Broadwater Road, Welwyn Garden City, AL7 3AX, UK 5 Current address: Argenta Discovery, 8/9 Spire Green Centre, Flex Meadow, Harlow, CM19 5TR, UK 6 Current address: Pharmaxis Ltd, 20 Rodborough Road, Locked Bag 5015, Frenchs Forest, NSW 2086, Australia 7 Current address: Science for Life Laboratory, Department of Medical Biochemistry & Biophysics, Karolinska Institute, S-171 21 Stockholm, Sweden 8 Current address: Astra Zeneca R&D Mölndal, Cardiovascular & Metabolic Diseases iMed, Pepparedsleden 1, SE-431 83 Mölndal, Sweden 9 These authors contributed equally to this work. Received 3 May 2013; revised 7 January 2014; accepted 14 January 2014; accepted article preview online 23 January 2014; advance online publication, 18 February 2014
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involving L- and Q-type calcium channels.15 Additionally, rat ileum treated in situ with an endogenous ligand for GPR119, oleoylethanolamide (OEA) induced GLP-1 release.12 In humans, activation of GPR119 by administration of a jejunal bolus of 2-oleoyl glycerol (a component of fatty acid digestion with affinity for GPR119) stimulated GLP-1 release,16 implicating GPR119 as a fat sensor. The L-cell-derived hormone, peptide YY (PYY), is predominantly expressed in the ileum and the colon,17 and circulating PYY increases or decreases after feeding or fasting, respectively.18 PYY activates Y1 and Y2 receptors to inhibit upper GI motility19 and inhibits water and electrolyte secretion in human colon,20,21 and its product, PYY(3–36) appears to reduce appetite.22 In support of the anti-secretory role of PYY in the colon, activation of GPR119 mechanisms results in anti-secretory effects via endogenous PYY-activating epithelial Y1 receptors.23 Postprandial blood glucose was also controlled by PYY, and both responses were absent from PYY-/- mice or their tissues.23 We synthesised PSN-GPR119 (N-(2-Hydroxyethyl)-4-{3-[1-(3isopropyl-[1,2,4] oxadiazol-5-yl)piperidin-4-yl]propoxy}-2-methylbenzamide (patent number, WO 2008/081205 A1), a potent small-molecule GPR119 agonist, and validated its potential to improve glucose tolerance in rodent models of diabetes and obesity. Our hypothesis was that GPR119 signalling involved secretion of K-cell- and L-cell-derived hormones and that the mechanisms involved were consistent in lean and diseased rodent models of diabetes and obesity. In order to define the GPR119 mechanisms in GI mucosa, we exploited the fact that PYY inhibits epithelial ion secretion in mammalian intestine and compared the downstream PYY-mediated pathways of PSN-GPR119. We extended these mechanistic studies to assess PSN-GPR119 activity in human colonic mucosal specimens.
MATERIALS AND METHODS Ethical approval Animal procedures were approved by the UK Home Office and undertaken in accordance with the Animals (Scientific Procedures) Act (1986). Human colon specimens were obtained from consenting patients undergoing bowel resection surgery with the approval of Guy’s and St Thomas’ NHS Foundation Trust, in accordance with The Human Tissue Act (2004).
GPR119-transfected HEK293 cell cAMP assay Stimulated cAMP accumulation was measured using an engineered HEK293 cell line expressing GPR119 as previously described.24 Monolayers were exposed to PSN-GPR119 (0.01–100 nM) for 30 min at 37 °C in buffer containing 1% dimethyl sulphoxide. Cells were lysed and cAMP was determined (AlphaScreen cAMP kit, Perkin Elmer, Cambridge, UK).
Measurement of GLP-1 secretion from GLUTag cells GLUTag cells were cultured in Dulbecco's modified Eagle medium containing 5.5 mM glucose. Cells were seeded onto 24-well plates coated with Matrigel (BD Biosciences, Oxford, UK) and used at 60–80% confluency. They were washed twice in nutrient-free bathing solution supplemented with 0.1 mM diprotin A and 0.1 % (w/v) BSA and incubated with PSNGPR119 (0.1 nM–3 μM) for 2 h at 37 °C. Media was assayed for GLP-1 using an active GLP-1 ELISA (Linco Research Inc., St Charles, MO, USA).
Measurement of insulin secretion from HIT-T15 cells HIT-T15 cells were cultured in RPMI1640 with 10% . Cells were seeded in 96-well plates and cultured for 24 h before assay. Cells were pre-incubated for 30 min in Krebs-Ringer-HEPES buffer (119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3, 1.2 mM KH2PO4, 10 mM HEPES) with 0.1% BSA. PSN-GPR119 was added to buffer supplemented with 5.6 mM glucose and incubated for 30 min. Media was assayed for insulin by ELISA (Mercodia, Uppsala, Sweden). International Journal of Obesity (2014) 1365 – 1373
Oral glucose tolerance test (OGTT) Animals were fed ab libitum and had free access to water and were maintained on a 12-h light:dark cycle. Oral glucose tolerance was assessed in overnight-fasted lean Sprague-Dawley (SD) rats (12 weeks old, n = 8), lean C57BL/6J mice (11–12 weeks old, n = 10), Zucker diabetic fatty (ZDF) rats (12 weeks old, n = 8) or obese ob/ob (leptin-deficient obese, B6.V-Lepob/J) mice (11–12 weeks old, n = 10) following acute treatment of PSN-GPR119. PSNGPR119 (10 or 30 mg kg−1) or vehicle (20% 2-hydroxy propyl-β-cyclodextrin) was administered by oral gavage 60 min (t = -60 min) before the glucose bolus that was delivered by oral gavage (2 g kg−1; t = 0 min). Blood samples (t = 0–250 min after glucose) were analysed using a commercially available glucose oxidase assay. Plasma glucose is presented as absolute values (mM) and reactive area under the curve (AUCreactive) was calculated by subtraction of the baseline AUC before glucose.
GPR119 expression in intestinal regions As GPR119 transcript expression is very low (L cells represent o 1% of the total mucosal cell population) intestinal RNA samples were pooled from five human male donors (BioChain, Newark, CA, USA), whereas individual samples from six male SD rats and three male C57BL/6J mice were analysed separately. Real-time PCR was performed using pre-validated primer / FAM-labelled GPR119 probe sets (Applied Biosystems, Paisley, UK) corresponding to GenBank accessions NM_178471.2 (human), NM_181751.2 (mouse) and BD274917.1/ Celera rCT63637.0 (rat). Data were normalised to the housekeeping genes, hypoxanthine phosphoribosyl transferase I and hydroxyl-methylbilane synthase, calculated using ΔCt.
Isolated rat intestinal perfusion and measurement of GIP, GLP-1 and PYY levels Male SD rats (250 g; Charles River, Margate, UK) were fed ad libitum, had free access to water and were maintained on a 12-h light:dark cycle. Loops of whole small intestine were isolated and perfused in single-pass mode as previously described.25 Briefly, the first 30 min was a control period during which Krebs–Henseleit (KH; in mM: 120 NaCl, 4.5 KCl, 1 MgSO4,1.8 Na2HPO4, 0.2 NaH2PO4, 25 NaHCO3, 1.25 CaCl2 and 5 D-glucose) was perfused at a flow rate of 1.6 ml min−1. After 30 min, the loop was perfused with a second experimental perfusate (identical to the first except for the addition of PSN-GPR119). During this period, loops were either perfused with KH containing PSN-GPR119 (0.1–100 μM) or 5 mM glucose (osmotically balanced to 25 mM sugar with mannitol) for 30 min, then switched to an identical perfusate containing PSN-GPR119 and 25 mM glucose. Serosal samples were assayed for glucose, total GIP (tGIP; Millipore Ltd, Oxford, UK), active GLP-1 (aGLP-1; Epitope Diagnostics Inc, San Diego, CA, USA) and total PYY (tPYY; Phoenix Pharmaceuticals, Burlingame, CA, USA) using commercially available ELISAs, as previously described.25 Tissue viability was indicated by maintenance of active glucose transport throughout the experiment. AUCreactive was calculated from time-courses in pg ml−1 (g dry weight)−1 × min and presented as mean ± s.e.m. (n ⩾ 4).
Mucosal electrophysiology Human colonic specimens from nine consenting patients undergoing intestinal resection for primary malignancy, or GI regions from rodents (Charles River; lean SD rats or C57BL/6J mice and diseased ZDF rats or ob/ ob mice) were stripped of smooth muscle and adjacent pieces of mucosa were bathed in KH buffer, mounted on Ussing chambers and voltageclamped at 0 mV, as described in detail previously.21,23,26 Electrogenic ion transport was measured as short-circuit current (ISC; in μA cm−2). PSN-GPR119 was added to the apical (for potency measurement) or basolateral reservoir, whereas peptides were added basolaterally. A concentration of 1 μM PSN-GPR119 gave near-maximal responses and was used to stimulate other GI areas. In mouse mucosa, vasoactive intestinal polypeptide (VIP, 10 nM) was used to pre-stimulate anion secretion, to optimise subsequent Gαi-coupled responses.23,26 PYY (10 nM) was a control activator of Y1 receptor-mediated responses through Gαi-coupled reductions in epithelial cAMP and consequent attenuation of Cl- secretion.27,28 Y1 responses were blocked with BIBO3304 ((R)-N-[[4(aminocarbonylamino methyl)-phenyl]methyl]-N2-(diphenylacetyl)-argininamide trifluoroacetate; 300 nM) and endogenous GLP-1 effects were abolished with 1 μM exendin(9–39). Mucosae were treated with both antagonists to block both endogenous peptide responses. The glucose dependence of agonist responses was tested using KH buffer (including 11.1 mM glucose) or buffer containing mannitol (11.1 mM). The Na+-glucose © 2014 Macmillan Publishers Limited
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Figure 1. PSN-GPR119 significantly improves oral glucose tolerance in lean C57BL/6J mice and SD rats, compared with ob/ob mice and ZDF rat models. Left time-courses depict dosing of PSN-GPR119 (either 10 or 30 mg kg−1, p.o.) at t = −60 min and glucose (3 g kg−1) at t = 0 min, and right glucose AUC reactive are calculated by subtracting the baseline glucose AUC before glucose delivery. Each point or bar is the mean ± 1 s. e.m. and statistical differences compared with vehicle controls were **Po 0.01, ***Po 0.001. cotransporter 1 (SGLT1) inhibitor phloridzin (50 μM) was used to show that only apical replacement of glucose reduced SGLT1 activity. The α2-agonist UK14 304 (5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine, 1 μM, basolateral) was used to reveal non-Y receptor Gαi-coupled reductions in ISC in rodent and human colon.
© 2014 Macmillan Publishers Limited
Data analysis AUCreactive data from perfused intestinal loops were calculated by subtracting the baseline 5 mM glucose (no compound) period from the PSN-GPR119-treated period. Statistical significance was determined using unpaired Student’s t-test where; *Po0.05, **P o0.01 and ***Po0.001 International Journal of Obesity (2014) 1365 – 1373
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1368 versus 5 mM glucose (no compound) control or; $Po0.05, $$P o0.01 and $$$ Po0.001 versus PSN-GPR119 (5 mM glucose). Plasma glucose was measured following in vivo glucose tolerance tests in millimolar. Comparisons were performed using one-way analysis of variance with Dunnett’s post hoc test; **Po 0.01 and ***Po0.001 versus vehicle. ISC data was analysed using GraphPad Prism, version 5.0 (GraphPad, La Jolla, CA, USA). Single comparisons were performed using Student’s t-test, whereas multiple comparisons utilised one-way analysis of variance with Dunnett’s post-hoc test; P o 0.05 was considered statistically different.
RESULTS GPR119 agonist activity, GLP-1 and insulin secretion in neuroendocrine cell lines PSN-GPR119 is a potent GPR119 agonist exhibiting an EC50 of 5.5 ± 2.0 nM for cAMP accumulation in hGPR119-transfected HEK293 cells (Supplementary Figure S1A). In GLUTag cells, PSNGPR119 increased GLP-1 secretion (EC50, 75 ± 19 nM, Supplementary Figure S1B) and induced insulin secretion from HIT-T15 cells (EC50, 90 ± 21 nM; Supplementary Figure S1C). Oral glucose tolerance tests In lean C57BL/6J mice, glucose tolerance following an oral glucose challenge improved dose-dependently after acute PSN-GPR119 (10 or 30 mg kg−1, p.o.) treatment (Figure 1). Blood glucose AUC was significantly lower following treatment with PSN-GPR119 compared with vehicle-treated animals (P o0.01; Figure 1). Despite a higher starting blood glucose concentration, the AUC for the glucose excursion following oral glucose challenge in ob/ ob mice was significantly decreased, notably to similar levels observed in the C57BL/6J mice (P o 0.001; Figure 1). Blood glucose levels following 30 mg kg−1 PSN-GPR119 treatment in lean SD and ZDF rats were significantly decreased by ~ 50% and ~ 65%, respectively, again significantly when compared with vehicle (P o 0.001 andP o 0.01 respectively; Figure 1). GPR119 mRNA expression in intestinal regions GPR119 transcripts were found in small intestinal and colonic regions from mice, rats and humans (Supplementary Figures S2A and C). In lean C57BL/6J mice and SD rats, GPR119 expression was highest in the distal gut and declined towards the duodenum, whereas human GPR119 expression was highest in the duodenum with slightly lower levels in jejunum, ileum and colon (Supplementary Figure S2C). GPR119 agonist induced GI hormone secretion from isolated loops of rat intestine Small intestines from fed male SD rats were perfused luminally for 30 min with PSN-GPR119 in 5 or 25 mM glucose to mimic pre- and post-prandial conditions. A concentration of 5 mM glucose was present as an energy source and to mimic the availability of glucose from the plasma before a meal. PSN-GPR119 concentration dependently stimulated tGIP, aGLP-1 and tPYY secretion in the presence of 5 and 25 mM glucose (P o 0.001 for all peptides after 1 and 10 μM PSN-GPR119; Figure 2). Consistent with the GLUTag model, ~ EC50 values with 5 mM glucose for tGIP, aGLP-1 and tPYY were 1.1, 0.9 and 1.1 μM, respectively, and were not significantly different in the presence 25 mM glucose. PSN-GPR119 and glucose stimulation of GI hormone secretion appeared to act additively. GPR119 agonism in lean and diseased rodent colon To investigate whether GPR119 signalling mechanisms were intact during metabolic disease, we exploited the finding that GPR119 activation causes endogenous PYY release and local epithelial Y1 International Journal of Obesity (2014) 1365 – 1373
Figure 2. Concentration responsiveness of PSN-GPR119 in lean SD rat small intestine perfused with 5 or 25 mM glucose. Rat small intestine was challenged luminally with 5 mM glucose in KH buffer for 30 min to equilibrate the preparation. Afterwards it was perfused for a further 30 min with an identical KH buffer containing either 5 or 25 mM and PSN-GPR119 (0.03–10 μM). Serosal secretion samples were analysed for total GIP, active GLP-1 and total PYY as indicated using commercially available ELISA kits. AUC reactive was calculated by subtracting the baseline AUC from the PSN-GPR119-treated AUC. Each bar is the mean ± 1 s.e.m. from six separate experiments and statistical differences are: *P o0.05, **P o0.01 and ***P o0.001 versus 5 mM glucose (no compound) control or $P o0.05, $$Po 0.01 and $$$P o0.001 versus PSN-GPR119 (5 mM glucose).
receptor-mediated anti-secretory responses23 using lean and diseased colonic tissue. First, lean C57BL/6J mice were significantly lighter than ob/ob mice of the same age (Po0.001), whereas the latter displayed significantly higher fasting blood glucose (Po0.001, Figure 3a) demonstrating obesity and hyperglycaemia. Apical PSN-GPR119 was equipotent in C57BL/6J colon (EC50 = 97.5 nM (38.5–246.9 nM)) and ob/ob colon (EC50 = 103.0 nM (50.5–210.3 nM); Figure 3b). Lean SD rats out-weighed their ZDF counterparts (Po0.001) but the latter exhibited significantly higher fasting blood glucose levels (Po0.001; Figure 3c). PSN-GPR119 potency was however similar in colonic mucosa from SD and ZDF rats (EC50 values were 126.6 nM (51.5–311.3 nM) versus 255.0 nM (34.8 nM–1.87 μM), respectively), and there were no significant differences in efficacy (Figure 3d). PSN-GPR119 sensitivity was greatest in the descending colon from lean and obese mice (P o 0.05; Supplementary Figure S3). There was no difference in PSN-GPR119 response size after apical or basolateral addition in any GI area. Subsequent PYY (10 nM) © 2014 Macmillan Publishers Limited
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Figure 3. GPR119 responses in C57BL/6J and ob/ob mice, SD and ZDF rats. (a and c) Weight and blood glucose measurements for the mice and rats used in these studies, and (b and d) concentration–response curves for PSN-GPR119 in colonic mucosa from C57BL/6J or ob/ob mice (b) and SD or ZDF rats. EC50 values are given in the text. Each bar or point is the mean ± s.e.m from the n numbers shown in parenthesis. ***P o0.001 (b and d) n = 4–5 and 4, respectively.
responses followed a similar pattern in each model (data not shown). In contrast, PSN-GPR119 responses were comparable throughout the small and large intestine from SD and ZDF rats (data not shown). Second, the receptor pharmacology was similar in lean and diseased rodent colon. Initially, Y1 receptor tone was revealed using BIBO3304, a competitive Y1 antagonist, as seen previously in C57BL/6J colon.26,29 BIBO3304 significantly increased basal ISC levels (Po 0.01), but the degree to which this occurred was reduced in ob/ob colon (Po 0.05, Figure 4a). Y1 antagonism abolished subsequent PSN-GPR119 responses in C57BL/6J and ob/ob mouse colon (Po0.01, Figure 4a) and responses to exogenous PYY in all groups (Po0.05). In C57BL/6J and ob/ob tissues, there were small exendin 4 increases in ISC, which were blocked by the GLP-1 receptor antagonist, exendin(9–39) (Po0.01, Figure 4a). In colon mucosa from SD and ZDF rats, Y1-mediated tone was similar, but interestingly a larger PSN-GPR119 response was observed in ZDF tissue (P o 0.05, Figure 4b). Consistent with the C57BL/6J and ob/ob data, PSN-GPR119 responses were abolished by BIBO3304 and there were small GLP-1 responses that were blocked by exendin(9–39) (Po 0.05 in ZDF colon; Figure 4b). Third, GPR119 responses were glucose-sensitive in mucosae from lean and diseased rodents. Apical replacement of glucose by mannitol reduced apical PSN-GPR119 responses significantly (P o 0.01) and subsequent phloridzin activity (Po 0.01; Figure 5), in line with the apical localisation of electrogenic SGLT1. Basolateral PSN-GPR119 responses were also glucose-sensitive (data not shown). Interestingly, phloridzin responses were significantly larger in ZDF compared with SD colon (P o 0.05; Figure 5b). Control PYY-Y1 and UK14 304 α2-mediated responses responses were glucose-insensitive. © 2014 Macmillan Publishers Limited
GPR119 agonism in human colon mucosa PSN-GPR119 added apically (Figure 6) or basolaterally (data not shown) caused long-lasting reductions in ISC (that were similar to PYY responses) with an EC50 of 71.3 (21.5–166.2) nM (Figure 6a). As seen in rodent colon mucosa, PSN-GPR119 responses were blocked by the Y1 antagonist, BIBO3304 (P o 0.05; Figure 6b), which also revealed Y1 tone, as seen previously.21,29 Furthermore, co-addition with the Y2 antagonist, BIIE0246 ((S)-N2-[[1-[2-[4-[(R,S)-5, 11-dihydro-6(6 h)-oxodibenz [b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl] cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1, 2,4-triazol-4-yl]ethyl]-argininamide) raised basal ISC levels further than BIBO3304 alone, and the combination of the two Y antagonists abolished subsequent PSN-GPR119 (P o0.05; Figure 6b) and PYY responses, but had no effect on UK14 304 α2-mediated responses anti-secretory responses (data not shown). Finally, PSN-GPR119 responses were glucose sensitive (P o 0.05; Figure 6c) and the effects of SGLT1 inhibitor, phloridzin were also reduced (Po 0.05) when apical glucose was replaced by mannitol. As seen in rodent mucosa, PYY-Y1 responses were not glucose sensitive in human colon.
DISCUSSION Few studies have shown intestinal efficacy with selective GPR119 agonists in normal and T2D rodent models9,11,12,30 and as yet none have compared this with efficacy in human intestinal tissue. Our study shows that luminal delivery of a GPR119 agonist stimulates K- and L-cell activity acutely in rat intestine. PSNGPR119 was highly potent, stimulating insulin and GLP-1 from secretory cell lines. As GPR119-activated insulin secretion is well International Journal of Obesity (2014) 1365 – 1373
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Figure 4. Blockade of Y1 receptors inhibits GPR119 agonism and PYY responses in descending colon mucosa from, in (a) mouse, (b) rat models. Vehicle (0.1% dimethyl sulphoxide, first bar) or Y1 antagonist BIBO3304 (300 nM, BIBO) was added in the absence or presence of 1 μM exendin (9–39) (BIBO+9–39). After vasoactive intestinal polypeptide (VIP) (30 nM, data not shown), PSN-GPR119 (1 μM, apical), PYY (10 nM) and exendin 4 (100 nM) were added sequentially and their anti-secretory effects pooled to give the means ± 1 s.e.m. (n, numbers in parenthesis). Control responses to exendin 4 (100 nM, Ex 4) are also included. Statistical differences between vehicle and antagonist-pretreated agonist mean responses are as shown. *Po 0.05, **P o0.01, ***P o0.001. Differences between lean and diseased rodent tissue responses are shown by a vertical line, *Po0.05.
established, we set out to determine the relative importance of this receptor on gut peptide release and to evaluate the peptide pharmacology involved in human colon. GPR119 expression was highest in mouse and rat colon, consistent with L-cell distribution along rodent GI tract. Interestingly, we observed a more uniform GPR119 distribution along the human GI tract with slightly elevated expression in the duodenum. GPR119 levels may be higher in the upper GI tract of humans relative to rodents; however, the source of the human gut RNA was pooled from multiple donors (because GPR119 transcript expression is low, L cells accounting for o1% of the total mucosal cell population), whereas the rodent intestinal RNA was sourced from individual animals, which may also account for some differences. In vivo, oral PSN-GPR119 improved glucose tolerance significantly in lean and diseased rodents. This is consistent with other studies showing that GPR119 agonism (acute or chronic) leads to effective glycaemic control.3,8,9,11,14,30 PSN-GPR119 (30 mg kg−1) was as efficacious (50–60% glucose lowering in an oral glucose tolerance test) in lean, as it was in rodents with T2D. The secretion profiles of GIP and GLP-1 in patients with T2D are well established; levels of GIP in systemic blood are normal in pre- and postprandial conditions. However, patients with T2D are insensitive to circulating GIP.31,32 Circulating levels of GLP-1, in contrast are reduced in T2D.32 The exact mechanism of L-cell impairment is unknown, but it is not thought to be via increased clearance of GLP-1.33 Exogenous administration has shown that the insulinotropic activity of GIP is defective, whereas that of GLP-1 remains in T2D. The insulinotropic, glucagonostatic and gastric emptying actions of GLP-1 are preserved in T2D.32,34,35 These data indicate International Journal of Obesity (2014) 1365 – 1373
that the contribution of each hormone towards glucose lowering in lean and diseased models may differ and in fact recent data in mice indicates L-cell activity is more important for acute regulation of glucose homeostasis.36 Unfortunately, we were unable to determine the relative contribution of GIP, GLP-1 or PYY to the improvement in glucose tolerance on our models. However, the net effect is that PSN-GPR119 ameliorates postprandial blood glucose concentrations in rodent models of diabetes and obesity indicating its potential to improve glucose tolerance in vivo. Luminal perfusion of PSN-GPR119 into loops of rat small intestine caused concentration-dependent increases in tGIP, aGLP-1 and tPYY secretion (an effect we have also seen with luminal OEA25). Peptide secretion was further enhanced by elevating luminal glucose from 5 to 25 mM, demonstrating the existence of further capacity for the K and L cells to secrete GIP, GLP-1 and PYY. GPR119 agonism and glucose acted in an additive manner, consistent with the recent report that GPR119 agonists stimulate GLP-1 secretion glucose independently.37 The notion that intestinal GPR119 agonism leads to an incretin effect is supported by studies showing that luminal OEA application in vivo caused significant GLP-1 release compared with i.v. administration of OEA at a 200-fold higher concentration.12 Another in-house small-molecule GPR119 agonist, PSN632408, has been shown to stimulate L-cell-derived PYY responses in human and murine colon.23 Apical or basolateral PSN632408 caused equivalent responses, through PYY activation of epithelial Y1 receptors. PSN632408 was less potent than PSN-GPR119 in the present study, exhibiting an EC50 of 5.7 μM in mouse colonic mucosa, whereas in human colonic mucosa, 10 μM PSN632408 © 2014 Macmillan Publishers Limited
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Figure 5. Glucose sensitivity of apical PSN-GPR119 (1 μM) in the presence of glucose (11.1 mM) both sides or following replacement with mannitol (11.1 mM) on the apical side only in C57BL/6J mice (a) and SD rats (b). Subsequent phloridzin (Phlor, 50 μM, apically) and PYY (10 nM) responses are also shown. Values are the mean − 1 s.e.m. from n numbers as shown. Statistical differences between responses obtained with glucose both sides versus mannitol apically, are shown, *Po0.05, **Po0.01, ***Po0.001. There were no differences between lean control and diseased mucosal responses except for phloridzin responses, which were significantly larger in ZDF than SD rat colon mucosa, *Po0.05 (vertical line).
caused a maximal mucosal response23 similar to that achieved here with 0.3–1 μM PSN-GPR119. To evaluate the efficacy of PSN-GPR119 during diabetes and obesity, we used ZDF rats and ob/ob mice as diseased counterparts to lean SD rats and C57BL/6J mice. Consistent with a 10-fold increase in potency over PSN632408, PSN-GPR119 (potency at mouse GPR119 receptors of 7.9 versus 0.18 μM, respectively) exhibited similar increased potency (5.7 versus 0.1 μM) in C57BL/6J colon mucosa. GPR119-induced PYY-Y1 signalling was consistent in diseased and lean rodent tissue and the responses to PSN-GPR119 were equivalent in each gut region. The lack of apparent sidedness to PSN-GPR119 mucosal responses was most likely due to the high permeability of PSN-GPR119, which precluded determination of the exact location of GPR119 (that is, apical alone or both apical and basolateral membranes). If GPR119 receptors are apically targeted, then their stimulation by gut-restricted agonists should limit off-target effects and minimise potential detrimental effects, for example, on fatty acid metabolism in skeletal muscle.38 Interestingly, we observed reduced Y1 tone in ob/ob colon, potentially a consequence of reduced PYY analogue, neuropeptide Y and Y1 colonic expression. Imai and co-workers have shown reduced neuropeptide Y and Y1 expression in pancreatic islets of ob/ob mice.39 Additionally, GPR119 responses (with 1 μM PSN-GPR119) were slightly larger in ZDF versus SD colon. Although GPR119 expression is known to alter in disease and is increased in islets from obese rats,13 it would be interesting to determine the receptor levels in ZDF and SD rat intestine. In lean and diseased mucosae, PSN-GPR119 responses were increased consistently in © 2014 Macmillan Publishers Limited
the presence of glucose, indicating that GPR119 agonism should reduce the risk of hypoglycemia in a diabetic population. However, in colonic preparations GPR119 agonism was not abolished when glucose was removed, suggesting that PSNGPR119 may exert some glucose-independent activity, as seen for GPR119 agonism in GLUTag cells and murine primary colonic cultures, but not in insulin-secreting pancreatic cells.37 We also observed larger phloridzin responses in ZDF compared with SD colon, potentially revealing alterations in colonic SGLT1 expression caused by disease. Normally, glucose is cleared from the lumen in the upper small intestine, so glucose reaching the colon in ZDF rats would manifest as larger responses to phloridzin. Upregulation of colonic SGLT1 activity indicates malabsorption of glucose in the ZDF model. Consistent with our in vivo studies, downstream peptide signalling pathways (PYY-mediated) were clearly functionally intact during disease, as the responses to PSN-GPR119 in both diabetic ZDF and obese and diabetic ob/ob models were maintained, in line with our hypothesis. The same PSN-GPR119initiated PYY-Y1 signalling occurred in human colon, and was comparable to the glucose-sensitive PSN632408 signalling described previously.23 These findings suggest that intestinal GPR119 stimulation has the potential for glucose lowering in man and may be comparable to that observed in rodent T2D models here and in other recent studies.30,40,41 In summary, PSN-GPR119 is a potent GPR119 tool compound that significantly lowers post-prandial glucose in lean and diseased models of T2D; luminal delivery of PSN-GPR119 to isolated loops of rat small intestine stimulates secretion of GIP, International Journal of Obesity (2014) 1365 – 1373
Pharmacology of a novel GPR119 agonist, PSN-GPR119 S Patel et al
1372 mimic more closely, the success of bariatric surgery in treating T2D through a gut-based therapeutic approach. GPR119 compounds are already in the clinic for the treatment of T2D and human data have been reported7,42 ( http://clinicaltrials.gov) showing modest glucose-lowering activity. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS SP, OJM, JW, MS, UWB and T-AC were employees of Prosidion Ltd at the time of the study, and we thank RenaSci Ltd (Nottingham, UK) for conducting the oral glucose tolerance test studies.
AUTHOR CONTRIBUTIONS SP and HMC were responsible for the conception and design of the experiments and writing of the article. OJM, IRT and JW were also responsible for collection, analysis, interpretation and presentation of the data. UWB and T-AC were responsible for the oral glucose tolerance test studies. MS was responsible for reviewing the article.
REFERENCES
Figure 6. The effect of PSN-GPR119 on normal human colon mucosa (a) concentration–response relationship for PSN-GPR119 (added apically, n = 3) and (b) Y1 receptor blockade (300 nM BIBO3304; +BIBO) inhibits GPR119 agonism (1 μM PSN-GPR119, apical) ± a Y2 receptor antagonist (1 μM BIIE0246; +BIBO & BIIE). (c) Glucose sensitivity of apical PSN-GPR119 (1 μM) responses in the presence of glucose both sides (11.1 mM) or, with apical mannitol (11.1 mM) in place of glucose. Subsequent phloridzin (Phlor, 50 μM, apically) and PYY (100 nM) responses are also shown. Values are the mean ± 1 s.e. m. from n numbers in parenthesis. Statistical differences are shown, *Po 0.05.
GLP-1 and PYY; and GPR119 agonism activates anti-secretory responses through PYY-mediated Y1 receptor activation in colonic mucosa of lean and T2D rodent models. PSN-GPR119 activity in human colon was comparable to that of lean and T2D rodent models and we conclude that L-cell-derived PYY, in addition to GIP and GLP-1, may contribute towards glucose lowering in man. The benefits of releasing more than one endogenous peptide from the enteroendocrine cell population in a glucose-dependent manner could offer a therapeutic advantage to modulate insulinotropic pathways with reduced risk of hypoglycemic, and International Journal of Obesity (2014) 1365 – 1373
1 Fredriksson R, Hoglund PJ, Gloriam DE, Lagerstrom MC, Schioth HB. Seven evolutionarily conserved human rhodopsin G protein-coupled receptors lacking close relatives. FEBS Lett 2003; 554: 381–388. 2 Overton HA, Babbs AJ, Doel SM, Fyfe MC, Gardner LS, Griffin G et al. Deorphanization of a G protein-coupled receptor for oleoylethanolamide and its use in the discovery of small-molecule hypophagic agents. Cell Metab 2006; 3: 167–175. 3 Chu ZL, Jones RM, He H, Carroll C, Gutierrez V, Lucman A et al. A role for beta-cell-expressed G protein-coupled receptor 119 in glycemic control by enhancing glucose-dependent insulin release. Endocrinology 2007; 148: 2601–2609. 4 Semple G, Fioravanti B, Pereira G, Calderon I, Uy J, Choi K et al. Discovery of the first potent and orally efficacious agonist of the orphan G protein-coupled receptor 119. J Med Chem 2008; 51: 5172–5175. 5 Yoshida S, Ohishi T, Matsui T, Shibasaki M. Identification of a novel GPR119 agonist, AS1269574, with in vitro and in vivo glucose-stimulated insulin secretion. Biochem Biophys Res Commun 2010; 400: 437–441. 6 Jones RM, Leonard JN, Buzard DJ, Lehmann J. GPR119 agonists for the treatment of type 2 diabetes. Expert Opin Ther Pat 2009; 19: 1339–1359. 7 Katz LB, Gambale JJ, Rothenburg PL, Vanapalli SR, Vaccaro N, Xi L et al. Effects of JNJ-38431055, a novel GPR119 receptor agonist, in randomized, double-blind, placebo-controlled studies in subjects with type 2 diabetes. Diabetes Obes Metab 2012; 14: 709–716. 8 Chu ZL, Carroll C, Chen R, Alfonso J, Gutierrez V, He H et al. N-oleoyldopamine enhances glucose homeostasis through the activation of GPR119. Mol Endocrinol 2010; 24: 161–170. 9 Flock G, Holland D, Seino Y, Drucker DJ. GPR119 regulates murine glucose homeostasis through incretin receptor-dependent and independent mechanisms. Endocrinology 2011; 152: 374–383. 10 Ning Y, O'Neill K, Lan H, Pang L, Shan LX, Hawes BE, Hedrick JA. Endogenous and synthetic agonists of GPR119 differ in signalling pathways and their effects on insulin secretion in MIN6c4 insulinoma cells. Br J Pharmacol 2008; 155: 1056–1065. 11 Chu ZL, Carroll C, Alfonso J, Gutierrez V, He H, Lucman A et al. A role for intestinal endocrine cell-expressed G protein-coupled receptor 119 in glycemic control by enhancing glucagon-like peptide-1 and glucose-dependent insulinotropic peptide release. Endocrinology 2008; 149: 2038–2047. 12 Lauffer LM, Iakoubov R, Brubaker PL. GPR119 is essential for oleoylethanolamideinduced glucagon-like peptide-1 secretion from the intestinal enteroendocrine L cell. Diabetes 2009; 58: 1058–1066. 13 Sakamoto Y, Inoue H, Kawakami S, Miyawaki K, Miyamoto T, Mizuta K, Itakura M. Expression and distribution of Gpr119 in the pancreatic islets of mice and rats: predominant localization in pancreatic polypeptide-secreting PP-cells. Biochem Biophys Res Commun 2006; 351: 474–480. 14 Lan H, Vassileva G, Corona A, Liu L, Baker H, Golovko A et al. GPR119 is required for physiological regulation of glucagon-like peptide-1 secretion but not for metabolic homeostasis. J Endocrinol 2009; 201: 219–230.
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1373 15 Rogers GJ, Tolhurst G, Ramzan A, Habib AM, Parker HE, Gribble FM, Reimann F. Electrical activity-triggered glucagon-like peptide-1 secretion from primary murine L-cells. J Physiol 2011; 589: 1081–1093. 16 Hansen KB, Rosenkilde MM, Knop FK, Wellner N, Diep TA, Rehfeld JF et al. 2-Oleoyl Glycerol is a GPR119 agonist and signals GLP-1 release in humans. J Clin Endocrinol Metab 2011; 96: E1409–E1417. 17 Sundler F, Ekblad E, Håkanson R. Localisation and colocalisation of GI peptides. In: Handbook of Experimental Pharmacology Gastrointestinal Regulatory Peptides Brown DR (ed) Springer-Verlag: Berlin, Germany, 1993; 106: pp 1–28. 18 Chan JL, Stoyneva V, Kelesidis T, Raciti P, Mantzoros CS. Peptide YY levels are decreased by fasting and elevated following caloric intake but are not regulated by leptin. Diabetologia 2006; 49: 169–173. 19 Tough IR, Forbes S, Tolhurst R, Ellis M, Herzog H, Bornstein J, Cox HM. Endogenous peptide YY and neuropeptide Y inhibit colonic ion transport, contractility and transit differentially via Y1 and Y2 receptors. Br J Pharmacol 2011; 164: 66–79. 20 Liu CD, Aloia T, Adrian TE, Newton TR, Bilchik AJ, Zinner MJ et al. Peptide YY: a potential proabsorptive hormone for the treatment of malabsorptive disorders. Am Surg 1996; 62: 232–236. 21 Cox HM, Tough IR. Neuropeptide Y, Y1, Y2 and Y4 receptors mediate Y agonist responses in isolated human colon mucosa. Br J Pharmacol 2002; 135: 1505–1512. 22 Batterham RL, Cohen MA, Ellis SM, Le Roux CW, Withers DJ, Frost GS et al. Inhibition of food intake in obese subjects by peptide YY3-36. N Engl J Med 2003; 349: 941–948. 23 Cox HM, Tough IR, Woolston AM, Zhang L, Nguyen AD, Sainsbury A, Herzog H. Peptide YY is critical for acylethanolamine receptor Gpr119-induced activation of gastrointestinal mucosal responses. Cell Metab 2010; 11: 532–542. 24 Overton HA, Babbs AJ, Doel SM, Fyfe CT, Gardiner LS, Griffin G et al. Deorphanization of a G protein-coupled receptor for oleoylethanolamide and its use in the discovery of small molecule hypophagic agents. Cell Metab 2006; 3: 167–175. 25 Mace OJ, Schindler M, Patel S. The regulation of K- and L cell activity by GLUT2 and the calcium-sensing receptor CasR in rat small intestine. J Physiol 2012; 590: 2917–2936. 26 Cox HM, Pollock EL, Tough IR, Herzog H. Multiple Y receptors mediate pancreatic polypeptide responses in mouse colon mucosa. Peptides 2001; 22: 445–452. 27 Cox HM, Cuthbert AW. Neuropeptide Y antagonises secretagogue evoked chloride transport in rat jejunal epithelium. Pflugers Arch 1988; 413: 38–42. 28 Cox HM, Cuthbert AW, Håkanson R, Wahlestedt C. The effect of neuropeptide Y and peptide YY on electrogenic ion transport in rat intestinal epithelia. J Physiol 1988; 398: 65–80. 29 Tough IR, Holliday ND, Cox HM. Y(4) receptors mediate the inhibitory responses of pancreatic polypeptide in human and mouse colon mucosa. J Pharmacol Exp Ther 2006; 319: 20–30.
30 Zhang M, Feng Y, Wang J, Zhao J, Li T, He M et al. High-throughput screening for GPR119 modulators identifies a novel compound with anti-diabetic efficacy in db/db mice. PLoS One 2013; 8: e63861. 31 Nauck M, Stockmann F, Ebert R, Creutzfeldt W. Reduced incretin effect in type 2 (non-insulin-dependent) diabetes. Diabetologia 1986; 29: 46–52. 32 Nauck MA, Heimesaat MM, Orskov C, Holst JJ, Ebert R, Creutzfeldt W. Preserved incretin activity of glucagon-like peptide 1 [7-36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus. J Clin Invest 1993; 91: 301–307. 33 Brubaker PL. Minireview: update on incretin biology: focus on glucagon-like peptide-1. Endocrinol 2010; 151: 1984–1989. 34 Willms B, Werner J, Holst JJ, Orskov C, Creutzfeldt W, Nauck MA. Gastric emptying, glucose responses, and insulin secretion after a liquid test meal: effects of exogenous glucagon-like peptide-1 (GLP-1)-(7-36) amide in type 2 (noninsulin-dependent) diabetic patients. J Clin Endocrinol Metab 1996; 81: 327–332. 35 Drucker DJ. Minireview: the glucagon-like peptides. Endocrinology 2001; 142: 521–527. 36 Pedersen J, Ugleholdt RK, Jorgenson SM, Windelov JA, Grunddal KV, Schwartz TW et al. Glucose metabolism is altered after loss of L cells and α-cells but not influenced by loss of K cells. Am J Physiol Endocrinol Metab 2012; 304: E60–E73. 37 Lan H, Lin HV, Wang CF, Wright MJ, Xu S, Kang L et al. GPR119 agonists mediate GLP-1 secretion from mouse enteroendocrine cells through glucose-independent pathways. Br J Pharmacol 2012; 165: 2799–2807. 38 Cornall LM, Mathai ML, Hryciw DH, McAinch AJ. Is GPR119 agonism an appropriate treatment modality for the safe amelioration of metabolic diseases? Expert Opin Investig Drugs 2013; 22: 487–498. 39 Imai Y, Patel HR, Hawkins EJ, Doliba NM, Matschinsky FM, Ahima RS. Insulin secretion is increased in pancreatic islets of neuropeptide Y-deficient mice. Endocrinology 2007; 148: 5716–5723. 40 Semple G, Ren A, Fioravanti B, Periera G, Calderon I, Choi K et al. Discovery of fused bicyclic agonists of the orphan G-protein coupled receptor GPR119 with in vivo activity in rodent models of glucose control. Bioorg Med Chem Letts 2013; 21: 3134–3141. 41 Ansarullah Yu L, Holstein M, DeRuyter B, Rabinovitch A, Guo Z. Stimulating β-cell regeneration by combining a GPR119 agonist with a DPP-IV inhibitor. PLoS One 2013; 8: e53345. 42 Goodman ML, Dow J, van Vliet AA, Hadi S, Karbiche D, Lockton JA. The novel GPR119-receptor agonist PSN821 shows glucose lowering and decreased energy intake in patients with T2DM after 14 days treatment. Diabetes 2011; 60: A273 (Abstract).
Supplementary Information accompanies this paper on International Journal of Obesity website (http://www.nature.com/ijo)
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International Journal of Obesity (2014) 1365 – 1373
ORIGINAL ARTICLE
Gastrointestinal hormonal responses on GPR119 activation in lean and diseased rodent models of type 2 diabetes S Patel1,3,9, OJ Mace1,4,9, IR Tough2,9, J White1,5, T-A Cock1,6, U Warpman Berglund1,7, M Schindler1,8 and HM Cox2 BACKGROUND: G protein-coupled receptor 119 (GPR119) has emerged as a potential target for the treatment of type 2 diabetes (T2D) and tool compounds have been critical in the evaluation of GPR119 functions. METHODS: We synthesised a novel small-molecule GPR119 agonist, PSN-GPR119, to study GPR119 signalling activities in cells overexpressing GPR119. We measured GPR119-stimulated peptide hormone release from intestinal loops and oral glucose tolerance in vivo from lean (C57BL/6J mouse or Sprague-Dawley (SD) rat) and diabetic (ob/ob mouse or ZDF rat) models. To evaluate the direct effects of GPR119 agonism on gastrointestinal (GI) tissue, we measured vectorial ion transport (measured as ISC; short-circuit current) across rodent GI mucosae and from normal human colon specimens. RESULTS: GPR119 activation by PSN-GPR119 increased cAMP accumulation in hGPR119-overexpressing HEK293 cells (EC50, 5.5 nM), stimulated glucagon-like peptide 1 (GLP-1) release from GLUTag cells (EC50, 75 nM) and insulin release from HIT-15 cells (EC50, 90 nM). In vivo, PSN-GPR119 improved glucose tolerance by ~50% in lean mice or rats and ~60% in the diabetic ob/ob mouse or ZDF rat models. Luminal addition of PSN-GPR119 to isolated loops of lean rat small intestine stimulated GLP-1, glucose insulinotropic peptide (GIP) and peptide YY (PYY) release under basal (5 mM) and high glucose (25 mM) conditions. Activation of GPR119 also reduced intestinal ion transport. Apical or basolateral PSN-GPR119 addition (1 μM) to lean or T2D rodent colon mucosae reduced ISC levels via PYY-mediated Y1 receptor agonism. The GPR119 response was glucose sensitive and was abolished by Y1 receptor antagonism. Similarly, in human colon, mucosa PSN-GPR119 acted via a Y1-specific mechanism. CONCLUSIONS: Our results show that functional GPR119 responses are similar in lean and diabetic rodent, and human colon; that GPR119 stimulation can result in glucose lowering through release of intestinal peptide hormones and that PSN-GPR119 is a useful tool compound for future studies. International Journal of Obesity (2014) 38, 1365–1373; doi:10.1038/ijo.2014.10 Keywords: GPR119; intestinal responses; incretins; peptide YY and Y1 receptors; glucagon-like peptide 1; type 2 diabetes rodent models
INTRODUCTION New improved anti-diabetic therapies would be advantageous to address the increasing epidemic of type 2 diabetes (T2D) and obesity. G protein-coupled receptor 119 (GPR119) is primarily expressed in gastrointestinal (GI) enteroendocrine cells and pancreatic β cells1–3 in rodents and humans, attracting significant interest as a therapeutic target for T2D. GPR119 agonists in vivo have shown impressive lowering of postprandial glucose in rodent models of T2D3–5 and GPR119 agonists have also demonstrated beneficial effects on body weight,2 offering potential advantages to obese T2D patients over therapeutics already in the market. As such, new GPR119 molecules have advanced into the clinic for the treatment of T2D.6,7 The ability of GPR119 agonism to improve glucose tolerance and reduce body weight shows that GPR119 biology warrants further investigation. GPR119 agonists have direct effects on
insulin-secreting cell lines and on rodent islets, which have been characterised extensively.5,8–10 Importantly, insulin secretion mediated by GPR119 is glucose dependent and agonism will not cause the hypoglycaemia associated with other diabetic agents.11,12 Furthermore, GPR119 expression is significantly increased in the islets of obese diabetic db/db mice when compared with islets from lean models13 adding credence to the potential therapeutic value of this receptor. In addition, GPR119 agonists have greater oral efficacy as compared with intravenous efficacy, suggesting that GPR119 may control glycaemia via additional incretin-based mechanisms.12 The secretion of endogenous anti-diabetic hormones, including glucose insulinotropic peptide (GIP) and glucagon-like peptide 1 (GLP-1), is a consequence of GPR119 agonism in vivo.9,14 In purified enteroendocrine cells and isolated murine intestinal L cells, GLP-1 secretion is mediated by signalling pathways
1 Prosidion Ltd, Oxford, UK and 2King's College London, Wolfson Centre for Age-Related Diseases, London, UK. Correspondence: Professor HM Cox, King’s College London, Wolfson Centre for Age-Related Diseases, Hodgkin Building, Guy’s Campus, Newcomen Street, London SE1 1UL, UK. E-mail: [email protected] 3 Current address: Bioelectronics R&D, GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY, UK. 4 Current address: Heptares Therapeutics, Broadwater Road, Welwyn Garden City, AL7 3AX, UK 5 Current address: Argenta Discovery, 8/9 Spire Green Centre, Flex Meadow, Harlow, CM19 5TR, UK 6 Current address: Pharmaxis Ltd, 20 Rodborough Road, Locked Bag 5015, Frenchs Forest, NSW 2086, Australia 7 Current address: Science for Life Laboratory, Department of Medical Biochemistry & Biophysics, Karolinska Institute, S-171 21 Stockholm, Sweden 8 Current address: Astra Zeneca R&D Mölndal, Cardiovascular & Metabolic Diseases iMed, Pepparedsleden 1, SE-431 83 Mölndal, Sweden 9 These authors contributed equally to this work. Received 3 May 2013; revised 7 January 2014; accepted 14 January 2014; accepted article preview online 23 January 2014; advance online publication, 18 February 2014
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involving L- and Q-type calcium channels.15 Additionally, rat ileum treated in situ with an endogenous ligand for GPR119, oleoylethanolamide (OEA) induced GLP-1 release.12 In humans, activation of GPR119 by administration of a jejunal bolus of 2-oleoyl glycerol (a component of fatty acid digestion with affinity for GPR119) stimulated GLP-1 release,16 implicating GPR119 as a fat sensor. The L-cell-derived hormone, peptide YY (PYY), is predominantly expressed in the ileum and the colon,17 and circulating PYY increases or decreases after feeding or fasting, respectively.18 PYY activates Y1 and Y2 receptors to inhibit upper GI motility19 and inhibits water and electrolyte secretion in human colon,20,21 and its product, PYY(3–36) appears to reduce appetite.22 In support of the anti-secretory role of PYY in the colon, activation of GPR119 mechanisms results in anti-secretory effects via endogenous PYY-activating epithelial Y1 receptors.23 Postprandial blood glucose was also controlled by PYY, and both responses were absent from PYY-/- mice or their tissues.23 We synthesised PSN-GPR119 (N-(2-Hydroxyethyl)-4-{3-[1-(3isopropyl-[1,2,4] oxadiazol-5-yl)piperidin-4-yl]propoxy}-2-methylbenzamide (patent number, WO 2008/081205 A1), a potent small-molecule GPR119 agonist, and validated its potential to improve glucose tolerance in rodent models of diabetes and obesity. Our hypothesis was that GPR119 signalling involved secretion of K-cell- and L-cell-derived hormones and that the mechanisms involved were consistent in lean and diseased rodent models of diabetes and obesity. In order to define the GPR119 mechanisms in GI mucosa, we exploited the fact that PYY inhibits epithelial ion secretion in mammalian intestine and compared the downstream PYY-mediated pathways of PSN-GPR119. We extended these mechanistic studies to assess PSN-GPR119 activity in human colonic mucosal specimens.
MATERIALS AND METHODS Ethical approval Animal procedures were approved by the UK Home Office and undertaken in accordance with the Animals (Scientific Procedures) Act (1986). Human colon specimens were obtained from consenting patients undergoing bowel resection surgery with the approval of Guy’s and St Thomas’ NHS Foundation Trust, in accordance with The Human Tissue Act (2004).
GPR119-transfected HEK293 cell cAMP assay Stimulated cAMP accumulation was measured using an engineered HEK293 cell line expressing GPR119 as previously described.24 Monolayers were exposed to PSN-GPR119 (0.01–100 nM) for 30 min at 37 °C in buffer containing 1% dimethyl sulphoxide. Cells were lysed and cAMP was determined (AlphaScreen cAMP kit, Perkin Elmer, Cambridge, UK).
Measurement of GLP-1 secretion from GLUTag cells GLUTag cells were cultured in Dulbecco's modified Eagle medium containing 5.5 mM glucose. Cells were seeded onto 24-well plates coated with Matrigel (BD Biosciences, Oxford, UK) and used at 60–80% confluency. They were washed twice in nutrient-free bathing solution supplemented with 0.1 mM diprotin A and 0.1 % (w/v) BSA and incubated with PSNGPR119 (0.1 nM–3 μM) for 2 h at 37 °C. Media was assayed for GLP-1 using an active GLP-1 ELISA (Linco Research Inc., St Charles, MO, USA).
Measurement of insulin secretion from HIT-T15 cells HIT-T15 cells were cultured in RPMI1640 with 10% . Cells were seeded in 96-well plates and cultured for 24 h before assay. Cells were pre-incubated for 30 min in Krebs-Ringer-HEPES buffer (119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3, 1.2 mM KH2PO4, 10 mM HEPES) with 0.1% BSA. PSN-GPR119 was added to buffer supplemented with 5.6 mM glucose and incubated for 30 min. Media was assayed for insulin by ELISA (Mercodia, Uppsala, Sweden). International Journal of Obesity (2014) 1365 – 1373
Oral glucose tolerance test (OGTT) Animals were fed ab libitum and had free access to water and were maintained on a 12-h light:dark cycle. Oral glucose tolerance was assessed in overnight-fasted lean Sprague-Dawley (SD) rats (12 weeks old, n = 8), lean C57BL/6J mice (11–12 weeks old, n = 10), Zucker diabetic fatty (ZDF) rats (12 weeks old, n = 8) or obese ob/ob (leptin-deficient obese, B6.V-Lepob/J) mice (11–12 weeks old, n = 10) following acute treatment of PSN-GPR119. PSNGPR119 (10 or 30 mg kg−1) or vehicle (20% 2-hydroxy propyl-β-cyclodextrin) was administered by oral gavage 60 min (t = -60 min) before the glucose bolus that was delivered by oral gavage (2 g kg−1; t = 0 min). Blood samples (t = 0–250 min after glucose) were analysed using a commercially available glucose oxidase assay. Plasma glucose is presented as absolute values (mM) and reactive area under the curve (AUCreactive) was calculated by subtraction of the baseline AUC before glucose.
GPR119 expression in intestinal regions As GPR119 transcript expression is very low (L cells represent o 1% of the total mucosal cell population) intestinal RNA samples were pooled from five human male donors (BioChain, Newark, CA, USA), whereas individual samples from six male SD rats and three male C57BL/6J mice were analysed separately. Real-time PCR was performed using pre-validated primer / FAM-labelled GPR119 probe sets (Applied Biosystems, Paisley, UK) corresponding to GenBank accessions NM_178471.2 (human), NM_181751.2 (mouse) and BD274917.1/ Celera rCT63637.0 (rat). Data were normalised to the housekeeping genes, hypoxanthine phosphoribosyl transferase I and hydroxyl-methylbilane synthase, calculated using ΔCt.
Isolated rat intestinal perfusion and measurement of GIP, GLP-1 and PYY levels Male SD rats (250 g; Charles River, Margate, UK) were fed ad libitum, had free access to water and were maintained on a 12-h light:dark cycle. Loops of whole small intestine were isolated and perfused in single-pass mode as previously described.25 Briefly, the first 30 min was a control period during which Krebs–Henseleit (KH; in mM: 120 NaCl, 4.5 KCl, 1 MgSO4,1.8 Na2HPO4, 0.2 NaH2PO4, 25 NaHCO3, 1.25 CaCl2 and 5 D-glucose) was perfused at a flow rate of 1.6 ml min−1. After 30 min, the loop was perfused with a second experimental perfusate (identical to the first except for the addition of PSN-GPR119). During this period, loops were either perfused with KH containing PSN-GPR119 (0.1–100 μM) or 5 mM glucose (osmotically balanced to 25 mM sugar with mannitol) for 30 min, then switched to an identical perfusate containing PSN-GPR119 and 25 mM glucose. Serosal samples were assayed for glucose, total GIP (tGIP; Millipore Ltd, Oxford, UK), active GLP-1 (aGLP-1; Epitope Diagnostics Inc, San Diego, CA, USA) and total PYY (tPYY; Phoenix Pharmaceuticals, Burlingame, CA, USA) using commercially available ELISAs, as previously described.25 Tissue viability was indicated by maintenance of active glucose transport throughout the experiment. AUCreactive was calculated from time-courses in pg ml−1 (g dry weight)−1 × min and presented as mean ± s.e.m. (n ⩾ 4).
Mucosal electrophysiology Human colonic specimens from nine consenting patients undergoing intestinal resection for primary malignancy, or GI regions from rodents (Charles River; lean SD rats or C57BL/6J mice and diseased ZDF rats or ob/ ob mice) were stripped of smooth muscle and adjacent pieces of mucosa were bathed in KH buffer, mounted on Ussing chambers and voltageclamped at 0 mV, as described in detail previously.21,23,26 Electrogenic ion transport was measured as short-circuit current (ISC; in μA cm−2). PSN-GPR119 was added to the apical (for potency measurement) or basolateral reservoir, whereas peptides were added basolaterally. A concentration of 1 μM PSN-GPR119 gave near-maximal responses and was used to stimulate other GI areas. In mouse mucosa, vasoactive intestinal polypeptide (VIP, 10 nM) was used to pre-stimulate anion secretion, to optimise subsequent Gαi-coupled responses.23,26 PYY (10 nM) was a control activator of Y1 receptor-mediated responses through Gαi-coupled reductions in epithelial cAMP and consequent attenuation of Cl- secretion.27,28 Y1 responses were blocked with BIBO3304 ((R)-N-[[4(aminocarbonylamino methyl)-phenyl]methyl]-N2-(diphenylacetyl)-argininamide trifluoroacetate; 300 nM) and endogenous GLP-1 effects were abolished with 1 μM exendin(9–39). Mucosae were treated with both antagonists to block both endogenous peptide responses. The glucose dependence of agonist responses was tested using KH buffer (including 11.1 mM glucose) or buffer containing mannitol (11.1 mM). The Na+-glucose © 2014 Macmillan Publishers Limited
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Figure 1. PSN-GPR119 significantly improves oral glucose tolerance in lean C57BL/6J mice and SD rats, compared with ob/ob mice and ZDF rat models. Left time-courses depict dosing of PSN-GPR119 (either 10 or 30 mg kg−1, p.o.) at t = −60 min and glucose (3 g kg−1) at t = 0 min, and right glucose AUC reactive are calculated by subtracting the baseline glucose AUC before glucose delivery. Each point or bar is the mean ± 1 s. e.m. and statistical differences compared with vehicle controls were **Po 0.01, ***Po 0.001. cotransporter 1 (SGLT1) inhibitor phloridzin (50 μM) was used to show that only apical replacement of glucose reduced SGLT1 activity. The α2-agonist UK14 304 (5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine, 1 μM, basolateral) was used to reveal non-Y receptor Gαi-coupled reductions in ISC in rodent and human colon.
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Data analysis AUCreactive data from perfused intestinal loops were calculated by subtracting the baseline 5 mM glucose (no compound) period from the PSN-GPR119-treated period. Statistical significance was determined using unpaired Student’s t-test where; *Po0.05, **P o0.01 and ***Po0.001 International Journal of Obesity (2014) 1365 – 1373
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1368 versus 5 mM glucose (no compound) control or; $Po0.05, $$P o0.01 and $$$ Po0.001 versus PSN-GPR119 (5 mM glucose). Plasma glucose was measured following in vivo glucose tolerance tests in millimolar. Comparisons were performed using one-way analysis of variance with Dunnett’s post hoc test; **Po 0.01 and ***Po0.001 versus vehicle. ISC data was analysed using GraphPad Prism, version 5.0 (GraphPad, La Jolla, CA, USA). Single comparisons were performed using Student’s t-test, whereas multiple comparisons utilised one-way analysis of variance with Dunnett’s post-hoc test; P o 0.05 was considered statistically different.
RESULTS GPR119 agonist activity, GLP-1 and insulin secretion in neuroendocrine cell lines PSN-GPR119 is a potent GPR119 agonist exhibiting an EC50 of 5.5 ± 2.0 nM for cAMP accumulation in hGPR119-transfected HEK293 cells (Supplementary Figure S1A). In GLUTag cells, PSNGPR119 increased GLP-1 secretion (EC50, 75 ± 19 nM, Supplementary Figure S1B) and induced insulin secretion from HIT-T15 cells (EC50, 90 ± 21 nM; Supplementary Figure S1C). Oral glucose tolerance tests In lean C57BL/6J mice, glucose tolerance following an oral glucose challenge improved dose-dependently after acute PSN-GPR119 (10 or 30 mg kg−1, p.o.) treatment (Figure 1). Blood glucose AUC was significantly lower following treatment with PSN-GPR119 compared with vehicle-treated animals (P o0.01; Figure 1). Despite a higher starting blood glucose concentration, the AUC for the glucose excursion following oral glucose challenge in ob/ ob mice was significantly decreased, notably to similar levels observed in the C57BL/6J mice (P o 0.001; Figure 1). Blood glucose levels following 30 mg kg−1 PSN-GPR119 treatment in lean SD and ZDF rats were significantly decreased by ~ 50% and ~ 65%, respectively, again significantly when compared with vehicle (P o 0.001 andP o 0.01 respectively; Figure 1). GPR119 mRNA expression in intestinal regions GPR119 transcripts were found in small intestinal and colonic regions from mice, rats and humans (Supplementary Figures S2A and C). In lean C57BL/6J mice and SD rats, GPR119 expression was highest in the distal gut and declined towards the duodenum, whereas human GPR119 expression was highest in the duodenum with slightly lower levels in jejunum, ileum and colon (Supplementary Figure S2C). GPR119 agonist induced GI hormone secretion from isolated loops of rat intestine Small intestines from fed male SD rats were perfused luminally for 30 min with PSN-GPR119 in 5 or 25 mM glucose to mimic pre- and post-prandial conditions. A concentration of 5 mM glucose was present as an energy source and to mimic the availability of glucose from the plasma before a meal. PSN-GPR119 concentration dependently stimulated tGIP, aGLP-1 and tPYY secretion in the presence of 5 and 25 mM glucose (P o 0.001 for all peptides after 1 and 10 μM PSN-GPR119; Figure 2). Consistent with the GLUTag model, ~ EC50 values with 5 mM glucose for tGIP, aGLP-1 and tPYY were 1.1, 0.9 and 1.1 μM, respectively, and were not significantly different in the presence 25 mM glucose. PSN-GPR119 and glucose stimulation of GI hormone secretion appeared to act additively. GPR119 agonism in lean and diseased rodent colon To investigate whether GPR119 signalling mechanisms were intact during metabolic disease, we exploited the finding that GPR119 activation causes endogenous PYY release and local epithelial Y1 International Journal of Obesity (2014) 1365 – 1373
Figure 2. Concentration responsiveness of PSN-GPR119 in lean SD rat small intestine perfused with 5 or 25 mM glucose. Rat small intestine was challenged luminally with 5 mM glucose in KH buffer for 30 min to equilibrate the preparation. Afterwards it was perfused for a further 30 min with an identical KH buffer containing either 5 or 25 mM and PSN-GPR119 (0.03–10 μM). Serosal secretion samples were analysed for total GIP, active GLP-1 and total PYY as indicated using commercially available ELISA kits. AUC reactive was calculated by subtracting the baseline AUC from the PSN-GPR119-treated AUC. Each bar is the mean ± 1 s.e.m. from six separate experiments and statistical differences are: *P o0.05, **P o0.01 and ***P o0.001 versus 5 mM glucose (no compound) control or $P o0.05, $$Po 0.01 and $$$P o0.001 versus PSN-GPR119 (5 mM glucose).
receptor-mediated anti-secretory responses23 using lean and diseased colonic tissue. First, lean C57BL/6J mice were significantly lighter than ob/ob mice of the same age (Po0.001), whereas the latter displayed significantly higher fasting blood glucose (Po0.001, Figure 3a) demonstrating obesity and hyperglycaemia. Apical PSN-GPR119 was equipotent in C57BL/6J colon (EC50 = 97.5 nM (38.5–246.9 nM)) and ob/ob colon (EC50 = 103.0 nM (50.5–210.3 nM); Figure 3b). Lean SD rats out-weighed their ZDF counterparts (Po0.001) but the latter exhibited significantly higher fasting blood glucose levels (Po0.001; Figure 3c). PSN-GPR119 potency was however similar in colonic mucosa from SD and ZDF rats (EC50 values were 126.6 nM (51.5–311.3 nM) versus 255.0 nM (34.8 nM–1.87 μM), respectively), and there were no significant differences in efficacy (Figure 3d). PSN-GPR119 sensitivity was greatest in the descending colon from lean and obese mice (P o 0.05; Supplementary Figure S3). There was no difference in PSN-GPR119 response size after apical or basolateral addition in any GI area. Subsequent PYY (10 nM) © 2014 Macmillan Publishers Limited
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Figure 3. GPR119 responses in C57BL/6J and ob/ob mice, SD and ZDF rats. (a and c) Weight and blood glucose measurements for the mice and rats used in these studies, and (b and d) concentration–response curves for PSN-GPR119 in colonic mucosa from C57BL/6J or ob/ob mice (b) and SD or ZDF rats. EC50 values are given in the text. Each bar or point is the mean ± s.e.m from the n numbers shown in parenthesis. ***P o0.001 (b and d) n = 4–5 and 4, respectively.
responses followed a similar pattern in each model (data not shown). In contrast, PSN-GPR119 responses were comparable throughout the small and large intestine from SD and ZDF rats (data not shown). Second, the receptor pharmacology was similar in lean and diseased rodent colon. Initially, Y1 receptor tone was revealed using BIBO3304, a competitive Y1 antagonist, as seen previously in C57BL/6J colon.26,29 BIBO3304 significantly increased basal ISC levels (Po 0.01), but the degree to which this occurred was reduced in ob/ob colon (Po 0.05, Figure 4a). Y1 antagonism abolished subsequent PSN-GPR119 responses in C57BL/6J and ob/ob mouse colon (Po0.01, Figure 4a) and responses to exogenous PYY in all groups (Po0.05). In C57BL/6J and ob/ob tissues, there were small exendin 4 increases in ISC, which were blocked by the GLP-1 receptor antagonist, exendin(9–39) (Po0.01, Figure 4a). In colon mucosa from SD and ZDF rats, Y1-mediated tone was similar, but interestingly a larger PSN-GPR119 response was observed in ZDF tissue (P o 0.05, Figure 4b). Consistent with the C57BL/6J and ob/ob data, PSN-GPR119 responses were abolished by BIBO3304 and there were small GLP-1 responses that were blocked by exendin(9–39) (Po 0.05 in ZDF colon; Figure 4b). Third, GPR119 responses were glucose-sensitive in mucosae from lean and diseased rodents. Apical replacement of glucose by mannitol reduced apical PSN-GPR119 responses significantly (P o 0.01) and subsequent phloridzin activity (Po 0.01; Figure 5), in line with the apical localisation of electrogenic SGLT1. Basolateral PSN-GPR119 responses were also glucose-sensitive (data not shown). Interestingly, phloridzin responses were significantly larger in ZDF compared with SD colon (P o 0.05; Figure 5b). Control PYY-Y1 and UK14 304 α2-mediated responses responses were glucose-insensitive. © 2014 Macmillan Publishers Limited
GPR119 agonism in human colon mucosa PSN-GPR119 added apically (Figure 6) or basolaterally (data not shown) caused long-lasting reductions in ISC (that were similar to PYY responses) with an EC50 of 71.3 (21.5–166.2) nM (Figure 6a). As seen in rodent colon mucosa, PSN-GPR119 responses were blocked by the Y1 antagonist, BIBO3304 (P o 0.05; Figure 6b), which also revealed Y1 tone, as seen previously.21,29 Furthermore, co-addition with the Y2 antagonist, BIIE0246 ((S)-N2-[[1-[2-[4-[(R,S)-5, 11-dihydro-6(6 h)-oxodibenz [b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl] cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1, 2,4-triazol-4-yl]ethyl]-argininamide) raised basal ISC levels further than BIBO3304 alone, and the combination of the two Y antagonists abolished subsequent PSN-GPR119 (P o0.05; Figure 6b) and PYY responses, but had no effect on UK14 304 α2-mediated responses anti-secretory responses (data not shown). Finally, PSN-GPR119 responses were glucose sensitive (P o 0.05; Figure 6c) and the effects of SGLT1 inhibitor, phloridzin were also reduced (Po 0.05) when apical glucose was replaced by mannitol. As seen in rodent mucosa, PYY-Y1 responses were not glucose sensitive in human colon.
DISCUSSION Few studies have shown intestinal efficacy with selective GPR119 agonists in normal and T2D rodent models9,11,12,30 and as yet none have compared this with efficacy in human intestinal tissue. Our study shows that luminal delivery of a GPR119 agonist stimulates K- and L-cell activity acutely in rat intestine. PSNGPR119 was highly potent, stimulating insulin and GLP-1 from secretory cell lines. As GPR119-activated insulin secretion is well International Journal of Obesity (2014) 1365 – 1373
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Figure 4. Blockade of Y1 receptors inhibits GPR119 agonism and PYY responses in descending colon mucosa from, in (a) mouse, (b) rat models. Vehicle (0.1% dimethyl sulphoxide, first bar) or Y1 antagonist BIBO3304 (300 nM, BIBO) was added in the absence or presence of 1 μM exendin (9–39) (BIBO+9–39). After vasoactive intestinal polypeptide (VIP) (30 nM, data not shown), PSN-GPR119 (1 μM, apical), PYY (10 nM) and exendin 4 (100 nM) were added sequentially and their anti-secretory effects pooled to give the means ± 1 s.e.m. (n, numbers in parenthesis). Control responses to exendin 4 (100 nM, Ex 4) are also included. Statistical differences between vehicle and antagonist-pretreated agonist mean responses are as shown. *Po 0.05, **P o0.01, ***P o0.001. Differences between lean and diseased rodent tissue responses are shown by a vertical line, *Po0.05.
established, we set out to determine the relative importance of this receptor on gut peptide release and to evaluate the peptide pharmacology involved in human colon. GPR119 expression was highest in mouse and rat colon, consistent with L-cell distribution along rodent GI tract. Interestingly, we observed a more uniform GPR119 distribution along the human GI tract with slightly elevated expression in the duodenum. GPR119 levels may be higher in the upper GI tract of humans relative to rodents; however, the source of the human gut RNA was pooled from multiple donors (because GPR119 transcript expression is low, L cells accounting for o1% of the total mucosal cell population), whereas the rodent intestinal RNA was sourced from individual animals, which may also account for some differences. In vivo, oral PSN-GPR119 improved glucose tolerance significantly in lean and diseased rodents. This is consistent with other studies showing that GPR119 agonism (acute or chronic) leads to effective glycaemic control.3,8,9,11,14,30 PSN-GPR119 (30 mg kg−1) was as efficacious (50–60% glucose lowering in an oral glucose tolerance test) in lean, as it was in rodents with T2D. The secretion profiles of GIP and GLP-1 in patients with T2D are well established; levels of GIP in systemic blood are normal in pre- and postprandial conditions. However, patients with T2D are insensitive to circulating GIP.31,32 Circulating levels of GLP-1, in contrast are reduced in T2D.32 The exact mechanism of L-cell impairment is unknown, but it is not thought to be via increased clearance of GLP-1.33 Exogenous administration has shown that the insulinotropic activity of GIP is defective, whereas that of GLP-1 remains in T2D. The insulinotropic, glucagonostatic and gastric emptying actions of GLP-1 are preserved in T2D.32,34,35 These data indicate International Journal of Obesity (2014) 1365 – 1373
that the contribution of each hormone towards glucose lowering in lean and diseased models may differ and in fact recent data in mice indicates L-cell activity is more important for acute regulation of glucose homeostasis.36 Unfortunately, we were unable to determine the relative contribution of GIP, GLP-1 or PYY to the improvement in glucose tolerance on our models. However, the net effect is that PSN-GPR119 ameliorates postprandial blood glucose concentrations in rodent models of diabetes and obesity indicating its potential to improve glucose tolerance in vivo. Luminal perfusion of PSN-GPR119 into loops of rat small intestine caused concentration-dependent increases in tGIP, aGLP-1 and tPYY secretion (an effect we have also seen with luminal OEA25). Peptide secretion was further enhanced by elevating luminal glucose from 5 to 25 mM, demonstrating the existence of further capacity for the K and L cells to secrete GIP, GLP-1 and PYY. GPR119 agonism and glucose acted in an additive manner, consistent with the recent report that GPR119 agonists stimulate GLP-1 secretion glucose independently.37 The notion that intestinal GPR119 agonism leads to an incretin effect is supported by studies showing that luminal OEA application in vivo caused significant GLP-1 release compared with i.v. administration of OEA at a 200-fold higher concentration.12 Another in-house small-molecule GPR119 agonist, PSN632408, has been shown to stimulate L-cell-derived PYY responses in human and murine colon.23 Apical or basolateral PSN632408 caused equivalent responses, through PYY activation of epithelial Y1 receptors. PSN632408 was less potent than PSN-GPR119 in the present study, exhibiting an EC50 of 5.7 μM in mouse colonic mucosa, whereas in human colonic mucosa, 10 μM PSN632408 © 2014 Macmillan Publishers Limited
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Figure 5. Glucose sensitivity of apical PSN-GPR119 (1 μM) in the presence of glucose (11.1 mM) both sides or following replacement with mannitol (11.1 mM) on the apical side only in C57BL/6J mice (a) and SD rats (b). Subsequent phloridzin (Phlor, 50 μM, apically) and PYY (10 nM) responses are also shown. Values are the mean − 1 s.e.m. from n numbers as shown. Statistical differences between responses obtained with glucose both sides versus mannitol apically, are shown, *Po0.05, **Po0.01, ***Po0.001. There were no differences between lean control and diseased mucosal responses except for phloridzin responses, which were significantly larger in ZDF than SD rat colon mucosa, *Po0.05 (vertical line).
caused a maximal mucosal response23 similar to that achieved here with 0.3–1 μM PSN-GPR119. To evaluate the efficacy of PSN-GPR119 during diabetes and obesity, we used ZDF rats and ob/ob mice as diseased counterparts to lean SD rats and C57BL/6J mice. Consistent with a 10-fold increase in potency over PSN632408, PSN-GPR119 (potency at mouse GPR119 receptors of 7.9 versus 0.18 μM, respectively) exhibited similar increased potency (5.7 versus 0.1 μM) in C57BL/6J colon mucosa. GPR119-induced PYY-Y1 signalling was consistent in diseased and lean rodent tissue and the responses to PSN-GPR119 were equivalent in each gut region. The lack of apparent sidedness to PSN-GPR119 mucosal responses was most likely due to the high permeability of PSN-GPR119, which precluded determination of the exact location of GPR119 (that is, apical alone or both apical and basolateral membranes). If GPR119 receptors are apically targeted, then their stimulation by gut-restricted agonists should limit off-target effects and minimise potential detrimental effects, for example, on fatty acid metabolism in skeletal muscle.38 Interestingly, we observed reduced Y1 tone in ob/ob colon, potentially a consequence of reduced PYY analogue, neuropeptide Y and Y1 colonic expression. Imai and co-workers have shown reduced neuropeptide Y and Y1 expression in pancreatic islets of ob/ob mice.39 Additionally, GPR119 responses (with 1 μM PSN-GPR119) were slightly larger in ZDF versus SD colon. Although GPR119 expression is known to alter in disease and is increased in islets from obese rats,13 it would be interesting to determine the receptor levels in ZDF and SD rat intestine. In lean and diseased mucosae, PSN-GPR119 responses were increased consistently in © 2014 Macmillan Publishers Limited
the presence of glucose, indicating that GPR119 agonism should reduce the risk of hypoglycemia in a diabetic population. However, in colonic preparations GPR119 agonism was not abolished when glucose was removed, suggesting that PSNGPR119 may exert some glucose-independent activity, as seen for GPR119 agonism in GLUTag cells and murine primary colonic cultures, but not in insulin-secreting pancreatic cells.37 We also observed larger phloridzin responses in ZDF compared with SD colon, potentially revealing alterations in colonic SGLT1 expression caused by disease. Normally, glucose is cleared from the lumen in the upper small intestine, so glucose reaching the colon in ZDF rats would manifest as larger responses to phloridzin. Upregulation of colonic SGLT1 activity indicates malabsorption of glucose in the ZDF model. Consistent with our in vivo studies, downstream peptide signalling pathways (PYY-mediated) were clearly functionally intact during disease, as the responses to PSN-GPR119 in both diabetic ZDF and obese and diabetic ob/ob models were maintained, in line with our hypothesis. The same PSN-GPR119initiated PYY-Y1 signalling occurred in human colon, and was comparable to the glucose-sensitive PSN632408 signalling described previously.23 These findings suggest that intestinal GPR119 stimulation has the potential for glucose lowering in man and may be comparable to that observed in rodent T2D models here and in other recent studies.30,40,41 In summary, PSN-GPR119 is a potent GPR119 tool compound that significantly lowers post-prandial glucose in lean and diseased models of T2D; luminal delivery of PSN-GPR119 to isolated loops of rat small intestine stimulates secretion of GIP, International Journal of Obesity (2014) 1365 – 1373
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1372 mimic more closely, the success of bariatric surgery in treating T2D through a gut-based therapeutic approach. GPR119 compounds are already in the clinic for the treatment of T2D and human data have been reported7,42 ( http://clinicaltrials.gov) showing modest glucose-lowering activity. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS SP, OJM, JW, MS, UWB and T-AC were employees of Prosidion Ltd at the time of the study, and we thank RenaSci Ltd (Nottingham, UK) for conducting the oral glucose tolerance test studies.
AUTHOR CONTRIBUTIONS SP and HMC were responsible for the conception and design of the experiments and writing of the article. OJM, IRT and JW were also responsible for collection, analysis, interpretation and presentation of the data. UWB and T-AC were responsible for the oral glucose tolerance test studies. MS was responsible for reviewing the article.
REFERENCES
Figure 6. The effect of PSN-GPR119 on normal human colon mucosa (a) concentration–response relationship for PSN-GPR119 (added apically, n = 3) and (b) Y1 receptor blockade (300 nM BIBO3304; +BIBO) inhibits GPR119 agonism (1 μM PSN-GPR119, apical) ± a Y2 receptor antagonist (1 μM BIIE0246; +BIBO & BIIE). (c) Glucose sensitivity of apical PSN-GPR119 (1 μM) responses in the presence of glucose both sides (11.1 mM) or, with apical mannitol (11.1 mM) in place of glucose. Subsequent phloridzin (Phlor, 50 μM, apically) and PYY (100 nM) responses are also shown. Values are the mean ± 1 s.e. m. from n numbers in parenthesis. Statistical differences are shown, *Po 0.05.
GLP-1 and PYY; and GPR119 agonism activates anti-secretory responses through PYY-mediated Y1 receptor activation in colonic mucosa of lean and T2D rodent models. PSN-GPR119 activity in human colon was comparable to that of lean and T2D rodent models and we conclude that L-cell-derived PYY, in addition to GIP and GLP-1, may contribute towards glucose lowering in man. The benefits of releasing more than one endogenous peptide from the enteroendocrine cell population in a glucose-dependent manner could offer a therapeutic advantage to modulate insulinotropic pathways with reduced risk of hypoglycemic, and International Journal of Obesity (2014) 1365 – 1373
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Supplementary Information accompanies this paper on International Journal of Obesity website (http://www.nature.com/ijo)
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International Journal of Obesity (2014) 1365 – 1373
