| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Arena Pharmaceuticals, San Diego, California 92121
Address all correspondence and requests for reprints to: James Leonard, Arena Pharmaceuticals, 6166 Nancy Ridge Drive, San Diego, California 92121. E-mail: jleonard{at}arenapharm.com.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
G protein-coupled receptors (GPCRs) are important modulators of GLP-1 release. GIP is a potent stimulator of GLP-1 release in most in vitro models (7, 8, 9). Nutrient stimulation of GIP release in the duodenum likely enhances in turn the timely release of ileal GLP-1, thus ensuring its effective contribution to enhanced β-cell function (9). In addition to GIP, a variety of neurotransmitters and neuropeptides stimulate GLP-1 release from isolated perfused ileum or colon (reviewed in Ref. 10), most notably gastrin-releasing peptide, calcitonin gene-related peptide, and acetylcholine, the latter of these acting via muscarinic receptors (8, 11, 12, 13, 14, 15, 16). Bile acids and free fatty acids may also act via GPCRs to stimulate GLP-1 release (16, 17, 18). Thus far, however, none of these mechanisms have been established as effective therapeutic strategies for the treatment of non-insulin-dependent diabetes mellitus. Non-insulin-dependent diabetes mellitus patients exhibit GIP resistance (19), whereas other GPCRs associated with GLP-1 release have not been well investigated in vivo.
Previously (20) we showed that the GPR119 agonist AR231453 acts directly on pancreatic β-cells to enhance glucose-dependent insulin release. AR231453 markedly improves oral glucose tolerance, but this effect is entirely lost in mice lacking GPR119. Interestingly, the effectiveness of AR231453 is somewhat reduced when glucose is delivered ip. These data suggest that GPR119 may also promote glycemic control via incretin-based mechanisms. Here we show that GPR119 expression is consistent with a role in GLP-1 release. Moreover, activation of GPR119 stimulates marked incretin hormone release in vitro and in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
For RT-PCR, the same human cDNA samples were incubated with the GPR119 forward primer 5'-CATTGCCGGGCTGTGGTTAGTGTC-3' and the GPR119 reverse primer 5'-GGCATAGATGAGTGGGTTGA-GCAG-3'. PCR was performed at 94 C for 2 min and then 36 cycles of 94 C for 30 min, 60 C for 30 min, and 72 C for 1 min, followed by 72 C for 7 min. The expected product from this amplification is 466 bp. A human S9 control reaction was run in parallel, using the S9 forward primer 5'-CCGGAGCTGGGTTTGTCG-3' and the S9 reverse primer 5'-AAGCGCTGATCCTGTTTATTTG-3'. The reaction conditions were the same as those used for GPR119, except that a 59 C annealing temperature was used and the number of amplification cycles was 25. The expected amplification size from this reaction is 641 bp.
RNase protection analysis of mouse GPR119 expression was performed by using mouse tissue total RNA purchased from BD CLONTECH (Palo Alto, CA) and a riboprobe containing a partial mouse GPR119 mRNA sequence. Briefly, a 255-bp fragment of mouse GPR119 cDNA (corresponding to 127 bp 5' untranslated region and 128 bp 5' open reading frame) was cloned into pCR II TOPO (Invitrogen, Carlsbad, CA). The plasmid was linearized by restriction enzyme BamH1 digestion and used as DNA template to synthesize a 356-bp 32P-labeled riboprobe (containing antisense sequence to the aforementioned region of mouse GPR119 and 101 bp of sequence derived from pCRII TOPO) via in vitro transcription using T7 RNA polymerase (Maxiscript kit; Ambion, Austin, TX). The probe was hybridized with 20 µg of total RNA, followed by digestion with RNase using reagents provided by a RPA III kit (Ambion). Protected probe fragments were separated on a 5% denaturing polyacrylamide gel and visualized by autoradiography.
Northern blot analysis of GPR119 in the mouse L-cell lines GLUTag (kindly provided by Dr. Daniel Drucker, University of Toronto, Toronto, Canada) and STC-1, and in the mouse β-cell lines MIN-6 (kindly provided by Dr. Jeffery Pessin, State University of New York-Stony Brook), HIT-T15, and NIT-1 (obtained from American Type Culture Collection, Manassas, VA) was conducted using 10 µg of total RNA isolated from the cultured cells and a 32P-labeled probe derived from mouse GPR119 cDNA. The hybridization signals were visualized by autoradiography.
In situ hybridization
Duodenum, ileum, and proximal colon were collected and frozen immediately in optimum cutting temperature compound in embedding molds over dry ice. Tissues were sectioned (12 µm) on a cryostat and collected on regular SuperFrost plus slides (VWR, West Chester, PA). The following plasmids were used for probe synthesis. For GPR119, the full-length mouse sequence was inserted into pCR4-TOPO (Invitrogen). For preproglucagon, a 544-bp fragment was inserted into pCRII-TOPO (Invitrogen). For GIP, a 400-bp PCR fragment at the 5' end was inserted into pCR4-TOPO. Sense and antisense 33P-radiolabeled (GPR119) or digoxigenin (DIG)-labeled (GLP-1, GIP) probes were generated using standard protocols. Tissue sections were fixed in 4% paraformaldehyde in phosphate buffer [0.1 M (pH 7.4)] for 30 min at room temperature, rinsed three times in 1x PBS, and acetylated in 0.1 M triethanolamine (pH 8.0) for 2 min and then briefly in the same buffer containing 0.25% acetic anhydride. Slides were then rinsed for 5 min in 1x PBS and dehydrated through graded alcohol concentrations and air dried. Radiolabeled probes were diluted in 2x hybridization buffer [1.2 M sodium chloride, 8 mM EDTA, 0.16 M Tris (pH 7.5), 2x Denhardt’s, 0.4% sodium dodecyl sulfate, 200 mM dithiothreitol, 500 µg/ml tRNA, 50 µg/ml polyA, 50 µg/ml polyC] to yield an approximate concentration of 16 x 106 cpm/slide. Salmon sperm DNA was added at a final concentration of 20 µg/slide and DIG-labeled probe was added to a final concentration of 500 ng/slide. Dextran sulfate/Formamide (20%) was added to give a 1:1 ratio with 2x hybridization buffer.
Diluted probe was placed on slides, coverslipped, and incubated at 55 C for 16–18 h in plastic trays humidified with 1x PBS. Coverslips were floated off with 1 mM dithiothreitol/4x saline sodium citrate (SSC) [600 mM sodium chloride and 60 mM sodium citrate (pH 7.2)], and sections were subsequently washed once in 4x SSC for 10 min, incubated in ribonuclease A (200 µg/ml) for 60 min in a 37 C water bath, and then rinsed in 2x, 1x, and 0.5x SSC for 5 min each. Sections were washed to a final stringency of 0.1x SSC at 65 C for 1 h and then washed twice in 0.1x SSC and then in TN [100 mM Tris (pH 7.5), 150 mM NaCl] for 5 min. Sections were then placed in 0.5% casein/TN blocking solution for 30 min and then incubated for 2 h with antidigoxigenin-AP antibody (Roche, Indianapolis, IN; no. 1093274) in 0.5% casein/TN solution. Sections were then washed three times, 2 min each in TN, and then three times for 5 min each in 100 mM Tris (pH 9.5), 100 mM NaCl, and 50 mM MgCl2. After the last wash, sections were incubated in color reagent [0.2 mg/ml levamisole, 3.4 µl/ml 4-nitro blue tetrazolium chloride (Roche; no. 1383213), 3.5 µl/ml 5-bromo-4-chloro-3-indoyl-phosphate, 4-toluidine salt (Roche; no. 1383221) in 100 mM Tris (pH 9.5), 100 mM NaCl, and 50 mM MgCl2 and 0.22 µM sterile filtered] for 20–30 min and reaction stopped in Tris/EDTA buffer for 30 min. Antibody was striped off by incubating sections in 0.1 M glycine and 0.5% Triton X-100 for 10 min and washed in water. Sections were fixed in 2.5% gluteraldehyde for 1–2 h and washed with water and then air dried. Once the section dried, they were exposed to x-ray sensitive film (Bio-Max; Eastman Kodak Co., Rochester, NY) for 2–7 d and dipped in photographic emulsion (Ilford Scientific K.5D emulsion in gel form, no. 17537; Polysciences, Warrington, PA) dried, and stored in a slide box with desiccant at 4 C for 4–8 wk, depending on the level of expression. After development of dipped slides following manufacturer recommendations (Kodak D19), sections were washed extensively in water and air dried and then mounted with coverslips for microscopic examination.
Membrane cAMP assay
GLUTag cells provided by Dr. Drucker were termed GLUTag-Fla. A subline of GLUTag cells was obtained by dilution cloning and termed GLUTag-Fro. Expression of GPR119 in these cells was determined by Northern blot as described above. In either case, GLUTag cells were plated at approximately 85% confluence in 15-cm tissue culture plate in DMEM medium supplemented with 10% fetal bovine serum and antibiotics. On the next day, cells were scraped off the culture plate in ice-cold scraping buffer [20 mM HEPES, 10 mM EDTA (pH7.4)], followed by centrifugation at 48,000 x g for 17 min at 4 C. Resultant pellet was washed with ice-cold membrane wash buffer [20 mM HEPES, 0.1 mM EDTA (pH7.4)] and subjected to centrifugation at 48,000 x g for 17 min at 4 C. The resultant membrane pellets were resuspended in ice-cold binding buffer [20 mM HEPES, 1 mM MgCl2, 100 mM NaCl (pH7.4)] and homogenized twice using polytron mixer PT3100 (Brinkman Instruments, Westbury, NY) at 7000 rpm for 10 sec. Plasma membrane protein concentration was determined by Bradford assay. Protein concentration of membrane preparation was adjusted to 0.2 mg/ml in the binding buffer. Compound stimulation of cAMP production with GLUTag membranes were assayed by using a Flash Plate adenylyl cyclase kit (NEN Life Science Products, Boston, MA). Briefly, GPR119 agonist AR231453 was freshly prepared at the desired concentration in 50 µl 2 x reconstitution buffer (20 mM phosphocreatine, 20 U/50 µl creatine phosphokinase, 20 µM GTP, 0.2 mM ATP, and 1 mM 3-isobutyl-1-methylxanthine) in a 96-well Flash Plate. Fifty microliters of membrane preparation described above were then added to the wells and incubated for 60 min at room temperature. Detection mix (100 µl volume) containing tracer cAMP was then added to the wells. Plates were incubated for an additional 2 h followed by washing and counting in a MicroBeta scintillation counter (Wallac, Turku, Finland). Values of cAMP per well were extrapolated from a standard cAMP curve, which was included within each assay plate.
GLP-1 in vitro secretion assay
GLUTag cells were plated in 24-well plates on d 1 in DMEM supplemented with 10% fetal bovine serum and antibiotics. Culture medium was replaced with DMEM (3 mM glucose) supplemented with 10% fetal bovine serum 24 h before analysis of GLP-1 secretion. For GLP-1 secretion, cells were washed twice with PBS and incubated with GPR119 agonist AR231453 at desired concentrations or with adenylyl cyclase stimulator forskolin at 1 µM in serum-free DMEM and 15 mM glucose for 1 h at 37 C and 5% CO2 in cell culture incubator. The supernatants were then collected and clarified by centrifugation at 500 x g and 4 C for 5 min. GLP-1 content in the supernatant was determined by an ELISA method with reagents purchased from Linco Research Laboratory [GLP-1 (active) ELISA kit, catalog no. EGLP-35K; St. Charles, MO].
In vivo experiments
C57BL/6 male mice were obtained from Harlan (Indianapolis, IN). GPR119-deficient mice were described previously (20) All in vivo animal protocols were approved by the Animal Welfare Committee of Arena Pharmaceuticals, Inc., and in adherence with government regulations. For oral glucose tolerance test, overnight fasted mice (n = 6/treatment) were given either vehicle or test compounds at desired doses via oral gavage. A glucose bolus (3 g/kg) was then delivered per orally. Plasma glucose levels were determined at desired time points over a 2-h period using blood (
5 µl) collected from tail nick and a glucose meter. For GLP-1 (active) and GIP (total) pharmacodynamic studies, vehicle or test compounds were administered orally to fasted animals, and after 30 min, a glucose bolus of 3 g/kg was administered orally. Blood was collected in Eppendorf tubes containing EDTA and a DPP-IV inhibitor (Linco Laboratory; catalog no. DPP4–010) at desired time points. Plasma samples were obtained via centrifugation at 500 x g for 20 min and assayed for active GLP-1 (as noted above) and total GIP (using Linco ELISA kit catalog no. EZRMGIP-55K).
Statistical analysis
Statistical analysis (t test) was performed using either Microsoft EXCEL (Redmond, WA) or Prism statistical methods (GraphPad, San Diego, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, A and B). The weak Taqman signal seen in most other tissues was most likely background because a similar signal is seen in mouse tissues not expressing human GPR119 (not shown). This conclusion is also consistent with the RT-PCR data, which was generated under nonquantitative conditions [36 cycles vs. cycle threshold (Ct) 31–33 cycles for Taqman on pancreatic and intestinal samples]. Thus, GPR119 is largely confined to pancreatic and intestinal tissues in humans, with extremely low or undetectable expression in all other human tissues examined.
|
). GPR119 levels in colon and small intestine were greater than those detected in pancreas and somewhat less than those seen in isolated islets. GPR119 was also highly expressed in GLP-1-producing GLUTag and STC-1 cells (Fig. 1D), suggesting that the gut expression of GPR119 might correspond to intestinal endocrine cells. Notably, GPR119 levels in these cells were similar to those seen in HIT-T15 insulinoma cells, a cell line previously shown to elicit a robust response to GPR119 agonists (20). Although GPR119 could not be detected in MIN6 cells by Northern blot, we were able to detect relatively low levels of GPR119 in these cells by Taqman (not shown). The expression of GPR119 expression in MIN6 cells may be variable, based on the data shown here and published data indicating both low and high levels of expression (21, 22).
Northern blot data indicated that all GPR119-positive cell lines contained a faint transcript migrating slightly faster than the major transcript of 2–2.5 kb. This is consistent with published data indicating that a minority of mouse GPR119 transcripts correspond to a splice variant lacking 233 bases in the 3'-untranslated region (22). The precise nature of the faint, slower migrating (3.5 kb) band is unknown.
To determine whether GPR119 was expressed in GLP-1 producing cells in vivo, we examined their colocalization in rat ileum, which corresponds to the rat intestinal subregion containing the greatest density of GLP-1-producing cells (23). DIG-labeled preproglucagon and 33P-labeled GPR119 probes were hybridized to rat ileal sections (Fig. 2, A and B). The full-length antisense GPR119 probe detected the receptor in most preproglucagon-expressing (GLP-1+) cells. By contrast, a sense GPR119 probe detected no significant hybridization to rat ileal sections (not shown), suggesting that the antisense signal corresponded to specific detection of GPR119. Eight sections from two rats were evaluated, with 74.6 ± 1.1% (298/408 overall) of ileal GLP-1+ cells also positive for GPR119.
|
). Colocalization was assessed using sections from wild-type (Fig. 2, C and D) and GPR119-deficient (Fig. 2, E and F) mice. No GPR119 hybridization signal was detected in sections from GPR119-deficient mice, whereas the signal was readily seen in preproglucagon-positive cells in wild-type littermates. As in rat ileum, the vast majority of preproglucagon-expressing cells in mouse colon were positive for GPR119 (90 ± 8%). These data imply that intestinal expression of GPR119 is largely endocrine in nature and indicate that a significant fraction of GLP-1-producing cells contain GPR119.
GPR119 agonist stimulates cAMP accumulation and GLP-1 release in GLUTag cells
To evaluate whether activation of GPR119 could directly stimulate the release of GLP-1, we first examined GPR119 agonists in GLUTag cells that endogenously express GPR119 (GLUTag-Fla) and in an isolated subline that does not express GPR119 at detectable levels (GLUTag-Fro). The GPR119 agonist AR231453 (20) significantly stimulated cAMP accumulation in GLUTag-Fla cells (Fig. 3A), with an EC50 of 4.3 nM. This is consistent with the potency of AR231453 in other cells containing GPR119 (20). Moreover, the efficacy of AR231453 approached that seen with forskolin. On the other hand, GLUTag-Fro cells did not respond to AR231453 (Fig. 3A).
|
) with an EC50 of 56 nM. As was seen with cAMP stimulation, the magnitude of these effects approximated those seen with forskolin. These data suggest that GPR119 could be a physiologically significant modulator of GLP-1 release.
GPR119 agonist increases plasma GLP-1 levels
To test this hypothesis in vivo, we evaluated the ability of AR231453 to increase plasma levels of GLP-1 (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) in C57Bl/6 mice. AR231453 is orally bioavailable and elicits significant improvement in glucose tolerance (20). Here AR231453 (10 mg/kg orally) stimulated a rapid, transient, and glucose-dependent increase in active GLP-1 levels (p < .0025 at 2 min), which was approximately 50% of the effect observed in mice treated with the DPP-IV inhibitor sitagliptin (Fig. 4A). Because GLP-1 has a very short half-life in vivo, we reasoned that the GLP-1-releasing effects of GPR119 agonists would be more evident in the presence of a DPP-IV inhibitor. Indeed, in sitagliptin-treated mice, AR231453 robustly enhanced plasma GLP-1 levels within 2 min after a glucose challenge (Fig. 4A). This effect was sustained for as long as 60 min (Fig. 4B). At 2 min after glucose challenge, the combined administration of AR231453 and sitagliptin increased plasma levels of GLP-1 (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) by 6-fold relative to control-treated animals. Interestingly, the combined delivery of these compounds stimulated GLP-1 levels by 4-fold before glucose administration. This suggests that basal secretion of GLP-1 is not strictly glucose dependent in mice and can be detected under conditions of enhanced secretion and reduced clearance.
|
, C and D). This indicates that GLP-1 stimulating activity of AR231453 is indeed mediated via GPR119, consistent with GPR119 expression in L-cells (see Fig. 2).
GLP-1 receptor blockade reduces glucose control by GPR119 agonist
In addition to these data indicating that activation of GPR119 enhances GLP-1 release, we previously showed that GPR119 agonists also stimulate insulin release via a direct action on the pancreatic β-cell (20). To assess whether the GLP-1 releasing effects contribute significantly to the overall control of glucose homeostasis by GPR119, we evaluated the efficacy of AR231453 in a glucose tolerance test, either alone or in combination with the GLP-1 receptor antagonist exendin (9–39) (25, 26, 27). At a dose known to fully antagonize GLP-1 signaling in mice (28), 25 nmol/kg exendin(9–39) significantly reduced the efficacy of AR231453 (Fig. 5). This was particularly evident 20 min after the glucose challenge, in which the effects of AR231453 are most robust, with a trend toward reduced efficacy also seen at 40 and 60 min after the glucose challenge (Fig. 5A). Consistent with these data, AR231453 in combination with the PBS vehicle inhibited the glycemic excursion by 34%, but this was reduced to 19% when AR231453 was administered in combination with exendin(9–39) (Fig. 5B).
|
). Remarkably, GPR119 was not detected in any GIP-positive cells. Similar data were obtained in mouse duodenal sections (not shown). Nevertheless, AR231453 triggered a sustained rise in plasma total GIP levels (Fig. 7A). This effect was already evident at the time of glucose delivery. Moreover, glucose-stimulated GIP release was significantly enhanced by AR231453 for at least 60 min after the oral glucose challenge. The effect on GIP was completely lost in GPR119-deficient mice (Fig. 7B), indicating that GPR119 mediates the GIP-stimulating action of AR231453. Based on the observation that GPR119 is undetectable in GIP-producing cells, this may be an indirect effect.
|
|
, A and D). When maximal doses of AR231453 and sitagliptin were administered together, this benefit was still evident but greatly diminished (not shown). In mice lacking GPR119, AR231453 coadministration had no impact on sitagliptin with regard to glucose tolerance (Fig. 8, B and C).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although our previously published data strongly suggest significant expression of GPR119 specifically in insulin-producing cells of the islet, recent published data from others suggest that pancreatic polypeptide-producing cells are the primary site of GPR119 expression in the islet (22). The reason for this apparent discrepancy is not known at present. However, specificity of our GPR119 reagents was established using mice that lack the receptor. The β-cell expression of GPR119 was also established by means of multiple independent approaches (20). On the other hand, we did not evaluate the expression of GPR119 in PP cells.
With regard to intestinal expression of GPR119, there is clearly substantial coexpression with preproglucagon in gut regions in which GLP-1 producing cells are known to be particularly abundant. However, a full characterization of GPR119 expression across various intestinal regions was not done. It remains very possible that GPR119 might be significantly expressed in other enteroendocrine cell types.
In vivo, AR231453 markedly elevated plasma GIP levels in mice, even before oral glucose delivery. GIP release is stimulated by nutrients (29, 30), including glucose (31, 32), but its release may not be strictly glucose dependent (33). Our data clearly show that both GPR119 agonists and glucose independently stimulate GIP release in vivo and that these agents cooperate to provide profound increases in plasma GIP. Interestingly, we have so far not detected colocalization of GIP and GPR119. Nonetheless, AR231453 stimulation of plasma GIP levels is a GPR119-dependent event because no such stimulation is seen in mice lacking the receptor. Thus, there may be unidentified sites of GPR119 expression that indirectly regulate GIP release. Alternatively, GPR119 may be present in GIP-producing cells at levels too low to detect by the in situ hybridization technique used here.
Numerous modulators of GLP-1 or GIP release have been identified (reviewed in Ref. 10), including GIP (7), gastrin-releasing peptide (15), calcitonin gene-related peptide (8), galanin (34), somatostatin (35), muscarinic agonists, and adrenergic agonists (11, 36). Whereas previous studies have largely relied on isolated intestinal preparations, immortalized cell lines, or anesthetized rats, to our knowledge this is the first study using oral delivery of a synthetic agonist to elicit significantly increased plasma incretin levels. Although the physiological ligand for GPR119 has not yet been identified with certainty, recent published data indicate that oleoylethanolamide (OEA) is a robust activator of the receptor (37). Intestinal OEA levels are markedly increased after a meal (38), consistent with a role for GPR119 as a regulator of incretin release. The data shown herein strongly argue that GPR119 should be considered a major regulator of the incretin system.
Both GLP-1, whose release is enhanced by GPR119, and OEA, an activator of GPR119, are known to reduce food intake (39, 40). This suggests that GPR119 agonists might also mediate feeding behavior. There are published data in support of this hypothesis using other synthetic GPR119 agonists (37). In our own studies, we have not seen GPR119-mediated effects on feeding behavior or body weight. Unpublished data with compounds significantly more potent than AR231453 in vivo (Bagnol, D., and J. Leonard) indicate that reductions in feeding occur only at very high doses (60–100 mg), far greater than those required for an effect on glucose tolerance. Moreover, these effects are retained in GPR119-deficient mice. It is possible that GPR119 agonists need significant access to certain central nervous system regions to impact food intake. Thus far, we have not obtained convincing data for expression of GPR119 in any region of the brain (e.g. see Fig. 1). However, we have not excluded whether GPR119 levels might be significantly regulated in the central nervous system under certain conditions. Finally, note that DDP-IV inhibition, which is more effective at sustaining plasma GLP-1 levels, does not raise plasma GLP-1 levels sufficiently to impact food intake or body weight (41). An interesting avenue of future investigation will be to assess whether combined administration of GPR119 agonists and DPP-IV inhibitors might increase GLP-1 levels enough to elicit bona fide effects on feeding.
A significant prediction, supported by these studies, is that incretin-releasing agents should functionally cooperate with approaches that prevent incretin degradation. Combined administration of AR231453 and sitagliptin led to significant increases in plasma GLP-1 and markedly improved oral glucose tolerance, relative to either compound alone. Oral glucose tolerance studies in GPR119-deficient mice clearly indicated that AR231453 must act via GPR119 to detect the benefit of combined administration. These data suggest that the general strategy of promoting GLP-1 release in combination with blocking its degradation may be an effective therapeutic approach to improve glucose homeostasis. Orally active GPR119 agonists may be particularly beneficial in this regard.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
First Published Online January 17, 2008
Abbreviations: DIG, Digoxigenin; DPP-IV, dipeptidyl peptidase IV; GIP, glucose-dependent insulinotropic peptide; GLP-1, glucagon-like peptide-1; GPCR, G protein-coupled receptor; OEA, oleoylethanolamide; SSC, saline sodium citrate; TN, Tris and NaCl.
Received July 16, 2007.
Accepted for publication January 8, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Nature 425:90–93[CrossRef][Medline]This article has been cited by other articles:
 |
|
 |
|
  S. M. Yoder, Q. Yang, T. L. Kindel, and P. Tso Stimulation of incretin secretion by dietary lipid: is it dose dependent? Am J Physiol Gastrointest Liver Physiol, August 1, 2009; 297(2): G299 - G305. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-i. Eiki, K. Saeki, N. Nagano, T. Iino, M. Yonemoto, Y. Takayenoki-Iino, S. Ito, T. Nishimura, Y. Sato, M. Bamba, et al. A selective small molecule glucagon-like peptide-1 secretagogue acting via depolarization-coupled Ca2+ influx J. Endocrinol., June 1, 2009; 201(3): 361 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Lan, G. Vassileva, A. Corona, L. Liu, H. Baker, A. Golovko, S. J Abbondanzo, W. Hu, S. Yang, Y. Ning, et al. GPR119 is required for physiological regulation of glucagon-like peptide-1 secretion but not for metabolic homeostasis J. Endocrinol., May 1, 2009; 201(2): 219 - 230. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. Lauffer, R. Iakoubov, and P. L. Brubaker GPR119 Is Essential for Oleoylethanolamide-Induced Glucagon-Like Peptide-1 Secretion From the Intestinal Enteroendocrine L-Cell Diabetes, May 1, 2009; 58(5): 1058 - 1066. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Ma, M. Bellon, J. M. Wishart, R. Young, L. A. Blackshaw, K. L. Jones, M. Horowitz, and C. K. Rayner Effect of the artificial sweetener, sucralose, on gastric emptying and incretin hormone release in healthy subjects Am J Physiol Gastrointest Liver Physiol, April 1, 2009; 296(4): G735 - G739. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Fujita, R. D. Wideman, M. Speck, A. Asadi, D. S. King, T. D. Webber, M. Haneda, and T. J. Kieffer Incretin release from gut is acutely enhanced by sugar but not by sweeteners in vivo Am J Physiol Endocrinol Metab, March 1, 2009; 296(3): E473 - E479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Tolhurst, F. Reimann, and F. M. Gribble Nutritional regulation of glucagon-like peptide-1 secretion J. Physiol., January 1, 2009; 587(1): 27 - 32. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Kim and J. M. Egan The Role of Incretins in Glucose Homeostasis and Diabetes Treatment Pharmacol. Rev., December 1, 2008; 60(4): 470 - 512. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-R. Berthoud Paying the Price for Eating Ice Cream: Is Excessive GLP-1 Signaling in the Brain the Culprit? Endocrinology, October 1, 2008; 149(10): 4765 - 4767. [Full Text] [PDF]
|
|
|
|
|
|
S. Edfalk, P. Steneberg, and H. Edlund Gpr40 Is Expressed in Enteroendocrine Cells and Mediates Free Fatty Acid Stimulation of Incretin Secretion Diabetes, September 1, 2008; 57(9): 2280 - 2287. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Costanzi, S. Neumann, and M. C. Gershengorn Seven Transmembrane-spanning Receptors for Free Fatty Acids as Therapeutic Targets for Diabetes Mellitus: Pharmacological, Phylogenetic, and Drug Discovery Aspects J. Biol. Chem., June 13, 2008; 283(24): 16269 - 16273. [Full Text] [PDF]
|
|
|
|
|
|
L. Lauffer, R. Iakoubov, and P. L. Brubaker GPR119: "Double-Dipping" for Better Glycemic Control Endocrinology, May 1, 2008; 149(5): 2035 - 2037. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
)] or a GLUTag subline lacking GPR119 [GLUTag-Fro (
)] were used in the cAMP assay. The response of these lines to 1 µM forskolin (Fsk) is also shown. Expression of GPR119 in GLUTag-Fla and GLUTag-Fro cells is shown by Northern blot on the right. B, GLP-1 secretion. GLUTag cells (corresponding to GLUTag-Fla) were treated with AR231453 at the indicated concentration, 1 µM forskolin (Fsk), or vehicle (basal), and GLP-1 secretion was assayed as described in Materials and Methods. Data are expressed as mean (n = 3) GLP-1 levels ± SEM. *, P < 0.05 and **, P < 0.01 denote a statistically significant difference between vehicle vs. compound-treated cells using a t test.
), 10 mg/kg AR231453 (
), or 1 mg/kg DPP-IV inhibitor sitagliptin (
). Glucose (3 g/kg, orally) was delivered 30 min later and blood samples were collected at the indicated times thereafter. Data are given as vertical scatter plot, with each data point representing plasma GLP-1 value from one individual mouse. D, GLP-1 release in GPR119-deficient mice. The experiment was carried out as in D except male GPR119-deficient mice were used.
, 25 nmol/kg, delivered ip), or treated with dosing vehicle in combination with PBS (
) or exendin(9–39) (
), or combination of AR231453 (1 mg/kg) and sitagliptin (0.1 mg/kg) (
). Animals were first dosed with compounds or vehicle 30 min (at time –30) before glucose bolus. Results are given in mean glucose levels (±SEM) from n = 6 animals in each group. Asterisks (**) and (*) denote statistical difference at P < 0.01 and < 0.05, respectively, in glucose levels between mice treated with DPP-IV inhibitor alone and mice treated with DPP-IV inhibitor in combination with AR231453. B and C, Lack of cooperativity between AR231453 and DPP-IV inhibitor in GPR119-deficient (KO) mice. The oral glucose tolerance test was performed as in A using male GPR119-deficient mice (C) or their wild-type (WT) littermates (B), treated with vehicle (