Intestinal Glucagon-like Peptide-1 Research Articles

小豆子葉部のα-グルコシダーゼ活性およびGLP-1分泌に与える影響

小豆子葉部のα-グルコシダーゼ活性およびGLP-1分泌に与える影響

Transformation of postingestive glucose responses after deletion of sweet taste receptor subunits or gastric bypass surgery

The glucose-dependent secretion of the insulinotropic hormone glucagon-like peptide-1 (GLP-1) is a critical step in the regulation of glucose homeostasis. Two molecular mechanisms have separately been suggested as the primary mediator of intestinal glucose-stimulated GLP-1 secretion (GSGS): one is a metabotropic mechanism requiring the sweet taste receptor type 2 (T1R2) + type 3 (T1R3) while the second is a metabolic mechanism requiring ATP-sensitive K(+) (K(ATP)) channels. By quantifying sugar-stimulated hormone secretion in receptor knockout mice and in rats receiving Roux-en-Y gastric bypass (RYGB), we found that both of these mechanisms contribute to GSGS; however, the mechanisms exhibit different selectivity, regulation, and localization. T1R3(-/-) mice showed impaired glucose and insulin homeostasis during an oral glucose challenge as well as slowed insulin granule exocytosis from isolated pancreatic islets. Glucose, fructose, and sucralose evoked GLP-1 secretion from T1R3(+/+), but not T1R3(-/-), ileum explants; this secretion was not mimicked by the K(ATP) channel blocker glibenclamide. T1R2(-/-) mice showed normal glycemic control and partial small intestine GSGS, suggesting that T1R3 can mediate GSGS without T1R2. Robust GSGS that was K(ATP) channel-dependent and glucose-specific emerged in the large intestine of T1R3(-/-) mice and RYGB rats in association with elevated fecal carbohydrate throughout the distal gut. Our results demonstrate that the small and large intestines utilize distinct mechanisms for GSGS and suggest novel large intestine targets that could mimic the improved glycemic control seen after RYGB.

Meal-contingent intestinal lymph sampling from awake, unrestrained rats

Standard procedures for intestinal lymph collection involve continuous, quantitative drainage of the lymph fluid in anesthetized or restrained animals that are often euthanized within 48 h. We here describe a novel technique for the nonocclusive cannulation of the major intestinal lymph duct in rats that allows for repetitive in vivo sampling of intestinal lymph from unrestrained, awake, and ad libitum-fed animals. The distinctive feature of this novel technique is that a 5- to 7-mm long piece of Vialon tubing (OD/ID: 0.8/0.7 mm) with a small hole in its wall is first implanted into the major intestinal lymph duct for stabilization. The tapered tip (OD: ≈0.1 mm) of the catheter is then inserted into the hole of the tubing and fixed in place with a polyamid suture and a drop of tissue glue. In our hands, catheters implanted this way remain patent for up to 6 wk after surgery. In an initial experiment we collected lymph from six adult rats before (0) and 15, 30, 45, 60, 75, 90, 120, and 180 min (120 μl, each) after the onset of isocaloric (12.5 kcal) low-fat (LF) or high-fat (HF) test meals and measured active glucagon-like peptide-1 (GLP-1). Intestinal lymphatic GLP-1 concentration increased (P < 0.05) from ≈4 pmol/l (0 min) to a peak of 33 ± 6 (means ± SE) or 22 ± 4 pmol/l at 15 (HF) or 30 min (LF) after meal onset and gradually returned to baseline levels by 180 min. With this new technique fewer animals are required to generate physiologically relevant data for various aspects of gastrointestinal physiology that involve the lymphatic system. Furthermore, the advantage of this system is that the animal can act as its own control when the effect of different experimental protocols is tested.

Perspective: TGR5 (Gpbar-1) in liver physiology and disease

Bile acids are signaling molecules with diverse endocrine functions. Bile acid effects are mediated through the nuclear receptor farnesoid X receptor (FXR), the G-protein coupled receptor TGR5 (Gpbar-1) and various other bile acid sensing molecules. TGR5 is almost ubiquitously expressed and has been detected in different non-parenchymal cells of human and rodent liver. Here, TGR5 has anti-inflammatory, anti-apoptotic and choleretic functions. Mice with targeted deletion of TGR5 are protected from the development of cholesterol gallstones. Administration of specific TGR5 agonists lowers serum and liver triglyceride levels thereby reducing liver steatosis. Furthermore, activation of TGR5 promotes intestinal glucagon-like peptide-1 (GLP-1) release, thereby modulating glucose homeostasis and energy expenditure in brown adipose tissue and skeletal muscle. Additionally, TGR5 exerts anti-inflammatory actions resulting in decreased liver injury in animal models of sepsis. These beneficial effects make TGR5 an attractive therapeutic target for metabolic diseases, such as diabetes, obesity, atherosclerosis and steatohepatitis.

When GLP-1 hits the liver: a novel approach for insulin resistance and NASH

nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum ranging from simple steatosis to steatohepatitis (NASH), increasing fibrosis and eventually, cirrhosis ([22][1]). Importantly, NASH accompanied by fibrosis and severe inflammation is the most relevant predictor for disease progression

Potential New Approaches to Modifying Intestinal GLP-1 Secretion in Patients with Type 2 Diabetes Mellitus : Focus on Bile Acid Sequestrants.

Type 2 diabetes mellitus is associated with a progressive decline in insulinproducing pancreatic β-cells, an increase in hepatic glucose production, and a decrease in insulin sensitivity. The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) stimulate glucose-induced insulin secretion; however, in patients with type 2 diabetes, the incretin system is impaired by loss of the insulinotropic effects of GIP as well as a possible reduction in secretion of GLP-1. Agents that modify GLP-1 secretion may have a role in the management of type 2 diabetes. The currently available incretin-based therapies, GLP-1 receptor agonists (incretin mimetics) and dipeptidyl peptidase-4 (DPP-4) inhibitors (CD26 antigen inhibitors) [incretin enhancers], are safe and effective in the treatment of type 2 diabetes. However, they may be unable to halt the progression of type 2 diabetes, perhaps because they do not increase secretion of endogenous GLP-1. Therapies that directly target intestinal L cells to stimulate secretion of endogenous GLP-1 could possibly prove more effective than treatment with GLP-1 receptor agonists and DPP-4 inhibitors. Potential new approaches to modifying intestinal GLP-1 secretion in patients with type 2 diabetes include G-protein-coupled receptor (GPCR) agonists, α-glucosidase inhibitors, peroxisome proliferator-activated receptor (PPAR) agonists, metformin, bile acid mimetics and bile acid sequestrants. Both the GPCR agonist AR231453 and the novel bile acid mimetic INT-777 have been shown to stimulate GLP-1 release, leading to increased insulin secretion and improved glucose tolerance in mice. Similarly, a study in insulin-resistant rats demonstrated that the bile acid sequestrant colesevelam increased GLP-1 secretion and improved glucose levels and insulin resistance. In addition, the bile acid sequestrant colestimide (colestilan) has been shown to increase GLP-1 secretion and decrease glucose levels in patients with type 2 diabetes; these results suggest that the glucose-lowering effects of bile acid sequestrants may be partly due to their ability to increase endogenous GLP-1 levels. Evidence suggests that GPCR agonists, α-glucosidase inhibitors, PPAR agonists, metformin, bile acid mimetics and bile acid sequestrants may represent a new approach to management of type 2 diabetes via modification of endogenous GLP-1 secretion.

A new animal model for intestinal lymph sampling for a new look at intestinal physiology

Standard procedures for intestinal lymph collection involve continuous, quantitative sampling of lymph fluid and anesthetized or restrained animals that are often sacrificed after 48 h. We here describe a novel technique for the non-occlusive cannulation of the major mesenteric lymph duct in rats that allows for repetitive in-vivo sampling of intestinal lymph from unrestrained, awake and ad libitum-fed animals. The distinctive feature of this novel technique is that a 5–7 mm long piece of VialonTM tubing (OD/ID: 0.8/0.7 mm) with a small hole in its wall is first implanted into the mesenteric lymph duct for stabilization. The tapered tip (OD: 0.1 mm) of the catheter is then inserted into the hole of the tubing and fixed in place with a polyamid suture and a drop of tissue adhesive. In our hands, catheters implanted this way remain patent for up to 6 weeks after surgery. In one experiment we collected lymph from 6 adult rats prior to (0) and 15, 30, 45, 60, 75, 90,120, and 180 min (120 μl, each) after the onset of isocaloric (12 kcal) low fat (LF) or high fat (HF) test meals and measured active glucagon-like peptide-1 (GLP-1). Intestinal lymphatic GLP-1 concentration increased (p 5 pMol (0 min) to a peak of 30 ± 5 (mean ± SEM) or 21 ± 5 pMol at 15 (HF) or 30 min (LF) after meal onset and gradually returned to baseline levels by 180 min. With this new technique few animals suffice to generate physiologically relevant data for various aspects of gastrointestinal physiology that involve the lymphatic system.

The separate and combined impact of the intestinal hormones, GIP, GLP-1, and GLP-2, on glucagon secretion in type 2 diabetes

Type 2 diabetes mellitus (T2DM) is associated with reduced suppression of glucagon during oral glucose tolerance test (OGTT), whereas isoglycemic intravenous glucose infusion (IIGI) results in normal glucagon suppression in these patients. We examined the role of the intestinal hormones glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and glucagon-like peptide-2 (GLP-2) in this discrepancy. Glucagon responses were measured during a 3-h 50-g OGTT (day A) and an IIGI (day B) in 10 patients with T2DM [age (mean ± SE), 51 ± 3 yr; body mass index, 33 ± 2 kg/m(2); HbA(1c), 6.5 ± 0.2%]. During four additional IIGIs, GIP (day C), GLP-1 (day D), GLP-2 (day E) and a combination of the three (day F) were infused intravenously. Isoglycemia during all six study days was obtained. As expected, no suppression of glucagon occurred during the initial phase of the OGTT, whereas significantly (P < 0.05) lower plasma levels of glucagon during the first 30 min of the IIGI (day B) were observed. The glucagon response during the IIGI + GIP + GLP-1 + GLP-2 infusion (day F) equaled the inappropriate glucagon response to OGTT (P = not significant). The separate GIP infusion (day C) elicited significant hypersecretion of glucagon, whereas GLP-1 infusion (day D) resulted in enhancement of glucagon suppression during IIGI. IIGI + GLP-2 infusion (day E) resulted in a glucagon response in the midrange between the glucagon responses to OGTT and IIGI. Our results indicate that the intestinal hormones, GIP, GLP-1, and GLP-2, may play a role in the inappropriate glucagon response to orally ingested glucose in T2DM with, especially, GIP, acting to increase glucagon secretion.

Electrical activity-triggered glucagon-like peptide-1 secretion from primary murine L-cells.

Glucagon like peptide 1 (GLP-1) based therapies are now widely used for the treatment of type 2 diabetes. Developing our understanding of intestinal GLP-1 release may facilitate the development of new therapeutics aimed at targeting the GLP-1 producing L-cells. This study was undertaken to characterise the electrical activity of primary L-cells and the importance of voltage gated sodium and calcium channels for GLP-1 secretion. Primary murine L-cells were identified and purified using transgenic mice expressing a fluorescent protein driven by the proglucagon promoter. Fluorescent L-cells were identified within primary colonic cultures for patch clamp recordings. GLP-1 secretion was measured from primary colonic cultures. L-cells purified by flow cytometry were used to measure gene expression by microarray and quantitative RT-PCR. Electrical activity in L-cells was due to large voltage gated sodium currents, inhibition of which by tetrodotoxin reduced both basal and glutamine-stimulated GLP-1 secretion. Voltage gated calcium channels were predominantly of the L-type, Q-type and T-type, by expression analysis, consistent with the finding that GLP-1 release was blocked both by nifedipine and ω-conotoxin MVIIC. We observed large voltage-dependent potassium currents, but only a small chromanol sensitive current that might be attributable to KCNQ1. GLP-1 release from primary L-cells is linked to electrical activity and activation of L-type and Q-type calcium currents. The concept of an electrically excitable L-cell provides a basis for understanding how GLP-1 release may be modulated by nutrient, hormonal and pharmaceutical stimuli.

Release of Satiety Hormones in Response to Specific Dietary Proteins Is Different between Human and Murine Small Intestinal Mucosa

Background/Aim: High protein diets are the most effective to stimulate cholecystokinin (CCK) and glucagon-like peptide 1 (GLP-1) release; however, which proteins are the most potent is not known. Here, the effects of specific dietary proteins on intestinal CCK and GLP-1 release were examined. Methods: Duodenal biopsies of 10 healthy male subjects and 10 male rats were taken and placed in an Ussing chamber system. The biopsies were exposed on the luminal side to buffer, egg protein, codfish protein, ovomucoid, pea protein, and wheat protein. After an exposure time of 2 h, samples were taken from the serosal side. Results: Pea protein and wheat protein increased CCK and GLP-1 release in human duodenal tissue, while codfish protein only increased CCK release. No elevated levels of CCK and GLP-1 were found after exposure of rat tissue to different proteins. Conclusion: Pea and wheat protein are the most potent stimulators of CCK and GLP-1 release in human duodenal tissue, and may therefore be good dietary additives in weight management.

M1718 Fructo-Oligosaccharide (FOS) Supplementation Increases Enteroendocrine L Cells Containing GLP-1 and SCFA Receptor GPR43 (Ffa2) in the Large Intestine

Background and aim: Glucagon-like peptide-1 (GLP-1) is known as an incretin and a satiety hormone released by enteroendocrine L-cells, and its plasma concentration is reported to increase by dietary non-digestible carbohydrates such as certain dietary fibers and oligosaccharides. However, the triggers and modulators of the GLP-1 release remain unclear. We have previously reported that the L-cells containing peptide YY (PYY) express short-chain fatty acid (SCFA) receptor GPR43 in human and rat colon. PYY is reported to be contained in the same granules together with GLP-1. In the present study, we therefore investigated 1) the coexpression of GLP-1 and GPR43 in human and rat lower intestine, and 2) the effects of a long-term feeding of fructo-oligosaccharide (FOS) supplemented diet on these expressions in rats. Methods: 1) The expression of GLP-1, 5-hydroxytriptamine (5-HT), and GPR43 in the enteroendocrine cells were quantified by immunohistochemistry in the colon and terminal ileum isolated from rat and surgical specimens of humans. 2) Control and FOS-supplemented rats were fed a non-digestible carbohydrate-free and 5% (w/w) FOScontaining diet (16 g/day), respectively. On the 28th day, the rats were killed. The tissue and content weights of cecum, and GLP-1-, 5-HT-, and GPR43-positive cells in the colon, cecum and terminal ileum were measured. Results: Immunohistochemical analysis confirmed that GPR43-positive enteroendocrine cells were GLP-1-containing L-cells, but not 5-HTcontaining enterochromaffin (EC) cells, both in human and rat colon and terminal ileum. Furthermore, compared to control, 4week FOS (5%) supplementation in rats significantly increased cecal tissue weight (but not its content weight) and the densities (cell number/ mm2 mucosa) of both GLP-1and GPR43-containing L-cells in particular the proximal colon. On the other hand, 5-HT-expressing EC cells were not affected by the FOS supplementation. Conclusion: The results showed that FOS supplementation increased the expression of GLP1and GPR43-containing enteroendocrine L-cells in the large intestine, suggesting that the prebiotic effects of FOS, such as improvement of the colonic environment to protect against pathogen and inflammation, is associated with the expression of the SCFA receptor GPR43. Therefore, it is possible that the long-term FOS ingestion contributes to the prevention of obesity and diabetes through PYY/GLP-1 release mediated by SCFA-stimulation of GPR43 in the large intestine.

M1719 Umami Receptor Activation Increases Duodenal Bicarbonate Secretion via GLP-2 Release in Rats

Background and aim: Glucagon-like peptide-1 (GLP-1) is known as an incretin and a satiety hormone released by enteroendocrine L-cells, and its plasma concentration is reported to increase by dietary non-digestible carbohydrates such as certain dietary fibers and oligosaccharides. However, the triggers and modulators of the GLP-1 release remain unclear. We have previously reported that the L-cells containing peptide YY (PYY) express short-chain fatty acid (SCFA) receptor GPR43 in human and rat colon. PYY is reported to be contained in the same granules together with GLP-1. In the present study, we therefore investigated 1) the coexpression of GLP-1 and GPR43 in human and rat lower intestine, and 2) the effects of a long-term feeding of fructo-oligosaccharide (FOS) supplemented diet on these expressions in rats. Methods: 1) The expression of GLP-1, 5-hydroxytriptamine (5-HT), and GPR43 in the enteroendocrine cells were quantified by immunohistochemistry in the colon and terminal ileum isolated from rat and surgical specimens of humans. 2) Control and FOS-supplemented rats were fed a non-digestible carbohydrate-free and 5% (w/w) FOScontaining diet (16 g/day), respectively. On the 28th day, the rats were killed. The tissue and content weights of cecum, and GLP-1-, 5-HT-, and GPR43-positive cells in the colon, cecum and terminal ileum were measured. Results: Immunohistochemical analysis confirmed that GPR43-positive enteroendocrine cells were GLP-1-containing L-cells, but not 5-HTcontaining enterochromaffin (EC) cells, both in human and rat colon and terminal ileum. Furthermore, compared to control, 4week FOS (5%) supplementation in rats significantly increased cecal tissue weight (but not its content weight) and the densities (cell number/ mm2 mucosa) of both GLP-1and GPR43-containing L-cells in particular the proximal colon. On the other hand, 5-HT-expressing EC cells were not affected by the FOS supplementation. Conclusion: The results showed that FOS supplementation increased the expression of GLP1and GPR43-containing enteroendocrine L-cells in the large intestine, suggesting that the prebiotic effects of FOS, such as improvement of the colonic environment to protect against pathogen and inflammation, is associated with the expression of the SCFA receptor GPR43. Therefore, it is possible that the long-term FOS ingestion contributes to the prevention of obesity and diabetes through PYY/GLP-1 release mediated by SCFA-stimulation of GPR43 in the large intestine.

A new life for bile acids

TGR5 is a G protein-coupled receptor expressed in brown adipose tissue and muscle, where its activation by bile acids triggers an increase in energy expenditure and attenuates diet-induced obesity. Using a combination of pharmacological and genetic gain- and loss-of-function studies invivo, we show here that TGR5 signaling induces intestinal glucagon-like peptide-1 (GLP-1) release, leading to improved liver and pancreatic function and enhanced glucose tolerance in obese mice. In addition, we show that the induction of GLP-1 release in enteroendocrine cells by 6alpha-ethyl-23(S)-methyl-cholic acid (EMCA, INT-777), a specific TGR5 agonist, is linked to an increase of the intracellular ATP/ADP ratio and a subsequent rise in intracellular calcium mobilization. Altogether, these data show that the TGR5 signaling pathway is critical in regulating intestinal GLP-1 secretion invivo, and suggest that pharmacological targeting of TGR5 may constitute a promising incretin-based strategy for the treatment of diabesity and associated metabolic disorders.

TGR5-Mediated Bile Acid Sensing Controls Glucose Homeostasis

TGR5 is a G protein-coupled receptor expressed in brown adipose tissue and muscle, where its activation by bile acids triggers an increase in energy expenditure and attenuates diet-induced obesity. Using a combination of pharmacological and genetic gain- and loss-of-function studies in vivo, we show here that TGR5 signaling induces intestinal glucagon-like peptide-1 (GLP-1) release, leading to improved liver and pancreatic function and enhanced glucose tolerance in obese mice. In addition, we show that the induction of GLP-1 release in enteroendocrine cells by 6alpha-ethyl-23(S)-methyl-cholic acid (EMCA, INT-777), a specific TGR5 agonist, is linked to an increase of the intracellular ATP/ADP ratio and a subsequent rise in intracellular calcium mobilization. Altogether, these data show that the TGR5 signaling pathway is critical in regulating intestinal GLP-1 secretion in vivo, and suggest that pharmacological targeting of TGR5 may constitute a promising incretin-based strategy for the treatment of diabesity and associated metabolic disorders.

Bile Acids Have the Gall to Function as Hormones

In this issue of Cell Metabolism, Thomas et al. (2009) show that specific activation of the bile-acid-activated G protein-coupled receptor TGR5 improves pancreatic and hepatic function and impairs the development of obesity following administration of a high-fat diet.

Cross Talk between the Insulin and Wnt Signaling Pathways: Evidence from Intestinal Endocrine L Cells

The proglucagon gene (glu) encodes the incretin hormone glucagon-like peptide-1 (GLP-1), produced in the intestinal endocrine L cells. We found previously that the bipartite transcription factor beta-catenin/T cell factor (cat/TCF), the major effector of the canonical Wnt signaling pathway, activates intestinal glu expression and GLP-1 production. We show here that 100 nm insulin stimulated glu expression and enhanced GLP-1 content in the intestinal GLUTag L cell line as well as in primary fetal rat intestinal cell cultures. Increased intestinal glu mRNA expression and GLP-1 content were also observed in vivo in hyperinsulinemic MKR mice. In the GLUTag cells, insulin-induced activation of glu expression occurred through the same TCF site that mediates cat/TCF activation. Phosphatidylinositol 3-kinase inhibition, but not protein kinase B inhibition, attenuated the stimulation by insulin. Furthermore, nuclear beta-catenin content in the intestinal L cells was increased by insulin. Finally, insulin enhanced the binding of TCF-4 and beta-catenin to the TCF site in the glu promoter G2 enhancer element, as determined by quantitative chromatin immunoprecipitation assay. Collectively, these findings indicate that enhancement of beta-catenin nuclear translocation and cat/TCF binding are among the mechanisms underlying cross talk between the insulin and Wnt signaling pathways in intestinal endocrine L cells.

The role of intestinal glucagon-like peptide 1 in satiety.

Glucagon-like peptide-1 (GLP-1), secreted by the distal intestine in response to nutrients entering the gut, is hypothesized to participate in the process of satiety. Therefore, we predicted that peripheral GLP-1 receptor (GLP-1-R) stimulation would reduce food intake by limiting meal size. To test this hypothesis, we continuously monitored rats’ chow intake after IP injection of either the GLP-1-R agonist Exendin-4 (Ex4; 1 μg/kg) or saline. Ex4 significantly reduced feeding for 4 h post-treatment by selectively decreasing meal size, with no effect on meal frequency. To determine whether nutrient-stimulated endogenous GLP-1 release plays a role in satiety, we examined the effects of GLP-1-R blockade on food intake after an intragastric nutrient preload. Rats fitted with intragastric catheters received a preload infusion of either saline or 10 kcal glucose (10 ml at 1 ml/min). Immediately after the infusion, they were injected with saline or Exendin 9–39 (Ex9, a GLP-1-R antagonist; 100 μg/kg, IP), and then presented with vanilla Ensure liquid diet for 2 h. The glucose preload significantly decreased Ensure intake, and Ex9 treatment reduced the magnitude of this preload-induced intake suppression by 40%. We observed similar effects of GLP-1-R blockade after an orally consumed preload of 12.5% sucrose solution (mean intake=10.42 kcal). The sucrose preload significantly reduced subsequent chow intake compared with the no-preload condition, and Ex9 (100 μg/kg IP) treatment completely blocked this preload effect. Taken together, these results implicate GLP-1 released from the intestine in response to ingested nutrients as a physiologically relevant satiety signal.

GLUT2 and the incretin receptors are involved in glucose-induced incretin secretion

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretins secreted in response to oral glucose ingestion by intestinal L and K cells, respectively. The molecular mechanisms responsible for intestinal cell glucose sensing are unknown but could be related to those described for β-cells, brain and hepatoportal sensors. We determined the role of GLUT2, GLP-1 or GIP receptors in glucose-induced incretins secretion, in the corresponding knockout mice. GLP-1 secretion was reduced in all mutant mice, while GIP secretion did not require GLUT2. Intestinal GLP-1 content was reduced only in GIP and GLUT2 receptors knockout mice suggesting that this impairment could contribute to the phenotype. Intestinal GIP content was similar in all mice studied. Furthermore, the impaired incretins secretion was associated with a reduced glucose-stimulated insulin secretion and an impaired glucose tolerance in all mice. In conclusion, both incretins secretion depends on mechanisms involving their own receptors and GLP-1 further requires GLUT2.

Role of glial cell-line derived neurotropic factor family receptor α2 in the actions of the glucagon-like peptides on the murine intestine

The intestinal glucagon-like peptides GLP-1 and GLP-2 inhibit intestinal motility, whereas GLP-2 also stimulates growth of the intestinal mucosa. However, the mechanisms of action of these peptides in the intestine remain poorly characterized. To determine the role of the enteric nervous system in the actions of GLP-1 and GLP-2 on the intestine, the glial cell line-derived neurotropic factor family receptor alpha(2) (GFRalpha2) knockout (KO) mouse was employed. The mice exhibited decreased cholinergic staining, as well as reduced mRNA transcripts for substance P-ergic excitatory motoneurons in the enteric nervous system (ENS) (P < 0.05). Examination of parameters of intestinal growth (including small and large intestinal weight and small intestinal villus height, crypt depth, and crypt cell proliferation) demonstrated no differences between wild-type and KO mice in either basal or GLP-2-stimulated mucosal growth. Nonetheless, KO mice exhibited reduced numbers of synaptophysin-positive enteroendocrine cells (P < 0.05), as well as a markedly impaired basal gastrointestinal (GI) transit rate (P < 0.05). Furthermore, acute administration of GLP-1 and GLP-2 significantly inhibited transit rates in wild-type mice (P < 0.05-0.01) but had no effect in GFRalpha2 KO mice. Despite these changes, expression of mRNA transcripts for the GLP receptors was not reduced in the ENS of KO animals, suggesting that GLP-1 and -2 modulate intestinal transit through enhancement of inhibitory input to cholinergic/substance P-ergic excitatory motoneurons. Together, these findings demonstrate a role for GFRalpha2-expressing enteric neurons in the downstream signaling of the glucagon-like peptides to inhibit GI motility, but not in intestinal growth.

Nutrient‐Stimulated Secretory Response and Mechanisms of the Incretins GLP‐1 and GIP Using Lymph as a Novel Assay Model

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretins produced in the intestine that play a central role in glucose metabolism and insulin secretion. Plasma GLP-1 and GIP are low, in the range of 5–20 pM, and difficult to assay accurately in rodents. We have established a novel method of assaying GLP-1 and GIP using the intestinal lymph fistula model. Using this model, we have now determined that GLP-1 and GIP levels are 5–6 times higher in lymph than portal vein plasma, reaching postprandial peaks of 300 and 50 pM respectively. Intraduodenal administration of isocaloric meal of Intralipid, dextrin (a glucose polymer), or Intralipid plus dextrin caused significant increases in peak intestinal lymphatic GLP-1 and GIP secretion both at their peak and over 6 hours (P < 0.001). Incretin secretion was more rapid in response to Intralipid than dextrin. The combination of Intralipid plus dextrin additively stimulated GLP-1 and potentiated GIP secretion. When the surfactant, Pluronic L-81, was given concurrently with nutrients, GLP-1 and GIP secretion was blunted demonstrating the importance of chylomicron formation in incretin secretion. In conclusion, we have demonstrated that intestinal lymph is enriched with GLP-1 and GIP and the potential for lymphatic sampling as a novel and powerful means of studying the secretion and secretory mechanism of gut hormones in vivo.