Role of MGAT2 and DGAT1 in the release of gut peptides after triglyceride ingestion

Triglyceride ingestion releases gut peptides from enteroendocrine cells located in the intestinal epithelia and provides feedback regulations of gastrointestinal function. The precise mechanisms sensing lipids in the intestinal wall, however, are not well characterized. In the current study, we investigated the release of gut peptides following oral triglyceride loading in mice deficient for monoacylglycerol acyltransferase 2 (MGAT2KO) and diacylglycerol acyltransferase 1 (DGAT1KO), enzymes that sequentially re-synthesize triglyceride to secrete as chylomicron at the small intestine. In wild-type (Wt) mice, oral triglyceride loading resulted in hypertriglycemia. In addition, plasma glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) were significantly increased 30 min after triglyceride loading, before decaying in 2 h. In MGAT2KO and DGAT1KO mice, oral triglyceride loading did not result in hypertriglycemia and the increase in GIP was significantly suppressed in both KO mouse strains. In contrast, the increases in plasma GLP-1 and PYY in both KO mouse strains were comparable to Wt mice 30 min after triglyceride loading, however, they remained elevated in DGAT1KO mice even 2 h after triglyceride loading. In parallel to the changes in GLP-1 and PYY, gastric emptying was delayed after oral triglyceride loading in MGAT2KO mice comparably to Wt type mice and was further delayed in DGAT1KO mice. STC-1 and GLUTag, GLP-1-producing intestinal endocrine L-cell lines, displayed a significant level of DGAT1 activity but not MGAT activity. These findings suggest that synthesis and/or secretion of triglyceride-rich lipoproteins play an important role in the release of GIP. Moreover, DGAT1 may directly regulate the release of GLP-1 and PYY in L-cells.     

The effects of duodenal peptides on glucagon-like peptide-1 secretion from the ileum. A duodeno--ileal loop?

Secretion of the gut hormone glucagon-like peptide-1 (GLP-1) is stimulated by meal ingestion. The response is rapid, suggesting a stimulatory pathway elicited from the upper gastrointestinal area. In pigs, we have been unable to demonstrate a neural stimulatory pathway, but GLP-1 secretion is regulated by local somatostatin secretion. In search for an endocrine pathway, we studied the effect of a range of concentrations of cholecystokinin octapeptide (26-33) (CCK 8), gastric inhibitory peptide 1-42 (GIP), secretin, motilin, calcitonin gene-related peptide (CGRP), and the modified amino acid, 5-hydroxytryptamine (serotonin, 5-HT) on GLP-1 and somatostatin release from isolated perfused segments of porcine ileum.GLP-1 secretion was stimulated by 1 nM CCK 8 and 10 nM GIP, but suppressed by 1 nM motilin and 1 microM 5-HT. Secretin and CGRP had no effect. Somatostatin secretion was stimulated by CCK 8 at 1 and 10 nM, by GIP at 1 and 10 nM and by 10 nM CGRP. Secretin, 5-HT and motilin had no effect on somatostatin secretion.We conclude that CCK 8 and GIP 1-42 stimulated GLP-1 secretion, but only in concentrations greatly exceeding normal postprandial concentrations. Thus, we find it unlikely that endocrine agents from the duodenum regulate GLP-1 secretion in pigs.     

Effect of glucagon-like peptide-1 on insulin secretion

The insulinotropic actions of two forms of glucagon-like peptide 1 (GLP-1) containing 31 and 37 amino acid residues on perfused rat pancreas were compared with that of gastric inhibitory polypeptide (GIP), hitherto the most potent intestinal insulinotropic polypeptide known. The smaller form, C-terminally amidated GLP-1-(7-36), strongly enhanced insulin secretion stimulated by 11.1 mM D-glucose at a concentration as low as 0.1 nM. Comparable effects of GIP and GLP-1-(1-37) on insulin secretion were observed at concentrations of 1.0 nM and 10.0 nM, respectively. At the doses tested, neither GLP-1s nor GIP had any effect on insulin secretion induced by 3.3 mM D-glucose. At a concentration of 1.0 nM, GLP-1-(7-36 amide) also enhanced insulin secretion induced by 5 mM L-arginine whereas at concentrations of up to 10.0 nM, GLP-1-(1-37) did not. The results show that the smaller form of GLP-1 is more strongly insulinotropic than GIP. These findings suggest that the smaller GLP-1 may have a physiologically more important role as a modulator of insulin release.     

Priming effect of glucagon-like peptide-1 (7-36) amide, glucose-dependent insulinotropic polypeptide and cholecystokinin-8 at the isolated perfused rat pancreas.

The priming effect of glucagon-like peptide-1 (7-36) amide (GLP-1 (7-36) amide), glucose-dependent insulin-releasing polypeptide (GIP) and cholecystokinin-8 (CCK-8) on glucose-induced insulin secretion from rat pancreas was investigated. The isolated pancreas was perfused in vitro with Krebs-Ringer bicarbonate buffer containing 2.8 mmol/l glucose. After 10 min this medium was supplemented with GLP-1 (7-36) amide, GIP or CCK-8 (10, 100, 1000 pmol/l) for 10 min. After an additional 10 min period with 2.8 mmol/l glucose alone, insulin secretion was stimulated with buffer containing 10 mmol/l glucose for 44 min. In control experiments the typical biphasic insulin response to 10 mmol/l glucose occurred. Pretreatment of the pancreas with GIP augmented insulin secretion: 10 pmol/l GIP enhanced only the first phase of the secretory response to 10 mmol/l glucose; 100 and 1000 pmol/l GIP stimulated both phases of hormone secretion. After exposure to CCK-8, enhanced insulin release during the first (at 10 and 1000 pmol/l CCK-8) and the second phase (at 1000 pmol/l) was observed. Priming with 100 pmol/l GLP-1 (7-36) amide significantly amplified the first and 1000 pmol/l GLP-1 (7-36) amide both secretion periods, 10 pmol/l GLP-1 (7-36) amide had no significant effect. All three peptide hormones influenced the first, quickly arising secretory response more than the second phase. Priming with forskolin (30 mM) enhanced the secretory response to 10 mM glucose plus 0.5 nM GLP-1 (7-36) amide 4-fold. With a glucose-responsive B-cell line (HIT cells), we investigated the hypothesis that the priming effect of GLP-1 (7-36) amide is mediated by the adenylate cyclase system. Priming with either IBMX (0.1 mM) or forskolin (2.5 microM) enhanced the insulin release after a consecutive glucose stimulation (5 mM). This effect was pronounced when GLP-1 (7-36) amide (100 pM) was added during glucose stimulation. Priming capacities of intestinal peptide hormones may be involved in the regulation of postprandial insulin release. The incretin action of these hormones can probably, at least in part, be explained by these effects. The priming effect of GLP-1 (7-36) amide is most likely mediated by the adenylate cyclase system.     

Insulin is secreted upon glucose stimulation by both gastrointestinal enteroendocrine K-cells and L-cells engineered with the preproinsulin gene

Transgenic mice carrying the human insulin gene driven by the K-cell glucose-dependent insulinotropic peptide (GIP) promoter secrete insulin and display normal glucose tolerance tests after their pancreatic p-cells have been destroyed. Establishing the existence of other types of cells that can process and secrete transgenic insulin would help the development of new gene therapy strategies to treat patients with diabetes mellitus. It is noted that in addition to GIP secreting K-cells, the glucagon-like peptide 1 (GLP-1) generating L-cells share/ many similarities to pancreatic p-cells, including the peptidases required for proinsulin processing, hormone storage and a glucose-stimulated hormone secretion mechanism. In the present study, we demonstrate that not only K-cells, but also L-cells engineered with the human preproinsulin gene are able to synthesize, store and, upon glucose stimulation, release mature insulin. When the mouse enteroendocrine STC-1 cell line was transfected with the human preproinsulin gene, driven either by the K-cell specific GIP promoter or by the constitutive cytomegalovirus (CMV) promoter, human insulin co-localizes in vesicles that contain GIP (GIP or CMV promoter) or GLP-1 (CMV promoter). Exposure to glucose of engineered STC-1 cells led to a marked insulin secretion, which was 7-fold greater when the insulin gene was driven by the CMV promoter (expressed both in K-cells and L-cells) than when it was driven by the GIP promoter (expressed only in K-cells). Thus, besides pancreatic p-cells, both gastrointestinal enteroendocrine K-cells and L-cells can be selected as the target cell in a gene therapy strategy to treat patients with type 1 diabetes mellitus.     

Somatostatin restrains the secretion of glucagon-like peptide-1 and -2 from isolated perfused porcine ileum

Suspecting that paracrine inhibition might influence neuronal regulation of the endocrine L cells, we studied the role of somatostatin (SS) in the regulation of the secretion of the proglucagon-derived hormones glucagon-like peptide-1 and -2 (GLP-1 and GLP-2). This was examined using the isolated perfused porcine ileum stimulated with acetylcholine (ACh, 10(-6) M), neuromedin C (NC, 10(-8) M), and electrical nerve stimulation (NS) with or without alpha-adrenergic blockade (phentolamine 10(-5) M), and perfusion with a high-affinity monoclonal antibody against SS. ACh and NC significantly increased GLP secretion, whereas NS had little effect. SS immunoneutralization increased GLP secretion eight- to ninefold but had little influence on the GLP responses to ACh, NC, and NS. Basal SS secretion (mainly SS28) was unaffected by NS alone. Phentolamine + NS and NC abstract strongly stimulated release mainly of SS14, whereas ACh had little effect. Infused intravascularly, SS14 weakly and SS28 strongly inhibited GLP secretion. We conclude that GLP secretion is tonically inhibited by a local release of SS28 from epithelial paracrine cells, whereas SS14, supposedly derived from enteric neurons, only weakly influences GLP secretion.     

The biology of incretin hormones

Gut peptides, exemplified by glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are secreted in a nutrient-dependent manner and stimulate glucose-dependent insulin secretion. Both GIP and GLP-1 also promote β cell proliferation and inhibit apoptosis, leading to expansion of β cell mass. GLP-1, but not GIP, controls glycemia via additional actions on glucose sensors, inhibition of gastric emptying, food intake and glucagon secretion. Furthermore, GLP-1, unlike GIP, potently stimulates insulin secretion and reduces blood glucose in human subjects with type 2 diabetes. This article summarizes current concepts of incretin action and highlights the potential therapeutic utility of GLP-1 receptor agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors for the treatment of type 2 diabetes.     

The role of the stomach in the control of appetite and the secretion of satiation peptides

It is widely accepted that gastric parameters such as gastric distention provide a direct negative feedback signal to inhibit eating; moreover, gastric and intestinal signals have been reported to synergize to promote satiation. However, there are few human data exploring the potential interaction effects of gastric and intestinal signals in the short-term control of appetite and the secretion of satiation peptides. We performed experiments in healthy subjects receiving either a rapid intragastric load or a continuous intraduodenal infusion of glucose or a mixed liquid meal. Intraduodenal infusions (3 kcal/min) were at rates comparable with the duodenal delivery of these nutrients under physiological conditions. Intraduodenal infusions of glucose elicited only weak effects on appetite and the secretion of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY). In contrast, identical amounts of glucose delivered intragastrically markedly suppressed appetite (P < 0.05) paralleled by greatly increased plasma levels of GLP-1 and PYY (≤3-fold, P < 0.05). Administration of the mixed liquid meal showed a comparable phenomenon. In contrast to GLP-1 and PYY, plasma ghrelin was suppressed to a similar degree with both intragastric and intraduodenal nutrients. Our data confirm that the stomach is an important element in the short-term control of appetite and suggest that gastric and intestinal signals interact to mediate early fullness and satiation potentially by increased GLP-1 and PYY secretions.     

Effects of intraduodenal glucose and fructose on antropyloric motility and appetite in healthy humans

Oral fructose empties from the stomach more rapidly and may suppress food intake more than oral glucose. The purpose of the study was to evaluate the effects of intraduodenal infusions of fructose and glucose on antropyloric motility and appetite. Ten healthy volunteers were given intraduodenal infusions of 25% fructose, 25% glucose, or 0.9% saline (2 ml/min for 90 min). Antropyloric pressures, blood glucose, and plasma insulin, gastric inhibitory peptide (GIP), and glucagon-like peptide-1 (GLP-1) were measured concurrently; a buffet meal was offered at the end of the infusion. Intraduodenal fructose and glucose suppressed antral waves (P < 0. 0005 for both), stimulated isolated pyloric pressure waves (P < 0.05 for both), and increased basal pyloric pressure (P = 0.10 and P < 0. 05, respectively) compared with saline, without any significant difference between them. Intraduodenal glucose increased blood glucose (P < 0.0005), as well as plasma insulin (P < 0.0005) and GIP (P < 0.005) more than intraduodenal fructose, whereas there was no difference in the GLP-1 response. Intraduodenal fructose suppressed food intake compared with saline (P < 0.05) and glucose (P = 0.07). We conclude that, when infused intraduodenally at 2 kcal/min for 90 min 1) fructose and glucose have comparable effects on antropyloric pressures, 2) fructose tends to suppress food intake more than glucose, despite similar GLP-1 and less GIP release, and 3) GIP, rather than GLP-1, probably accounts for the greater insulin response to glucose than fructose.     

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.     

Increased postprandial responses of GLP-1 and GIP in patients with chronic pancreatitis and steatorrhea following pancreatic enzyme substitution

We aimed to investigate how assimilation of nutrients affects the postprandial responses of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) and to evaluate the effect of pancreatic enzyme substitution (PES) on insulin secretion in patients with chronic pancreatitis (CP) and pancreatic exocrine insufficiency (PEI). Eight male patients with CP and PEI were studied. Blood was sampled frequently on two separate days after ingestion of a liquid meal with and without PES, respectively. Eight healthy male subjects served as a control group. beta-Cell responsiveness was estimated as changes in insulin secretion rates in response to changes in postprandial plasma glucose (PG). There was no difference in the PG incremental area under curve (AUC) for patients with and without PES [406 +/- 100 vs. 425 +/- 80 mM.4 h (mean +/- SE), P = 0.8]. The response of total GLP-1 was higher after PES (AUC: 7.8 +/- 1.2 vs. 5.3 +/- 0.6 nM.4 h, P = 0.01), as was the response of total GIP (AUC: 32.7 +/- 7.5 vs. 21.1 +/- 8.3 nM.4 h, P = 0.01). Concurrently, both plasma insulin, plasma C-peptide, and total insulin secretion increased after PES (AUC: 17.7 +/- 4.2 vs. 13.6 +/- 2.9 nM.4 h, P = 0.02; 237 +/- 31.4 vs. 200 +/- 27.4 nM.4 h, P = 0.005; and 595 +/- 82 vs. 497 +/- 80 pmol.kg(-1).4 h, P = 0.01, respectively). beta-Cell responsiveness to glucose was not significantly different on the two study days for patients with CP. These results suggest that the secretion of GLP-1 and GIP is under influence of the digestion and absorption of nutrients in the small intestine and that PES increases insulin secretion.     

Dietary resistant starch upregulates total GLP-1 and PYY in a sustained day-long manner through fermentation in rodents

Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are anti-diabetes/obesity hormones secreted from the gut after meal ingestion. We have shown that dietary-resistant starch (RS) increased GLP-1 and PYY secretion, but the mechanism remains unknown. RS is a fermentable fiber that lowers the glycemic index of the diet and liberates short-chain fatty acids (SCFAs) through fermentation in the gut. This study investigates the two possible mechanisms by which RS stimulates GLP-1 and PYY secretion: the effect of a meal or glycemic index, and the effect of fermentation. Because GLP-1 and PYY secretions are stimulated by nutrient availability in the gut, the timing of blood sample collections could influence the outcome when two diets with different glycemic indexes are compared. Thus we examined GLP-1 and PYY plasma levels at various time points over a 24-h period in RS-fed rats. In addition, we tested proglucagon (a precursor to GLP-1) and PYY gene expression patterns in specific areas of the gut of RS-fed rats and in an enteroendocrine cell line following exposure to SCFAs in vitro. Our findings are as follows. 1) RS stimulates GLP-1 and PYY secretion in a substantial day-long manner, independent of meal effect or changes in dietary glycemia. 2) Fermentation and the liberation of SCFAs in the lower gut are associated with increased proglucagon and PYY gene expression. 3) Glucose tolerance, an indicator of increased active forms of GLP-1 and PYY, was improved in RS-fed diabetic mice. We conclude that fermentation of RS is most likely the primary mechanism for increased endogenous secretions of total GLP-1 and PYY in rodents. Thus any factor that affects fermentation should be considered when dietary fermentable fiber is used to stimulate GLP-1 and PYY secretion.     

Cellular glucose availability and glucagon-like peptide-1

Glucagon-like peptide (GLP)-1 and gastric inhibitory polypeptide (GIP, glucose-dependent insulinotropic polypeptide) are produced in enteroendocrine L-cells and K-cells, respectively. They are known as incretins because they potentiate postprandial insulin secretion. Although unresponsiveness of type 2 diabetes (T2D) patients to GIP has now been reconsidered, GLP-1 mimetics and inhibitors of the GLP-1 degradation enzyme dipeptidyl peptidase (DPP)-4 have now been launched as drugs against T2D. The major roles of GLP-1 in T2D are reduction of appetite, gastric motility, glucagon secretion, enhancement of insulin secretion and β-cell survival. For insulin secretion and peripheral insulin function, GLP-1 and its mimetics sensitise β-cells to glucose; accelerate blood glucose withdrawal, in-cell glucose utilisation and glycogen synthesis in insulin-sensitive tissues; and assist in the function and survival of neurons mainly using glucose as an energy source. Taken together, GLP-1 acts to potentiate glucose availability of various cells or tissues to assist with their essential functions and/or survival. Herein, we review the signalling pathways and clinical relevance of GLP-1 in enhancing cellular glucose availability. On the basis of our recent research results, we also describe a mechanism that regulates GLP-1 for glucokinase activity. Because diabetic tissues including β-cells resist glucose, GLP-1 may be useful for treating T2D.     

What do we know about the secretion and degradation of incretin hormones?

The incretin hormones, glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted from endocrine cells located in the intestinal mucosa, and act to enhance meal-induced insulin secretion. GIP and GLP-1 concentrations in the plasma rise rapidly after food ingestion, and the presence of unabsorbed nutrients in the intestinal lumen is a strong stimulus for their secretion. Nutrients can stimulate release of both hormones by direct contact with the K-cell (GIP) and L-cell (GLP-1), and this may be the most important signal. However, nutrients also stimulate GLP-1 and GIP secretion indirectly via other mechanisms. Incretin hormone secretion can be modulated neurally, with cholinergic muscarinic, beta-adrenergic and peptidergic (gastrin-releasing peptide, GRP) fibres generally having positive effects, while secretion is restrained by alpha-adrenergic and somatostatinergic fibres. Hormonal factors may also influence incretin hormone secretion. Somatostatin exerts a local inhibitory effect on the activity of both K- and L-cells via a paracrine mechanism, while, in rodents at least, GIP from the proximal intestine has a stimulatory effect on GLP-1 secretion, possibly mediated via a neural loop involving GRP. Once they have been released, both GLP-1 and GIP are subject to rapid degradation. The ubiquitous enzyme, dipeptidyl peptidase IV (DPP IV) cleaves N-terminally, removing a dipeptide and thereby inactivating both peptides, because the N-terminus is crucial for receptor binding. Subsequently, the peptides may be degraded by other enzymes and extracted in an organ-specific manner. The intact peptides are inactivated during passage across the hepatic bed and further metabolised by the peripheral tissues, while the kidney is important for the final elimination of the metabolites.     

Two incretin hormones GLP-1 and GIP: comparison of their actions in insulin secretion and β cell preservation

Gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are the two primary incretin hormones secreted from the intestine upon ingestion of glucose or nutrients to stimulate insulin secretion from pancreatic β cells. GIP and GLP-1 exert their effects by binding to their specific receptors, the GIP receptor (GIPR) and the GLP-1 receptor (GLP-1R), which belong to the G-protein coupled receptor family. Receptor binding activates and increases the level of intracellular cAMP in pancreatic β cells, thereby stimulating insulin secretion glucose-dependently. In addition to their insulinotropic effects, GIP and GLP-1 have been shown to preserve pancreatic β cell mass by inhibiting apoptosis of β cells and enhancing their proliferation. Due to such characteristics, incretin hormones have been gaining mush attention as attractive targets for treatment of type 2 diabetes, and indeed incretin-based therapeutics have been rapidly disseminated worldwide. However, despites of plethora of rigorous studies, molecular mechanisms underlying how GIPR and GLP-1R activation leads to enhancement of glucose-dependent insulin secretion are still largely unknown. Here, we summarize the similarities and differences of these two incretin hormones in secretion and metabolism, their insulinotropic actions and their effects on pancreatic β cell preservation. We then try to discuss potential of GLP-1 and GIP in treatment of type 2 diabetes.     

Incretin and islet hormonal responses to fat and protein ingestion in healthy men

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) regulate islet function after carbohydrate ingestion. Whether incretin hormones are of importance for islet function after ingestion of noncarbohydrate macronutrients is not known. This study therefore examined integrated incretin and islet hormone responses to ingestion of pure fat (oleic acid; 0.88 g/kg) or protein (milk and egg protein; 2 g/kg) over 5 h in healthy men, aged 20-25 yr (n=12); plain water ingestion served as control. Both intact (active) and total GLP-1 and GIP levels were determined as was plasma activity of dipeptidyl peptidase-4 (DPP-4). Following water ingestion, glucose, insulin, glucagon, GLP-1, and GIP levels and DPP-4 activity were stable during the 5-h study period. Both fat and protein ingestion increased insulin, glucagon, GIP, and GLP-1 levels without affecting glucose levels or DPP-4 activity. The GLP-1 responses were similar after protein and fat, whereas the early (30 min) GIP response was higher after protein than after fat ingestion (P<0.001). This was associated with sevenfold higher insulin and glucagon responses compared with fat ingestion (both P<0.001). After protein, the early GIP, but not GLP-1, responses correlated to insulin (r(2)=0.86; P=0.0001) but not glucagon responses. In contrast, after fat ingestion, GLP-1 and GIP did not correlate to islet hormones. We conclude that, whereas protein and fat release both incretin and islet hormones, the early GIP secretion after protein ingestion may be of primary importance to islet hormone secretion.     

Signalling pathways involved in the detection of peptones by murine small intestinal enteroendocrine L-cells

Glucagon like peptide-1 is an insulinotropic hormone released from intestinal L-cells in response to food ingestion. Here, we investigated mechanisms underlying the sensing of peptones by primary small intestinal L-cells. Meat, casein and vegetable-derived peptones (5 mg/ml), the L-amino acids Phe, Trp, Gln and Ala (20 mM each), and the dipeptide glycine-sarcosine (20 mM) stimulated GLP-1 secretion from primary cultures prepared from the small intestine. Further mechanistic studies were performed with meat peptone, and revealed the elevation of intracellular calcium in L-cells. Inhibition of the calcium sensing receptor (CaSR), transient receptor potential (TRP) channels and Q-type voltage gated calcium channels (VGCC) significantly attenuated peptone-stimulated GLP-1 release and reduced intracellular Ca²⁺ responses. CaSR inhibition also attenuated the GLP-1 secretory response to Gln. Targeting these pathways in L-cells could be used to increase endogenous production of GLP-1 and offer exploitable avenues for the development of therapeutics to treat diabetes and obesity.     

Inhibition of gastric emptying by acarbose is correlated with GLP-1 response and accompanied by CCK release

We investigated the effect of acarbose, an alpha-glucosidase and pancreatic alpha-amylase inhibitor, on gastric emptying of solid meals of varying nutrient composition and plasma responses of gut hormones. Gastric emptying was determined with scintigraphy in healthy subjects, and all studies were performed with and without 100 mg of acarbose, in random order, at least 1 wk apart. Acarbose did not alter the emptying of a carbohydrate-free meal, but it delayed emptying of a mixed meal and a carbohydrate-free meal given 2 h after sucrose ingestion. In meal groups with carbohydrates, acarbose attenuated responses of plasma insulin and glucose-dependent insulinotropic polypeptide (GIP) while augmenting responses of CCK, glucagon-like peptide-1 (GLP-1), and peptide YY (PYY). With mixed meal + acarbose, area under the curve (AUC) of gastric emptying was positively correlated with integrated plasma response of GLP-1 (r = 0.68, P < 0.02). With the carbohydrate-free meal after sucrose and acarbose ingestion, AUC of gastric emptying was negatively correlated with integrated plasma response of GIP, implying that prior alteration of carbohydrate absorption modifies gastric emptying of a meal. The results demonstrate that acarbose delays gastric emptying of solid meals and augments release of CCK, GLP-1, and PYY mainly by retarding/inhibiting carbohydrate absorption. Augmented GLP-1 release by acarbose appears to play a major role in the inhibition of gastric emptying of a mixed meal, whereas CCK and PYY may have contributory roles.     

Lipid derivatives activate GPR119 and trigger GLP-1 secretion in primary murine L-cells

Aims/hypothesisGlucagon-like peptide-1 (GLP-1) is an incretin hormone derived from proglucagon, which is released from intestinal L-cells and increases insulin secretion in a glucose dependent manner. GPR119 is a lipid derivative receptor present in L-cells, believed to play a role in the detection of dietary fat. This study aimed to characterize the responses of primary murine L-cells to GPR119 agonism and assess the importance of GPR119 for the detection of ingested lipid.MethodsGLP-1 secretion was measured from murine primary cell cultures stimulated with a panel of GPR119 ligands. Plasma GLP-1 levels were measured in mice lacking GPR119 in proglucagon-expressing cells and controls after lipid gavage. Intracellular cAMP responses to GPR119 agonists were measured in single primary L-cells using transgenic mice expressing a cAMP FRET sensor driven by the proglucagon promoter.ResultsL-cell specific knockout of GPR119 dramatically decreased plasma GLP-1 levels after a lipid gavage. GPR119 ligands triggered GLP-1 secretion in a GPR119 dependent manner in primary epithelial cultures from the colon, but were less effective in the upper small intestine. GPR119 agonists elevated cAMP in ∼70% of colonic L-cells and 50% of small intestinal L-cells.Conclusions/interpretationGPR119 ligands strongly enhanced GLP-1 release from colonic cultures, reflecting the high proportion of colonic L-cells that exhibited cAMP responses to GPR119 agonists. Less GPR119-dependence could be demonstrated in the upper small intestine. In vivo, GPR119 in L-cells plays a key role in oral lipid-triggered GLP-1 secretion.     

Both GLP-1 and GIP are insulinotropic at basal and postprandial glucose levels and contribute nearly equally to the incretin effect of a meal in healthy subjects

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are both incretin hormones regulating postprandial insulin secretion. Their relative importance in this respect under normal physiological conditions is unclear, however, and the aim of the present investigation was to evaluate this. Eight healthy male volunteers (mean age: 23 (range 20-25) years; mean body mass index: 22.2 (range 19.3-25.4) kg/m2) participated in studies involving stepwise glucose clamping at fasting plasma glucose levels and at 6 and 7 mmol/l. Physiological amounts of either GIP (1.5 pmol/kg/min), GLP-1(7-36)amide (0.33 pmol/kg/min) or saline were infused for three periods of 30 min at each glucose level, with 1 h "washout" between the infusions. On a separate day, a standard meal test (566 kcal) was performed. During the meal test, peak insulin concentrations were observed after 30 min and amounted to 223+/-27 pmol/l. Glucose+saline infusions induced only minor increases in insulin concentrations. GLP-1 and GIP infusions induced significant and similar increases at fasting glucose levels and at 6 mmol/l. At 7 mmol/l, further increases were seen, with GLP-1 effects exceeding those of GIP. Insulin concentrations at the end of the three infusion periods (60, 150 and 240 min) during the GIP clamp amounted to 53+/-5, 79+/-8 and 113+/-15 pmol/l, respectively. Corresponding results were 47+/-7, 95+/-10 and 171+/-21 pmol/l, respectively, during the GLP-1 clamp. C-peptide responses were similar. Total and intact incretin hormone concentrations during the clamp studies were higher compared to the meal test, but within physiological limits. Glucose infusion alone significantly inhibited glucagon secretion, which was further inhibited by GLP-1 but not by GIP infusion. We conclude that during normal physiological plasma glucose levels, glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide contribute nearly equally to the incretin effect in humans, because their differences in concentration and potency outweigh each other.