Proapelin is processed extracellularly in a cell line-dependent manner with clear modulation by proprotein convertases.

Apelin is a peptide hormone that binds to a class A GPCR (the apelin receptor/APJ) to regulate various bodily systems. Upon signal peptide removal, the resulting 55-residue isoform, proapelin/apelin-55, can be further processed to 36-, 17-, or 13-residue isoforms with length-dependent pharmacological properties. Processing was initially proposed to occur intracellularly. However, detection of apelin-55 in extracellular fluids indicates that extracellular processing may also occur. To test for this, apelin-55 was applied exogenously to HEK293A cells overexpressing proprotein convertase subtilisin kexin 3 (PCSK3), the only apelin processing enzyme identified thus far, and to differentiated 3T3-L1 adipocytes, which endogenously express apelin, PCSK3 and other proprotein convertases. Analysis of culture media constituents from each cell type by high performance liquid chromatography-mass spectrometry and western blot demonstrated a time-dependent decrease in apelin-55 levels. This decrease was partially, but not fully, attenuated by PCSK inhibitor treatment in both cell lines. Comparison of the resulting apelin-55-derived peptide profile between the two cell lines demonstrated distinct processing patterns, with apelin-36 production apparent in 3T3-L1 adipocytes vs. detection of the prodomain of a shorter isoform (likely the apelin-13 prodomain, observed after additional proteolytic processing) in PCSK3-transfected HEK293A cells. Extracellular processing of apelin, with distinct cell type dependence, provides an alternative mechanism to regulate isoform-mediated physiological effects of apelin.

VIP regulates CFTR membrane expression and function in Calu-3 cells by increasing its interaction with NHERF1 and P-ERM in a VPAC1- and PKCε-dependent manner.

Vasoactive intestinal peptide (VIP) is a topical airway gland secretagogue regulating fluid secretions, primarily by stimulating cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride secretion that contributes to the airways innate defense mechanism. We previously reported that prolonged VIP stimulation of pituitary adenylate cyclase-activating peptide receptors (VPAC1) in airway cells enhances CFTR function by increasing its membrane stability. In the present study, we identified the key effectors in the VIP signaling cascade in the human bronchial serous cell line Calu-3. Using immunocytochemistry and in situ proximity ligation assays, we found that VIP stimulation increased CFTR membrane localization by promoting its colocalization and interaction with the scaffolding protein Na(+)/H(+) exchange factor 1 (NHERF1), a PDZ protein known as a positive regulator for CFTR membrane localization. VIP stimulation also increased phosphorylation, by protein kinase Cε of the actin-binding protein complex ezrin/radixin/moesin (ERM) and its interaction with NHERF1 and CFTR complex. On the other hand, it reduced intracellular CFTR colocalization and interaction with CFTR associated ligand, another PDZ protein known to compete with NHERF1 for CFTR interaction, inducing cytoplasmic retention and lysosomal degradation. Reducing NHERF1 or ERM expression levels by specific siRNAs prevented the VIP effect on CFTR membrane stability. Furthermore, iodide efflux assays confirmed that NHERF1 and P-ERM are necessary for VIP regulation of the stability and sustained activity of membrane CFTR. This study shows the cellular mechanism by which prolonged VIP stimulation of airway epithelial cells regulates CFTR-dependent secretions.

Cystic fibrosis transmembrane conductance regulator dysfunction in VIP knockout mice.

Vasoactive intestinal peptide (VIP), a neuropeptide, controls multiple functions in exocrine tissues, including inflammation, and relaxation of airway and vascular smooth muscles, and regulates CFTR-dependent secretion, which contributes to mucus hydration and local innate defense of the lung. We had previously reported that VIP stimulates the VPAC1 receptor, PKCϵ signaling cascade, and increases CFTR stability and function at the apical membrane of airway epithelial cells by reducing its internalization rate. Moreover, prolonged VIP stimulation corrects the molecular defects associated with F508del, the most common CFTR mutation responsible for the genetic disease cystic fibrosis. In the present study, we have examined the impact of the absence of VIP on CFTR maturation, cellular localization, and function in vivo using VIP knockout mice. We have conducted pathological assessments and detected signs of lung and intestinal disease. Immunodetection methods have shown that the absence of VIP results in CFTR intracellular retention despite normal expression and maturation levels. A subsequent loss of CFTR-dependent chloride current was measured in functional assays with Ussing chamber analysis of the small intestine ex vivo, creating a cystic fibrosis-like condition. Interestingly, intraperitoneal administration of VIP corrected tissue abnormalities, close to the wild-type phenotype, as well as associated defects in the vital CFTR protein. The results show in vivo a primary role for VIP chronic exposure in CFTR membrane stability and function and confirm in vitro data.

Intranasal insulin treatment ameliorates spatial memory, muscular strength, and frailty deficits in 5xFAD mice.

The 5xFAD mouse model shows age-related weight loss as well as cognitive and motor deficits. Metabolic dysregulation, especially impaired insulin signaling, is also present in AD. This study examined whether intranasal delivery of insulin (INI) at low (0.875 U) or high (1.750 U) doses would ameliorate these deficits compared to saline in 10-month-old female 5xFAD and B6SJL wildtype (WT) mice. INI increased forelimb grip strength in the wire hang test in 5xFAD mice in a dose-dependent manner but did not improve the performance of 5xFAD mice on the balance beam. High INI doses reduced frailty scores in 5xFAD mice and improved spatial memory in both acquisition and reversal probe trials in the Morris water maze. INI increased swim speed in 5xFAD mice but had no effect on object recognition memory or working memory in the spontaneous alternation task, nor did it improve memory in the contextual or cued fear memory tasks. High doses of insulin increased the liver, spleen, and kidney weights and reduced brown adipose tissue weights. P-Akt signaling in the hippocampus was increased by insulin in a dose-dependent manner. Altogether, INI increased strength, reduced frailty scores, and improved visual spatial memory. Hypoglycemia was not present after INI, however alterations in tissue and organ weights were present. These results are novel and important as they indicate that intra-nasal insulin can reverse cognitive, motor and frailty deficits found in this mouse model of AD.

VIP reduction in the pancreas of F508del homozygous CF mice and early signs of Cystic Fibrosis Related Diabetes (CFRD).

Vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide with potent anti-inflammatory, bronchodilatory and immunomodulatory functions, is secreted by intrinsic neurons innervating all exocrine glands, including the pancreas, in which it exerts a regulatory function in the secretion of insulin and glucagon. Cystic fibrosis-related diabetes (CFRD) is the most common co-morbidity associated with cystic fibrosis (CF), impacting approximately 50% of adult patients. We recently demonstrated a 50% reduction of VIP abundance in the lungs, duodenum and sweat glands of C57Bl/6 CF mice homozygous for the F508del-CFTR disease-causing mutation. VIP deficiency resulted from a reduction in VIPergic and cholinergic innervation, starting before signs of CF disease were observed. As VIP functions as a neuromodulator with insulinotropic effect on pancreatic beta cells, we sought to study changes in VIP in the pancreas of CF mice. Our goal was to examine VIP content and VIPergic innervation in the pancreas of 8- and 17-week-old F508del-CFTR homozygous mice and to determine whether changes in VIP levels would contribute to CFRD development. Our data showed that a decreased amount of VIP and reduced innervation are found in CF mice pancreas, and that these mice also exhibited reduced insulin secretion, up-regulation of glucagon production and high random blood glucose levels compared to same-age wild-type mice. We propose that low level of VIP, due to reduced innervation of the CF pancreas and starting at an early disease stage, contributes to changes in insulin and glucagon secretion that can lead to CFRD development.

Disrupted local innervation results in less VIP expression in CF mice tissues.

Vasoactive Intestinal Peptide (VIP) is the major physiological agonist of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) chloride channel activity. VIP functions as a neuromodulator and neurotransmitter secreted by neurons innervating all exocrine glands. VIP is also a potent vasodilator and bronchodilator that regulates exocrine gland secretions, contributing to local innate defense by stimulating the movement of water and chloride transport across intestinal and tracheobronchial epithelia. Previous human studies have shown that the rich intrinsic neuronal networks for VIP secretion around exocrine glands could be lost in tissues from patients with cystic fibrosis. Our research has since confirmed, in vitro and in vivo, the need for chronic VIP exposure to maintain functional CFTR chloride channels at the cell surface of airways and intestinal epithelium, as well as normal exocrine tissues morphology [1]. The goal of the present study was to examine changes in VIP in the lung, duodenum and sweat glands of 8- and 17-weeks old F508del/F508del mice and to investigate VIPergic innervation in the small intestine of CF mice, before important signs of the disease development. Our data show that a low amount of VIP is found in CF tissues prior to tissue damage. Moreover, we found a specific reduction in VIPergic and cholinergic innervation of the small intestine. The general innervation of the primary and secondary myenteric plexus was lost in CF tissues, with the presence of enlarged ganglionic cells in the tertiary layer. We propose that low amount of VIP in CF tissues is due to a reduction in VIPergic and cholinergic innervation and represents an early defect that constitutes an aggravating factor for CF disease progression.

Age related weight loss in female 5xFAD mice from 3 to 12 months of age.

In addition to cognitive decline, patients with Alzheimer's disease (AD) exhibit sensory, motor, and neuropsychiatric deficits. Many AD patients also show weight loss, suggesting that AD may involve a metabolic syndrome. The 5xFAD mouse model shows age-related weight loss compared to wildtype controls, and thus may exhibit metabolic dysfunction. This longitudinal study measured age-related weight loss in female 5xFAD and B6SJL/JF2 wild-type mice from 3 to 12 months of age, and examines some of the behavioural and physiological phenotypes in these mice that have been proposed to contribute to this weight loss. Because some mice had to be singly housed during the study, we also examined genotype by housing interactions. The 5xFAD mice weighed less and ate less than WT littermates starting at 6 months of age, exhibited less home cage activity, had higher frailty scores, less white adipose tissue, and lower leptin expression. At 9 and 12 months of age, heavier 5xFAD mice performed better on the rotarod, suggesting that metabolic deficits which begin between 6 and 9 months of age may exacerbate the behavioural deficits in 5xFAD mice. These results indicate that the 5xFAD mouse is a useful model to study the behavioural and metabolic changes in AD.

The effect of dairy products and non-dairy snacks on food intake, subjective appetite and cortisol levels in children: a randomized control study.

Dairy snacks are available in various physical forms and their consumption is linked to improved metabolic health. The objective of this study was to determine the effect of dairy snacks of different physical forms on short-term food intake (FI), subjective appetite, and the stress hormone, cortisol, in children. Following a repeated-measures crossover design, 40 children aged 9-14 years randomly consumed 1 of 5 isoenergetic (180 kcal) snacks per study session. These snacks included solid (potato chips, cookies, and cheese), semi-solid (Greek yogurt), and fluid (2% fat milk) snacks. FI was measured 120 min after snack consumption. Subjective appetite was measured at 0 (immediately before the snack), 15, 30, 45, 60, 90, and 120 min. Salivary cortisol (n = 18) was measured after the Greek yogurt and cookie snacks at 0, 30, 60, 90, and 120 min. FI did not differ between snacks (P = 0.15). The Greek yogurt (P < 0.0001) and cheese (P = 0.0009) snacks reduced average appetite compared with the 2% fat milk snack. Salivary cortisol levels were not affected by snack (P = 0.84). This study demonstrates that dairy snacks are as effective as other popular snacks at influencing subsequent FI. However, solid and semi-solid dairy snacks are more effective at repressing subjective appetite than a fluid dairy snack. Registered at ClinicalTrials.gov (NCT02484625). Novelty: Milk, Greek yogurt and cheese have a similar effect on short-term food intake in children as popular potato chips and cookie snacks. Solid, semi-solid and liquid snacks have a similar effect on short-term food intake in children.

Bariatric surgery induces a new gastric mucosa phenotype with increased functional glucagon-like peptide-1 expressing cells.

Glucagon-Like Peptide-1 (GLP-1) undergoes rapid inactivation by dipeptidyl peptidase-4 (DPP4) suggesting that target receptors may be activated by locally produced GLP-1. Here we describe GLP-1 positive cells in the rat and human stomach and found these cells co-expressing ghrelin or somatostatin and able to secrete active GLP-1 in the rats. In lean rats, a gastric load of glucose induces a rapid and parallel rise in GLP-1 levels in both the gastric and the portal veins. This rise in portal GLP-1 levels was abrogated in HFD obese rats but restored after vertical sleeve gastrectomy (VSG) surgery. Finally, obese rats and individuals operated on Roux-en-Y gastric bypass and SG display a new gastric mucosa phenotype with hyperplasia of the mucus neck cells concomitant with increased density of GLP-1 positive cells. This report brings to light the contribution of gastric GLP-1 expressing cells that undergo plasticity changes after bariatric surgeries, to circulating GLP-1 levels.

Effects of cultured dairy and nondairy products added to breakfast cereals on blood glucose control, satiation, satiety, and short-term food intake in young women.

Breakfast cereals are often consumed with dairy products or nondairy alternatives; however, the effect of the combination on blood glucose and food intake control is not well investigated. In a randomized, crossover study, 24 healthy women (age: 22.7 ± 2.5 years; body mass index: 22.1 ± 1.5 kg/m2) consumed, to satiation, 1 of 3 treatments: Greek yogurt with granola (150 kcal, 9.2 g protein, 2.6 g fat, 2.0 g dietary fibre, and 21.5 g available carbohydrate/100 g); cultured coconut product with granola (146 kcal, 3.2 g protein, 3.2 g fat, 5.6 g dietary fibre, and 21.9 g available carbohydrate/100 g); or water control. The data were analyzed with repeated-measures ANOVA. The 2 h blood glucose iAUC was 52% lower after the dairy compared with nondairy treatment (P < 0.0001). While there were no differences in food intake between the caloric treatments consumed to satiation, protein intake was 3 times higher and fibre intake was 4 times lower after the dairy compared with nondairy treatment. Both caloric treatments resulted in similar suppression of ad libitum food intake at 2 h (P < 0.003) and subjective appetite over 2 h (P < 0.0001) compared with water. The cumulative food intake over 2 h was lower after water (P < 0.05). The 1.8-fold increase in postprandial insulin after dairy compared with nondairy treatment may explain the reduction in blood glucose without an increase in subsequent energy intake. Novelty Blood glucose in young females is lower after a breakfast with granola in a high-protein cultured dairy than when in a high-fibre nondairy cultured product. Subjective appetite over 2 h and food intake 2 h later was similarly lower after both breakfasts but cumulative intake was higher compared with breakfast skipping.

Molecular cloning, ontogeny and tissue distribution of zebrafish (Danio rerio) prohormone convertases: pcsk1 and pcsk2.

Prohormone convertase subtilisin/kexin (PCSK) enzymes are a family of nine related serine proteases, found in a multitude of tissues, and responsible for the maturation of a variety of protein and peptide precursors. Pcsk1 and Pcsk2 are found within dense core secretory granules in endocrine and neuroendocrine cells and are responsible for cleaving several hormones and neuropeptide precursors. In this work, we cloned and sequenced the cDNA of pcsk1 and pcsk2 from zebrafish (Danio rerio). pcsk1 is a 2268bp ORF, whose 755 amino acid protein product is identical to that predicted from the genome sequence. pcsk2 is a 1941bp ORF, encoding a 646 amino acid peptide. Both Pcsk1 and Pcsk2 display high degrees of similarity to their counterparts in other species, including the conservation of the catalytic triad and other essential residues. The brain contained the highest expression levels of both pcsk1 (1.49+/-0.21) (displayed as ratio to EF-1a), and pcsk2 (0.23+/-0.04). Both transcripts were also detectable in the fore, mid and distal gut. pcsk1 and 2 were detectable at 4.5h post-fertilization, and while pcsk1 expression increased throughout development (0.12+/-0.01 maximum at 3 days post-fertilization), pcsk2 expression was highest at day 5 post-fertilization (0.03+/-0.01), and decreased prior. For the first time, we have identified and characterized a pcsk1 transcript in fish. We have also identified and characterized the pcsk2 transcript in zebrafish, and have assessed the tissue distribution and ontogeny of both.

Effect of dairy and nondairy snacks on postprandial blood glucose regulation in 9-14-year-old children.

In adults, dairy consumption improves short-term blood glucose regulation. It is unknown if these short-term benefits extend to children of different weight statuses. The objective of this study was to investigate the effect of a dairy and nondairy snack in both normal-weight (NW) and overweight/obese (OW/OB) children on blood glucose regulation and food intake (FI). In a repeated-measures crossover design, 11 NW and 7 OW/OB children (age: 9-14 years), consumed, in random order, a dairy (Greek yogurt, 198.9 g, 171 kcal, 0 g fat, 17 g protein) or nondairy (mini sandwich-type cookies, 37.5 g, 175 kcal, 7.5 g fat, 1.3 g protein) snack containing 25 g of available carbohydrates. Ad libitum FI was measured 120 min after snack consumption. Blood glucose, insulin, C-peptide, and glucagon-like peptide-1 (GLP-1) were measured at 0 min (before the snack), and at 30, 60, 90, and 120 min after snack consumption. Insulin secretion was calculated from deconvolution of C-peptide. Hepatic insulin extraction was calculated as C-peptide divided by insulin. FI did not differ between snacks (P = 0.55). Mean blood glucose was lower (P < 0.001) and insulin higher (P < 0.0001) in the 120 min after consuming the dairy snack. C-Peptide concentrations (P = 0.75) and insulin secretion (P = 0.37) were not different between snacks. The increase in insulin was explained by reduced hepatic insulin extraction (P < 0.01). Consumption of the dairy snack also increased mean GLP-1 concentrations (P < 0.001). In conclusion, consumption of a dairy snack by NW and OW/OB children results in reduced postprandial blood glucose concentrations and elevated circulating insulin compared with a nondairy snack possibly because of delayed hepatic insulin extraction.

Differences of pancreatic expression of 7B2 between C57BL/6J and C3H/HeJ mice and genetic polymorphisms at its locus (Sgne1).

C57BL/6 (B6) mice develop glucose intolerance with age, whereas C3H/He (C3H) mice do not. In this study, we examined whether this differential glucose homeostasis was associated with differences of proteolytic activation of pancreatic prohormones. Radioimmunoassays showed comparable levels of fasting plasma insulin between the two strains but a significantly lower glucagon level in B6 mice. Pulse-chase analysis of glucagon biosynthesis in isolated pancreatic islets revealed that proglucagon was less efficiently processed in B6 mice. Because proprotein convertase (PC)2 and its 7B2 helper protein are required for this processing, we quantified islet mRNA levels by RT-PCR and protein levels by immunoblotting. The levels of proPC2 mRNA were similar between the two strains, but B6 protein extracts contained less of the mature PC2. In contrast, 7B2 mRNA and protein levels were both significantly lower in B6 pancreas. Sequencing of the 7B2 gene promoter and cDNA in the two strains revealed seven single nucleotide polymorphisms and one dinucleotide insertion/deletion in the cDNA as well as a single nucleotide polymorphism and two insertions/deletions in the promoter. Differential expression of 7B2 may contribute to the difference between B6 and C3H mice not only in glucagon production and secretion but also in glucose tolerance.

The role of ghrelin in the regulation of glucose homeostasis.

Ghrelin is a 28-amino acid (aa) stomach-derived peptide discovered in 1999 as the endogenous ligand for growth hormone secretagogue-receptor (GHS-R). Ghrelin-producing cells constitute a distinct group of endocrine cells dispersed throughout the gastric mucosa and to a lesser extent in the small intestine and the endocrine pancreas. Ghrelin plasma levels rise during fasting and chronic caloric restriction to stimulate food intake and fat storage and to prevent life-threatening falls in blood glucose. Plasma ghrelin levels decrease after a meal is consumed and in conditions of energy surplus (such as obesity). Ghrelin has emerged as a key player in the regulation of appetite and energy homeostasis. Ghrelin achieves these functions through binding the ghrelin receptor GHS-R in appetite-regulating neurons and in peripheral metabolic organs including the endocrine pancreas. Ghrelin levels are negatively correlated with body mass index (BMI) and insulin resistance. In addition, ghrelin secretion is impaired in obesity and insulin resistance. Several studies highlight an important role for ghrelin in glucose homeostasis. Genetic, immunological, and pharmacological blockade of ghrelin signaling resulted in improved glucose tolerance and insulin sensitivity. Furthermore, exogenous ghrelin administration was shown to decrease glucose-induced insulin release and increase glucose level in both humans and rodents. GHS-R was shown to be expressed in pancreatic ß-cells and ghrelin suppressed insulin release via a Ca2+-mediated pathway. In this review, we provide a detailed summary of recent advances in the field that focuses on the role of insulin and insulin resistance in the regulation of ghrelin secretion and on the role of ghrelin in glucose-stimulated insulin secretion (GSIS).

Peripheral exendin-4 and peptide YY(3-36) synergistically reduce food intake through different mechanisms in mice.

Glucagon-like peptide-1(7-36NH2) (GLP-1) and peptide YY(3-36NH2) (PYY(3-36NH2)) are cosecreted from the intestine in response to nutrient ingestion. Peripheral administration of GLP-1 or PYY(3-36NH2) decreases food intake (FI) in rodents and humans; however, the exact mechanisms by which these peptides regulate FI remain unclear. Male C57BL/6 mice were injected (ip) with exendin-4(1-39) (Ex4, a GLP-1 receptor agonist) and/or PYY(3-36NH2) (0.03-3 microg), and FI was determined for up to 24 h. Ex4 and PYY(3-36NH2) alone decreased FI by up to 83 and 26%, respectively (P < 0.05-0.001), whereas a combination of the two peptides (0.06 microg Ex4 plus 3 microg PYY(3-36NH2)) further reduced FI for up to 8 h in a synergistic manner (P < 0.05-0.001). Ex4 and/or PYY(3-36NH2) delayed gastric emptying by a maximum of 19% (P < 0.01-0.001); however, there was no significant effect on locomotor activity nor was there induction of taste aversion. Capsaicin pretreatment prevented the inhibitory effect of Ex4 on FI (P < 0.05), but had no effect on the anorexigenic actions of PYY(3-36NH2). Similarly, exendin-4(9-39) (a GLP-1 receptor antagonist) partially abolished Ex4-induced anorexia (P < 0.05), but did not affect the satiation produced by PYY(3-36NH2). Conversely, BIIE0246 (a Y2 receptor antagonist) completely blocked the anorexigenic effects of PYY(3-36NH2) (P < 0.001), but had no effect on Ex4-induced satiety. Thus, Ex4 and PYY(3-36NH2) suppress FI via independent mechanisms involving a GLP-1 receptor-dependent, sensory afferent pathway (Ex4) and a Y2-receptor mediated pathway (PYY(3-36NH2)). These findings suggest that administration of low doses of Ex4 together with PYY(3-36NH2) may increase the suppression of FI without inducing significant side effects.

Role of leptin in the regulation of glucagon-like peptide-1 secretion.

Glucagon-like peptide-1 (GLP-1), released from intestinal endocrine L cells, is a potent insulinotropic hormone. GLP-1 secretion is diminished in obese patients. Because obesity is linked to abnormal leptin signaling, we hypothesized that leptin may modulate GLP-1 secretion. Leptin significantly stimulated GLP-1 secretion (by up to 250% of control) from fetal rat intestinal cells, a mouse L cell line (GLUTag), and a human L cell line (NCI-H716) in a dose-dependent manner (P < 0.05-0.001). The long form of the leptin receptor was shown to be expressed, and leptin induced the phosphorylation of STAT3 in the three cell types. The leptin receptor was also expressed by rodent and human intestinal L cells, and leptin (1 mg/kg i.p.) significantly stimulated GLP-1 secretion in rats and ob/ob mice. To determine the effect of leptin resistance on GLP-1 secretion, C57BL/6 mice were fed a high-fat (45%) or low-fat (10%) diet for 8 weeks. Mice on the high-fat diet became obese; developed glucose intolerance, hyperinsulinemia, and hyperleptinemia; and were leptin resistant. Mice on the high-fat diet also had twofold lower basal plasma GLP-1 and a diminished GLP-1 response to oral glucose, by 28.5 +/- 5.0% (P < 0.05). These results show for the first time that leptin stimulates GLP-1 secretion from rodent and human intestinal L cells, and they suggest that leptin resistance may account for the decreased levels of GLP-1 found in obese humans.

Muscarinic receptors control glucagon-like peptide 1 secretion by human endocrine L cells.

Glucagon-like peptide 1 (GLP-1) released from distal intestinal endocrine L cells after food intake is a potent glucose-dependent stimulant of insulin secretion. Plasma levels of GLP-1 rise rapidly after nutrient ingestion through an indirect mechanism triggered from the proximal intestine and involving the vagus nerve. Our previous studies showed the involvement of M1 muscarinic receptors expressed by the L cells in the regulation of postprandial GLP-1 secretion in rats. The goal of this study was to explore the involvement of muscarinic receptors in the regulation of GLP-1 secretion by human L cells using a newly described human L cell line (NCI-H716). Phorbol 12-myristate 13-acetate (positive control) stimulated GLP-1 secretion to 252 +/- 38% of the control (P < 0.001). Bethanechol, a nonselective muscarinic agonist, significantly stimulated GLP-1 secretion to 187 +/- 20% of the control (P < 0.01, n = 8). Pirenzepine (M1 antagonist; 10-1000 microM) and gallamine (M2 antagonist; 10-1000 microM) completely inhibited bethanechol-induced GLP-1 secretion, whereas 4-diphenylacetoxy-N-methylpiperidine (M3 antagonist) had no effect on bethanechol-stimulated GLP-1 secretion. McN-A-343 (M1 muscarinic agonist) dose dependently stimulated GLP-1 secretion (to 252 +/- 50% of control at 1000 microM; P < 0.01), whereas oxotremorine (M3 agonist) had no effect. M1, M2, and M3 muscarinic receptors were shown to be expressed in NCI-H716 cells by Western blot, immunohystochemistry, and RT-PCR. Expression of the M1, M2, and M3 muscarinic receptor subtypes was also confirmed in paraffin-embedded human small intestine sections by double immunofluorescent staining. These results demonstrate the role of M1 and M2 muscarinic receptors expressed by human L cells in the control of GLP-1 secretion.

Muscarinic receptors control postprandial release of glucagon-like peptide-1: in vivo and in vitro studies in rats.

Plasma levels of glucagon-like peptide-1 (GLP-1) rise rapidly after nutrient ingestion through an indirect mechanism triggered from the proximal intestine and involving the vagus nerve that stimulates the L cell in the distal gut. The role of muscarinic receptors in this pathway was thus investigated using the anesthetized rat and fetal rat intestinal cells (FRIC) in culture. GLP-1 secretion from the distal gut increased 5-fold after 3 ml corn oil were placed into the proximal duodenum (P < 0.001). Atropine (a nonspecific muscarinic receptor antagonist) completely inhibited fat-induced GLP-1 secretion in vivo (P < 0.01). Pirenzepine (an M1 muscarinic receptor antagonist) also inhibited fat-induced GLP-1 secretion in vivo, by 91 +/- 6% (P < 0.01). Gallamine (an M2 muscarinic receptor antagonist) and 4-diphenylacetoxy-N-methylpiperidine (an M3 muscarinic receptor antagonist) had no effect. Incubating FRIC cultures with bethanechol (a muscarinic receptor agonist) stimulated GLP-1 secretion to 200 +/- 22% of control (P < 0.01). Pirenzepine and gallamine significantly inhibited bethanechol-stimulated GLP-1 secretion, by 96 +/- 12% and 98 +/- 8%, respectively (P < 0.01). Unexpectedly, 4-diphenylacetoxy-N-methylpiperidine stimulated GLP-1 secretion by FRIC cells, to 324 +/- 52% of the control value (P < 0.01). Double immunofluorescent staining using GLP-1 and M1, M2, and M3 muscarinic receptor antibodies showed expression of the three subtypes of muscarinic receptors by the L cells in rat ileal sections and FRIC cultures. These results demonstrate the role of M1 muscarinic receptors expressed by L cells in the control of postprandial secretion of GLP-1. M2 muscarinic receptors also seem to play a role in controlling GLP-1 secretion by fetal, but not adult, L cells.

Glucagon-like peptide-2 receptor activation in the rat intestinal mucosa.

Glucagon-like peptide-2 (GLP-2) increases small intestinal growth and function in rodents and human subjects. GLP-2 exerts its effects through a seven-transmembrane domain, G protein-coupled receptor (GLP-2R), stimulating cAMP generation and activating protein kinase A signaling in heterologous cell lines transfected with the GLP-2R. As intestinal cell lines expressing the GLP-2R have not been identified, we developed methods for studying GLP-2R signaling in the rat small intestinal mucosa in vitro. Isolated rat intestinal mucosal cells expressed mRNA transcripts for the GLP-2R, as well as for chromogranin A and beta-tubulin III, markers for enteroendocrine and neural cells, respectively. cAMP production in response to [Gly2]GLP-2, a degradation-resistant analog of GLP-2, was maximal at 10-11 m (268 +/- 93% of control, P < 0.001), with reduced cAMP accumulation observed at higher doses. The cAMP response was diminished by pretreatment with 10-9 m GLP-2, and was abolished by pretreatment with 10-6 m GLP-2 (P < 0.05), indicating receptor desensitization. GLP-2 treatment of isolated mucosal cells increased 3H-thymidine incorporation (to 128 +/- 8% of controls, P < 0.05), and this was prevented by inhibition of the protein kinase A pathway with H89. In contrast, GLP-2 did not affect p44/p42 MAPK phosphorylation or the levels of cytosolic calcium in the mucosal cell preparation. These results provide the first evidence that activation of the endogenous rat mucosal GLP-2 receptor is linked to activation of a cAMP/protein kinase A-dependent, growth-promoting pathway in vitro.

Apelin, a new enteric peptide: localization in the gastrointestinal tract, ontogeny, and stimulation of gastric cell proliferation and of cholecystokinin secretion.

Apelin is a recently discovered peptide that is the endogenous ligand for the APJ receptor. The aim of this study was to characterize apelin expression (mRNA levels) in the rat gastrointestinal tract and pancreas, to localize distribution of apelin peptide-containing cells in the stomach by immunohistochemistry, and to characterize the ontogeny of gastric apelin expression and peptide and the influence of apelin on gastric cell proliferation in vitro. Additionally, the effect of apelin on cholecystokinin (CCK) secretion and the involvement of MAPK, protein kinase C, and changes in intracellular Ca(2+) in apelin-induced CCK secretion in vitro were examined. Northern analysis showed a maximal apelin expression in the stomach with a lower expression level in the intestine. Apelin expression was not detected in the pancreas. Immunohistochemistry revealed abundant apelin-positive cells in the glandular epithelium of the stomach. The ontogeny study showed a higher apelin expression in the fetal and postnatal rat stomachs when compared with the adult stomach. In contrast to apelin expression, apelin peptide was not detected in the rat stomach until 20 d of age and then increased progressively with age. Apelin was shown to stimulate gastric cell proliferation in vitro. Apelin also stimulated CCK secretion from a murine enteroendocrine cell line (STC-1); apelin-stimulated CCK secretion is mediated through MAPK but not by intracellular Ca(2+) signaling. Together, these data indicate that apelin is an important new stomach peptide with a potential physiological role in the gastrointestinal tract.