Glucagon-like peptide-1 receptor co-agonists for treating metabolic disease.

BACKGROUND: Glucagon-like peptide-1 receptor (GLP-1R) agonists are approved to treat type 2 diabetes and obesity. They elicit robust improvements in glycemic control and weight loss, combined with cardioprotection in individuals at risk of or with pre-existing cardiovascular disease. These attributes make GLP-1 a preferred partner for next-generation therapies exhibiting improved efficacy yet retaining safety to treat diabetes, obesity, non-alcoholic steatohepatitis, and related cardiometabolic disorders. The available clinical data demonstrate that the best GLP-1R agonists are not yet competitive with bariatric surgery, emphasizing the need to further improve the efficacy of current medical therapy. SCOPE OF REVIEW: In this article, we discuss data highlighting the physiological and pharmacological attributes of potential peptide and non-peptide partners, exemplified by amylin, glucose-dependent insulinotropic polypeptide (GIP), and steroid hormones. We review the progress, limitations, and future considerations for translating findings from preclinical experiments to competitive efficacy and safety in humans with type 2 diabetes and obesity. MAJOR CONCLUSIONS: Multiple co-agonist combinations exhibit promising clinical efficacy, notably tirzepatide and investigational amylin combinations. Simultaneously, increasing doses of GLP-1R agonists such as semaglutide produces substantial weight loss, raising the bar for the development of new unimolecular co-agonists. Collectively, the available data suggest that new co-agonists with robust efficacy should prove superior to GLP-1R agonists alone to treat metabolic disorders.

Rapid selection of a novel GLP-1/GIP dual receptor agonist with prolonged glycemic control and weight loss in rodent animals.

BACKGROUND: Glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) co-agonists have emerged as treatment options for reversing diabetes and obesity. Here, we screened the high potency receptor-biased GLP-1R agonists via a newly designed high-throughput GLP-1R extracellular domain (ECD)-based system and demonstrated its in vitro and in vivo therapeutic characters. METHODS: Twelve 9-mer peptides (named XEL1-XEL12) which were screened from a large phage-displayed peptide library were fused to the N-terminus of GIP (3-30) to generate another twelve fusion peptides, termed XEL13-24. Using the six lysine-altered XEL17 as leading sequences, eighteen fatty chain modified fusion peptides were further assessed via in vitro GLP-1R/GIPR-based cell assay. Moreover, the acute and long-acting in vivo effects of selected candidate on diabetic db/db mice and diet-induced obesity (DIO) rats were both carefully evaluated. RESULTS: XEL17 exhibited balanced activation potency on GLP-1R/GIPR in stable cell lines, and further assessment was performed to evaluate the XEL32, a fatty chain modified XEL17 derivative. Preclinical pharmacodynamic results in diabetic db/db mice demonstrated that XEL32 held outstanding insulinotropic and glucose-lowering activities. In addition, protracted antidiabetic effects of XEL32 were also proved by the hypoglycemic test and multiple oral glucose tolerance test. Furthermore, chronic treatment of XEL32 in DIO rats exhibited outstanding beneficial effects on body weight control, fat loss, food intake control, hemoglobin A1C (HbA1C) reduction as well as the glucose tolerance. CONCLUSIONS: XEL32, as a novel GLP-1/GIP dual receptor agonist, may supply efficient glycemic control and weight loss.

Applications of peptide hormone ligands for the treatment of dumping and short bowel syndrome.

Dumping syndrome is a common and debilitating complication of upper gastrointestinal surgery. Accelerated gastric emptying and dysregulated secretion of gastrointestinal (GI) hormones are involved in its pathophysiology. Pasireotide, a novel somatostatin analogue, improved dumping in a phase-2 study. Preliminary data suggest that the glucagon-like peptide-1 (GLP-1) analogue liraglutide can also improve dumping. Short bowel syndrome is the most common cause of intestinal failure and involves not only a loss of mucosal absorptive area but also hypersecretion and accelerated transit. GLP-2 is the best studied hormone involved in intestinal adaptation. An increasing body of evidence demonstrates that the GLP-2 analogue teduglutide reduces parenteral support needs. New GLP-2 analogues and analogues of other GI hormones such as liraglutide are being investigated as promising treatments in short bowel syndrome.

Treatment of Diabetes and Obesity by Rationally Designed Peptide Agonists Functioning at Multiple Metabolic Receptors.

Obesity and its comorbidities such as type 2 diabetes constitute major worldwide health threats, and the identification of an effective medical intervention has emerged as a global priority. The limited effectiveness of historical, anti-obesity treatments is commonly attributed to the complexity of the disease and the redundancy of metabolic regulatory mechanisms that sustain body weight. At the forefront of obesity research is the development of combinational drug therapies that simultaneously target multiple regulatory pathways, which promote dysfunctional metabolism. Recently, molecularly crafted unimolecular "multi-agonism" of balanced activity at 3 key receptors involved in metabolism and specifically the glucagon-like peptide (GLP)-1 receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor and glucagon receptor was reported as superior to conventional monoagonist therapy. These mixed peptide agonists are designed to pharmacologically integrate the insulinotropic and anorexigenic effects of GLP-1, the thermogenic and lipolytic activities of glucagon, and the insulinotropic and insulin sensitizing properties of GIP. The molecular mechanism of these purposefully promiscuous ligands is not completely understood, however, recent studies in pancreatic beta cells point to the prospect of a complex signaling network that can magnify the signaling of multi-agonist ligands. The activation of this signalosome might explain the additional therapeutic benefit inherent to simultaneous cellular activation through multiple metabolic receptors.

Common variants of GIP are associated with visceral fat accumulation in Japanese adults.

Animal studies have demonstrated that glucose-dependent insulinotropic polypeptide (GIP) and GIP receptor (GIPR) contribute to the etiology of obesity. In humans, genomewide association studies have identified single nucleotide polymorphisms (SNPs) in the GIPR gene that are strongly associated with body mass index (BMI); however, it is not clear whether genetic variations in the GIP gene are involved in the development of obesity. In the current study, we assessed the impact of GIP SNPs on obesity-related traits in Japanese adults. Six tag SNPs were tested for associations with obesity-related traits in 3,013 individuals. Multiple linear regression analyses showed that rs9904288, located at the 3'-end of GIP, was significantly associated with visceral fat area (VFA). Moreover, rs1390154 and rs4794008 showed significant associations with plasma triglyceride levels and hemoglobin A1c levels, respectively. Among the significant SNPs, rs9904288 and rs1390154 were independently linked with SNPs in active enhancers of the duodenum mucosa, the main GIP-secreting tissue. The haplotypes of these two SNPs exhibited stronger associations with VFA. Numbers of VFA-increasing alleles of rs9904288 and BMI-increasing alleles of previously identified GIPR SNPs showed a strong additive effect on VFA, waist circumference, and BMI in the subject population. These novel results support the notion that the GIP-GIPR axis plays a role in the etiology of central obesity in humans, which is characterized by the accumulation of visceral fat.

Physiologic and pharmacologic modulation of glucose-dependent insulinotropic polypeptide (GIP) receptor expression in beta-cells by peroxisome proliferator-activated receptor (PPAR)-gamma signaling: possible mechanism for the GIP resistance in type 2 diabetes.

OBJECTIVE: We previously showed that peroxisome proliferator-activated receptor (PPAR)-gamma in beta-cells regulates pdx-1 transcription through a functional PPAR response element (PPRE). Gene Bank blast for a homologous nucleotide sequence revealed the same PPRE within the rat glucose-dependent insulinotropic polypeptide receptor (GIP-R) promoter sequence. We investigated the role of PPARgamma in GIP-R transcription. RESEARCH DESIGN AND METHODS: Chromatin immunoprecipitation assay, siRNA, and luciferase gene transcription assay in INS-1 cells were performed. Islet GIP-R expression and immunohistochemistry studies were performed in pancreas-specific PPARgamma knockout mice (PANC PPARgamma(-/-)), normoglycemic 60% pancreatectomy rats (Px), normoglycemic and hyperglycemic Zucker fatty (ZF) rats, and mouse islets incubated with troglitazone. RESULTS: In vitro studies of INS-1 cells confirmed that PPAR-gamma binds to the putative PPRE sequence and regulates GIP-R transcription. In vivo verification was shown by a 70% reduction in GIP-R protein expression in islets from PANC PPARgamma(-/-) mice and a twofold increase in islets of 14-day post-60% Px Sprague-Dawley rats that hyperexpress beta-cell PPARgamma. Thiazolidinedione activation (72 h) of this pathway in normal mouse islets caused a threefold increase of GIP-R protein and a doubling of insulin secretion to 16.7 mmol/l glucose/10 nmol/l GIP. Islets from obese normoglycemic ZF rats had twofold increased PPARgamma and GIP-R protein levels versus lean rats, with both lowered by two-thirds in ZF rats made hyperglycemic by 60% Px. CONCLUSIONS: Our studies have shown physiologic and pharmacologic regulation of GIP-R expression in beta-cells by PPARgamma signaling. Also disruption of this signaling pathway may account for the lowered beta-cell GIP-R expression and resulting GIP resistance in type 2 diabetes.

Chemical modification of class II G protein-coupled receptor ligands: frontiers in the development of peptide analogs as neuroendocrine pharmacological therapies.

Recent research and clinical data have begun to demonstrate the huge potential therapeutic importance of ligands that modulate the activity of the secretin-like, Class II, G protein-coupled receptors (GPCRs). Ligands that can modulate the activity of these Class II GPCRs may have important clinical roles in the treatment of a wide variety of conditions such as osteoporosis, diabetes, amyotrophic lateral sclerosis and autism spectrum disorders. While these receptors present important new therapeutic targets, the large glycoprotein nature of their cognate ligands poses many problems with respect to therapeutic peptidergic drug design. These native peptides often exhibit poor bioavailability, metabolic instability, poor receptor selectivity and resultant low potencies in vivo. Recently, increased attention has been paid to the structural modification of these peptides to enhance their therapeutic efficacy. Successful modification strategies have included d-amino acid substitutions, selective truncation, and fatty acid acylation of the peptide. Through these and other processes, these novel peptide ligand analogs can demonstrate enhanced receptor subtype selectivity, directed signal transduction pathway activation, resistance to proteolytic degradation, and improved systemic bioavailability. In the future, it is likely, through additional modification strategies such as addition of circulation-stabilizing transferrin moieties, that the therapeutic pharmacopeia of drugs targeted towards Class II secretin-like receptors may rival that of the Class I rhodopsin-like receptors that currently provide the majority of clinically used GPCR-based therapeutics. Currently, Class II-based drugs include synthesized analogs of vasoactive intestinal peptide for type 2 diabetes or parathyroid hormone for osteoporosis.

The role of peptide YY in appetite regulation and obesity.

The last decade has witnessed a marked increase in our understanding of the importance of gut hormones in the regulation of energy homeostasis. In particular, the discovery that the gut hormone peptide YY 3-36 (PYY3-36) reduced feeding in obese rodents and humans fuelled interest in the role of PYY3-36 in body weight regulation. Pharmacological and genetic approaches have revealed that the Y2-receptor mediates the anorectic effects of PYY3-36 whilst mechanistic studies in rodents identified the hypothalamus, vagus and brainstem regions as potential sites of action. More recently, using functional brain imaging techniques in humans, PYY3-36 was found to modulate neuronal activity within hypothalamic and brainstem, and brain regions involved in reward processing. Several lines of evidence suggest that low circulating PYY concentrations predispose towards the development and or maintenance of obesity. Subjects with reduced postprandial PYY release exhibit lower satiety and circulating PYY levels that correlate negatively with markers of adiposity. In addition, mice lacking PYY are hyperphagic and become obese. Conversely, chronic PYY3-36 administration to obese rodents reduces adiposity, and transgenic mice with increased circulating PYY are resistant to diet-induced obesity. Moreover, there is emerging evidence that PYY3-36 may partly mediate the reduced appetite and weight loss benefits observed post-gastric bypass surgery. Taken together these findings, coupled with the retained responsiveness of obese subjects to the effects of PYY3-36, suggest that targeting the PYY system may offer a therapeutic strategy to help treat obesity.

Pancreatic polypeptide is secreted from and controls differentiation through its specific receptors in osteoblastic MC3T3-E1 cells.

Although the neuropeptide Y (NPY) family has been demonstrated to control bone metabolism, the role of pancreatic polypeptide (PP), which has structural homology with NPY and peptide YY (PYY) to share the NPY family receptors, in peripheral bone tissues has remained unknown. In the present study, we studied the regulatory roles of PP and its Y receptors using MC3T3-E1 cells, a murine transformed osteoblastic cell line, as a model for osteoblastic differentiation. We found that (1) PP mRNA was detected and increased during cell-contact-induced differentiation in MC3T3-E1 cells; (2) the immunoreactivity of PP was detected by radioimmunoassay and increased in culture medium during differentiation; (3) all the types of NPY family receptor mRNAs (Y1, Y2, Y4, Y5, and y6) were found to increase during differentiation; (4) PP stimulated differentiation in MC3T3-E1 cells in terms of ALP mRNA and BMP-2 mRNA. These findings suggested that MC3T3-E1 cells produce and secrete PP, which may in turn stimulate the differentiation of MC3T3-E1 through its specific receptors in an autocrine manner.

Incretin receptors for glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide are essential for the sustained metabolic actions of vildagliptin in mice.

OBJECTIVE: Dipeptidyl peptidase-4 (DPP4) inhibitors lower blood glucose in diabetic subjects; however, the mechanism of action through which these agents improve glucose homeostasis remains incompletely understood. Although glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic polypeptide (GIP) represent important targets for DPP4 activity, whether additional substrates are important for the glucose-lowering actions of DPP4 inhibitors remains uncertain. RESEARCH DESIGN AND METHODS: We examined the efficacy of continuous vildagliptin administration in wild-type (WT) and dual incretin receptor knockout (DIRKO) mice after 8 weeks of a high-fat diet. RESULTS: Vildagliptin had no significant effect on food intake, energy expenditure, body composition, body weight gain, or insulin sensitivity in WT or DIRKO mice. However, glycemic excursion after oral glucose challenge was significantly reduced in WT but not in DIRKO mice after vildagliptin treatment. Moreover, vildagliptin increased levels of glucose-stimulated plasma insulin and reduced levels of cholesterol and triglycerides in WT but not in DIRKO mice. Vildagliptin treatment reduced the hepatic expression of genes important for cholesterol synthesis and fatty acid oxidation, including phospho-mevalonate kinase (Mvk), acyl-coenzyme dehydrogenase medium chain (Acadm), mevalonate (diphospho)decarboxylase (Mvd), and Acyl-CoA synthetase (Acsl1), in WT but not in DIRKO mice. However, vildagliptin also reduced levels of hepatic mRNA transcripts for farnesyl di-phosphate transferase (Fdft1), acetyl coenzyme A acyltransferase 1 (Acaa1), and carnitine palmitoyl transferase 1 (Cpt 1) in DIRKO mice. No direct effect of GLP-1 receptor agonists was detected on cholesterol or triglyceride synthesis and secretion in WT hepatocytes. CONCLUSIONS: These findings illustrate that although GLP-1 and GIP receptors represent the dominant molecular mechanisms for transducing the glucoregulatory actions of DPP4 inhibitors, prolonged DPP4 inhibition modulates the expression of genes important for lipid metabolism independent of incretin receptor action in vivo.

Metabolic effects of sub-chronic ablation of the incretin receptors by daily administration of (Pro3)GIP and exendin(9-39)amide in obese diabetic (ob/ob) mice.

Effects of chemical ablation of the GIP and GLP-1 receptors on metabolic aspects of obesity-diabetes were investigated using the stable receptor antagonists (Pro3)GIP and exendin(9-39)amide. Ob/ob mice received a daily i.p. injection of saline vehicle, (Pro3)GIP, exendin(9-39)amide or a combination of both peptides over a 14-day period. Non-fasting plasma glucose levels were significantly (p<0.05) lower in (Pro3)GIP-treated mice compared to control mice after just 9 days of treatment. (Pro3)GIP-treated mice also displayed significantly lower plasma glucose concentrations in response to feeding and intraperitoneal administration of either glucose or insulin (p<0.05 to p<0.001). The (Pro3)GIP-treated group also exhibited significantly (p<0.05) reduced pancreatic insulin content. Acute administration of exendin(9-39)amide immediately prior to re-feeding completely annulled the beneficial effects of sub-chronic (Pro3)GIP treatment, but non-fasting concentrations of active GLP-1 were unchanged. Combined sub-chronic administration of (Pro3GIP) with exendin(9-39)amide revealed no beneficial effects. Similarly, daily administration of exendin(9-39)amide alone had no significant effects on any of the metabolic parameters measured. These studies highlight an important role for GIP in obesity-related forms of diabetes, suggesting the possible involvement of GLP-1 in the beneficial actions of GIP receptor antagonism.

Chemical ablation of gastric inhibitory polypeptide receptor action by daily (Pro3)GIP administration improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure in obesity-related diabetes.

Glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide [GIP]) is an important incretin hormone secreted by endocrine K-cells in response to nutrient ingestion. In this study, we investigated the effects of chemical ablation of GIP receptor (GIP-R) action on aspects of obesity-related diabetes using a stable and specific GIP-R antagonist, (Pro3)GIP. Young adult ob/ob mice received once-daily intraperitoneal injections of saline vehicle or (Pro3)GIP over an 11-day period. Nonfasting plasma glucose levels and the overall glycemic excursion (area under the curve) to a glucose load were significantly reduced (1.6-fold; P < 0.05) in (Pro3)GIP-treated mice compared with controls. GIP-R ablation also significantly lowered overall plasma glucose (1.4-fold; P < 0.05) and insulin (1.5-fold; P < 0.05) responses to feeding. These changes were associated with significantly enhanced (1.6-fold; P < 0.05) insulin sensitivity in the (Pro3)GIP-treated group. Daily injection of (Pro3)GIP reduced pancreatic insulin content (1.3-fold; P < 0.05) and partially corrected the obesity-related islet hypertrophy and beta-cell hyperplasia of ob/ob mice. These comprehensive beneficial effects of (Pro3)GIP were reversed 9 days after cessation of treatment and were independent of food intake and body weight, which were unchanged. These studies highlight a role for GIP in obesity-related glucose intolerance and emphasize the potential of specific GIP-R antagonists as a new class of drugs for the alleviation of insulin resistance and treatment of type 2 diabetes.

In depth analysis of the N-terminal bioactive domain of gastric inhibitory polypeptide.

Gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal regulator of insulin release and glucose homeostasis following a meal. Strategies have been undertaken to delineate the bioactive domains of GIP with the intention of developing small molecular weight GIP mimetics. The molecular cloning of receptors for GIP and the related hormone GLP-1 (glucagon-like peptide-1) has allowed examination of the characteristics of incretin analogs in transfected cell models. The current report examines the N-terminal bioactive domain of GIP residing in residues 1-14 by alanine scanning mutagenesis and N-terminal substitution/modification. Further studies examined peptide chimeras of GIP and GLP-1 designed to localize bioactive determinants of the two hormones. The alanine scan of the GIP(1-14) sequence established that the peptide was extremely sensitive to structural perturbations. Only replacement of amino acids 2 and 13 with those found in glucagon failed to dramatically reduce receptor binding and activation. Of four GIP(1-14) peptides modified by the introduction of DP IV-resistant groups, a peptide with a reduced bond between Ala2 and Glu3 demonstrated improved receptor potency compared to native GIP(1-14). The peptide chimera studies supported recent results on the importance of a mid-region helix for bioactivity of GIP, and confirmed existence of two separable regions with independent intrinsic receptor binding and activation properties. Furthermore, peptide chimeras showed that binding of GLP-1 also involves both N- and C-terminal domains, but that it apparently contains only a single bioactive domain in its N-terminus. Together, these results should facilitate development of incretin based therapies using rational drug design for potential treatment of diabetes.

Gut peptides in the treatment of diabetes mellitus.

It has been known for at least one century that agents secreted from the intestine during meal absorption regulates glucose assimilation. Extensive research during the past three decades has identified two gut hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP, also known as gastric inhibitory polypeptide) that are important in postprandial glucose metabolism. Both peptides are incretins; they are secreted during carbohydrate absorption and increase insulin secretion. Since they are potent insulin secretagogues, GIP and GLP-1 have received considerable attention as potential diabetes therapeutics. However, only GLP-1 exerts insulinotropic properties when administered to patients with Type 2 diabetes. Both GLP-1 and GIP are rapidly inactivated in the circulation by the enzyme dipeptidyl peptidase IV (DPP-IV). The application of GLP-1 into clinical practice has been delayed due to the need to develop compounds that overcome this rapid inactivation. Two approaches have been taken to utilise the insulinotropic and glucose-lowering actions of GLP-1 as an antidiabetic agent: the development of DPP-IV-resistant analogues and the inhibition of DPP-IV. This review focuses on the physiology of GLP-1 and GIP and the advances that have been made thus far in developing treatments based on these physiological incretins for Type 2 diabetes.

CpG methylation and transcription factors Sp1 and Sp3 regulate the expression of the human secretin receptor gene.

The human secretin receptor (hSR) is an important glycoprotein receptor for regulating the secretion of pancreatic bicarbonate, water, and electrolytes. In this study we investigated the transcriptional regulation of the hSR gene. A minimal 106-bp promoter was identified, and it contains two GC boxes (GC box-A, -240 to -226; and GC box-B, -203 to -194, from the translation start site). EMSA and supershift analyses showed that both GC boxes interact with Sp1 and Sp3 transcription factors. Transient transfection in pancreas-derived human pancreatic ductule carcinoma (PANC)-1 and bovine pancreatic duct-1 cells showed that mutation of either GC box-A or -B reduced the promoter strength by 56-67%, whereas mutation of both GC boxes caused more than 90% reduction of promoter activity. Cotransfections of the hSR promoter with Sp1 and Sp3 expression vectors in Sp-deficient Drosophila SL-2 Schneider cells further demonstrated that the ratio of Sp1 to Sp3 is the key mechanism to modulate hSR gene expression. The methylation statuses of 27 CpG sites within the promoter region (-400 to -151 bp) were assessed in various human pancreas and liver cell lines. The hSR promoter is unmethylated (CAPAN-1, human pancreatic adenocarcinoma) or partially methylated (PANC-1 and HPAC, human pancreatic adenocarcinoma) in hSR-expressing cell lines but is completely methylated in hSR nonexpressing HepG2 cells. Methyltransferase inhibitor 5-aza-2'deoxycytidine increased hSR gene expression level in PANC-1 cells and induced hSR gene expression in HepG2 cells. Together, our study shows that, in addition to Sp1 and Sp3, promoter methylation also plays a role in the regulation of hSR gene expression.

Interaction of the growth hormone-releasing peptides ghrelin and growth hormone-releasing peptide-6 with the motilin receptor in the rabbit gastric antrum.

The structural relationship between the motilin and the growth hormone secretagogue receptor (GHS-R), and between their respective ligands, motilin and ghrelin, prompted us to investigate whether ghrelin and the GHS-R agonist growth hormone-releasing peptide-6 (GHRP-6), could interact with the motilin receptor. The interaction was evaluated in the rabbit gastric antrum with binding studies on membrane preparations and with contraction studies on muscle strips in the presence of selective antagonists under conditions of electrical field stimulation (EFS) or not. Binding studies indicated that the affinity (pK(d)) for the motilin receptor was in the order of ghrelin (4.23 +/- 0.07) < GHRP-6 (5.54 +/- 0.08) < motilin (9.13 +/- 0.03). The interaction of ghrelin with the motilin receptor requires the octanoyl group. Motilin induced smooth muscle contractile responses but ghrelin and GHRP-6 were ineffective. EFS elicited on- and off-responses that were increased by motilin already at 10(-9) M, but not by 10(-5) M ghrelin. In contrast, GHRP-6 also enhanced the on- and off-responses. The motilin antagonist Phe-cyclo[Lys-Tyr(3-tBu)-betaAla-] trifluoroacetate (GM-109) blocked the effect of GHRP-6 on the off-responses but not on the on-responses. Under nonadrenergic noncholinergic conditions, the effects of motilin and GHRP-6 on the on-responses were abolished; those on the off-responses were preserved. All responses were blocked by neurokinin (NK)(1) and NK(2) antagonists. In conclusion, ghrelin is unable to induce contractions via the motilin receptor. However, GHRP-6 enhances neural contractile responses, partially via interaction with the motilin receptor on noncholinergic nerves with tachykinins as mediator, and partially via another receptor that may be a GHS-R subtype on cholinergic nerves that corelease tachykinins.

Ghrelin and growth hormone (GH) secretagogues potentiate GH-releasing hormone (GHRH)-induced cyclic adenosine 3',5'-monophosphate production in cells expressing transfected GHRH and GH secretagogue receptors.

GHRH stimulates GH secretion from somatotroph cells of the anterior pituitary via a pathway that involves GHRH receptor activation of adenylyl cyclase and increased cAMP production. The actions of GHRH to release GH can be augmented by the synthetic GH secretagogues (GHS), which bind to a distinct G protein-coupled receptor to activate phospholipase C and increase production of the second messengers calcium and diacylglycerol. The stomach peptide ghrelin represents an endogenous ligand for the GHS receptor, which does not activate the cAMP signaling pathway. This study investigates the effects of GHS and ghrelin on GHRH-induced cAMP production in a homogenous population of cells expressing the cloned GHRH and GHS receptors. Each epitope-tagged receptor was shown to be appropriately expressed and to functionally couple to its respective second messenger pathway in this heterologous cell system. Although activation of the GHS receptor alone had no effect on cAMP production, coactivation of the GHS and GHRH receptors produced a cAMP response approximately twice that observed after activation of the GHRH receptor alone. This potentiated response is dose dependent with respect to both GHRH and GHS, is dependent on the expression of both receptors, and was observed with a variety of peptide and nonpeptide GHS compounds as well as with ghrelin-(1-5). Pharmacological inhibition of signaling molecules associated with GHS receptor activation, including G protein betagamma-subunits, phospholipase C, and protein kinase C, had no effect on GHS potentiation of GHRH-induced cAMP production. Importantly, the potentiation appears to be selective for the GHRH receptor. Treatment of cells with the pharmacological agent forskolin elevated cAMP levels, but these levels were not further increased by GHS receptor activation. Similarly, activation of two receptors homologous to the GHRH receptor, the vasoactive intestinal peptide and secretin receptors, increased cAMP levels, but these levels were not further increased by GHS receptor activation. Based on these findings, we speculate that direct interactions between the GHRH and GHS receptors may explain the observed effects on signal transduction.

Demonstration of a functional motilin receptor in TE671 cells from human cerebellum.

BACKGROUND: Our laboratory has described the presence of motilin receptors in the rabbit cerebellum. We discovered its presence in the human TE671 cell line, which is of cerebellar origin. METHODS: Cytosolic Ca(2+) fluxes were monitored on a confocal microscope in cells loaded with Indo-1 and stimulated with motilin under various conditions. Binding studies were performed with 125I-[Nle(13)]porcine motilin. Using primers, PCR for the motilin receptor was performed. RESULTS: Cells responded to motilin after 45+/-20 s. At different concentrations of motilin (10(-8), 10(-7), 10(-6.5), 10(-6) and 10(-5) M) the percentage of responding cells was 0+/-0, 0.6+/-1.5, 4.9+/-4.7, 21.7+/-15 and 35.7+/-12, respectively. The response was blocked by the motilin antagonists [Phe(3), Nle(13)]po-motilin (0.8+/-1.8%) and GM-109 (0.0+/-0.0%) and mimicked by the agonist ABT-229 (23.6+/-15%). After stimulation with motilin, ABT-229 or [Phe(3),Leu(13)]po-motilin, but not with the antagonist GM-109, cells were desensitized. The response to motilin persisted in Ca(2+)-free solution (22.8+/-14.7%), was not affected by nifedipine (44+/-11%) but was abolished by incubation with thapsigargin (0+/-0%). Neither ryanodine, nor a previous stimulation with caffeine (0+/-0%) in Ca(2+)-free Krebs, nor both could block the response to motilin (28, 32.0+/-5.7, 41.3+/-6.1%, respectively). Binding studies revealed two binding sites for motilin, with a pK(d) of 8.9+/-0.05 and 6.11+/-0.61 (n=4). There were 100 times more low than high affinity receptors per cell. The presence of receptor mRNA was confirmed by PCR. CONCLUSION: Functional motilin receptors are present in TE671 cells. The response requires intracellular IP(3)-sensitive Ca(2+) stores. These cells may serve as a model of the central motilin receptor.

Vagus nerve modulates secretin binding sites in the rat forestomach.

Secretin is well known for its inhibitory action on gastric motility. It has been reported that secretin in a physiological dose inhibits gastric motility through mediation by the vagal afferent pathway. Secretin also elicited relaxation of carbachol-stimulated rat forestomach muscle strips by binding to its receptors, suggesting a direct action on this peripheral tissue. We hypothesized that vagal input may affect the action of secretin by modulating the level of secretin receptor in the forestomach. Several treatments, including vagal ligation, vagotomy, perivagal application of capsaicin or colchicine, intravenous infusion of tetrodotoxin, and intraperitoneal injection of atropine, were performed to investigate their effects on secretin receptor binding to forestomach membranes. Specific binding of 125I-labeled secretin to forestomach membranes was significantly decreased (45%) by vagal ligation, vagotomy (50%), or perivagal colchicine treatment (40%). On the contrary, specific binding of 125I-secretin was not affected by perivagal capsaicin treatment, intravenous infusion of tetrodotoxin, or intraperitoneal injection of atropine. By Scatchard analysis of the binding data, the capacity of the high-affinity binding sites in forestomach membranes was found to decrease significantly after vagal ligation compared with membranes from the sham-operated group. However, the affinity at the high-affinity binding sites, the binding parameters of the low-affinity binding sites, and binding specificity were not changed. Vagal ligation but not perivagal capsaicin treatment reduced the inhibitory effect of secretin on bethanechol-stimulated contraction of isolated forestomach muscle strips, causing a right shift in the dose-response curve. These results suggest that vagal input through axonal transport plays a significant role on secretin action by modulating the capacity of secretin binding sites (but not affinity or specificity), at least in rat forestomach.