Overexpression of glucagon-like peptide-1 receptor in an insulin-secreting cell line enhances glucose responsiveness

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the VIP (vasoactive intestinal polypeptide) family of peptides, has been demonstrated in neurons of the sensory system. PACAP expression of these neurons is sensitive to nerve damages such as nerve crush or axotomy. In the present study, PACAP expression in the mesencephalic trigeminal nucleus of the rat was examined after transsection of the main trunk of the masseteric nerve. The primary sensory neurons of the nucleus are considered to have purely proprioceptive functions. By quantitative in situ hybridization using a PACAP [35S]cRNA probe, an increase in PACAP mRNA was observed on the side ipsilateral to transsection already after 3 h and the expression reached a peak 24 h after surgery after which the levels gradually decreased during the next 14 days. A low and constant expression of PACAP mRNA could be seen on the side contralateral to transsection. PACAP immunoreactivity was demonstrated on the ipsilateral side after 18 h, using a specific monoclonal PACAP antibody. Co-existence of PACAP with NPY and galanin was demonstrated 7 days after transsection. Analysis of the masseteric nerve by radioimmunoassay on transsected and normal nerve stumps revealed an increase of PACAP-38 immunoreactivity in the nerve proximal to the transsection compared to the normal side (15.3 vs. 6.1 pmol/g wt). The results suggest that PACAP has a role in the early phase of adaptation to nerve injury in the proprioceptive neurons.     

Rapid oscillations in plasma glucagon-like peptide-1 (GLP-1) in humans: cholinergic control of GLP-1 secretion via muscarinic receptors

The mechanisms involved in the rapid glucagon-like peptide-1 (GLP-1) release following glucose ingestion are poorly defined. Besides a direct intestinal stimulation of L cells, humoral and neuronal mechanisms have been discussed. We investigated the temporal pattern of GLP-1 release in five healthy men (aged 27.8 +/- 3.6 yr, body mass index, 23.4 +/- 1.2 kg/m2) after an overnight fast for 60 min under basal conditions and for 60 min after an oral glucose load (OGL; 100 g) in both the presence and absence of atropine (80 ng/kg min, iv). Blood was sampled every 2 min, and data were evaluated for the temporal pattern of GLP-1 secretion by several computer-assisted programs (deconvolution, Pulsar analysis, and Fourier transformation). With all methods a pulsatile pattern of plasma GLP-1 levels with a frequency of five to seven per h was detected; this remained unchanged in the different metabolic states and during atropine treatment. Glucose and GLP-1 plasma levels showed a parallel increase after OGL (OGL without atropine = control: 8.4 +/- 2.9 and 7.9 +/- 3.0 min, respectively). Atropine infusion delayed this increase significantly (16.8 +/- 8.07 and 17.4 +/- 6.61 min, respectively; P < 0.02). In contrast to plasma glucose concentrations (82.7 +/- 0.3% of control; P < 0.05), atropine infusion reduced the integrated GLP-1 pulse amplitude to 56.0 +/- 11.3% of the control levels (P < 0.05). In conclusion, GLP-1 is secreted in a pulsatile manner with a frequency comparable to that of pancreatic hormones. Mean GLP-1 plasma concentrations increase after OGL due to augmented GLP-1 pulse amplitudes but not frequency. The differential effect of atropine on glucose and GLP-1 plasma levels suggest a direct cholinergic muscarinic control of L cells.     

Structure-function analysis of a series of glucagon-like peptide-1 analogs

We have used NMR in conjunction with measurements of functional bioactivity to define the receptor-binding structure of glucagon-like peptide-1 (GLP-1.) Identification of the important residues for binding was accomplished by the substitution of amino acids at sites that seemed likely, from an examination of the amino acid sequence and from previously published observations, to be important in the three-dimensional (3D) structure of the molecule. Identification of the receptor-bound conformation of GLP-1, because it is a flexible peptide, required constraint of the peptide backbone into a predetermined 3D structure. Constraint was achieved by the introduction of disulfide bonds and specific side chain-side chain cross-links. The biological relevance of the synthetic structure of each rigidified peptide was assessed by measurement of its ability to bind to the receptor present on RINm5F cells and to elicit a functional response, cyclic AMP production. NMR solution structures were obtained for the most biologically relevant of these analogs. The results of this study indicated that the residues necessary for the biological activity of GLP-1 occupy approximately three equally-spaced regions of the peptide 3D structure, at the corners of an equilateral triangle whose sides are, at a minimum estimate, 12-15A.     

Inhibition of intestinal glucagon-like immunoreactivity (GLI) secretion by somatostatin in man

This work was undertaken to investigate the effect of somatostatin on intestinal glucagon-like immunoreactivity (GLI) secretion in man. In normal subjects GLI release is slightly stimulated by oral glucose while this sugar evokes a much greater GLI response in gastrectomized patients. Therefore, our study was performed in a group of such patients (N = 6). As expected, in the control experiments glucose ingestion elicited a clear-cut elevation of GLI plasma levels as measured with two antisera, 78J and R-8 (maximal peaks: 340% and 150% above basal values, respectively). Somatostatin infusion did not modify fasting GLI concentrations but completely abolished GLI response to glucose. Termination of the infusion was followed by a rebound of circulating GLI. The well-known suppressor effect of somatostatin on glucagon and insulin secretion was also detected. Finally, during somatostatin infusion the initial elevation of blood sugar after oral glucose, in the absence of insulin response, appeared considerably delayed. Our data demonstrate that somatostatin behaves as a potent inhibitory agent of GLI secretion in man. A retarding effect of somatostatin on glucose absorption is also compatible with our results.     

High potency antagonists of the pancreatic glucagon-like peptide-1 receptor

GLP-1-(7-36)-amide and exendin-4-(1-39) are glucagon-like peptide-1 (GLP-1) receptor agonists, whereas exendin-(9-39) is the only known antagonist. To analyze the transition from agonist to antagonist and to identify the amino acid residues involved in ligand activation of the GLP-1 receptor, we used exendin analogs with successive N-terminal truncations. Chinese hamster ovary cells stably transfected with the rat GLP-1 receptor were assayed for changes in intracellular cAMP caused by the test peptides in the absence or presence of half-maximal stimulatory doses of GLP-1. N-terminal truncation of a single amino acid reduced the agonist activity of the exendin peptide, whereas N-terminal truncation of 3-7 amino acids produced antagonists that were 4-10-fold more potent than exendin-(9-39). N-terminal truncation of GLP-1 by 2 amino acids resulted in weak agonist activity, but an 8-amino acid N-terminal truncation inactivated the peptide. Binding studies performed using 125I-labeled GLP-1 confirmed that all bioactive peptides specifically displaced tracer with high potency. In a set of exendin/GLP-1 chimeric peptides, substitution of GLP-1 sequences into exendin-(3-39) produced loss of antagonist activity with conversion to a weak agonist. The results show that receptor binding and activation occur in separate domains of exendin, but they are more closely coupled in GLP-1.     

Amidated and non-amidated glucagon-like peptide-1 (GLP-1): non-pancreatic effects (cephalic phase acid secretion) and stability in plasma in humans

The incretin and enterogastrone hormone, GLP-1, occurs in an amidated (GLP-1 (7-36) amide; 75%) and a glycine-extended (GLP-1 (7-37); 25%) form. Their effects on the endocrine pancreas are similar and their overall (mainly renal) elimination rates appear to equal. Assuming that they might differentially affect non-pancreatic targets we investigated the effect of GLP-1 (7-37) infused at 0.7 pmol/kg/min on sham-feeding induced acid secretion in six healthy volunteers. The infusion increased the plasma concentrations from 16+/-2 pmol/l to 45+/-2 pmol/l. This was associated with a 61+/-14% decrease in acid output compared to saline and was not significantly different from that previously observed with GLP-1 (7-36) amide infused at the same rate. We then compared the degradation of the two forms in human plasma at 37 degrees C in vitro. T1/2 values were 32+/-3 (7-37) and 42+/-2 min (7-36) amide (P=0.007). The difference in metabolism persisted after addition of diprotin A, an inhibitor of dipeptidyl peptidase IV, the enzyme responsible for the initial degradation of GLP-1 in plasma, and broader enzyme inhibitors. Thus, the only effect of the amidation of GLP-1 seems to be to enhance its survival in plasma.     

Relation between gastric emptying of glucose and plasma concentrations of glucagon-like peptide-1

-Dopamine, via D1-like receptors, stimulates the activity of both protein kinase A (PKA) and protein kinase C (PKC), which results in inhibition of renal sodium transport. Since D1-like receptors differentially regulate sodium transport in normotensive and hypertensive rats, they may also differentially regulate PKC expression in these rat strains. Thus, 2 different D1-like agonists (fenoldopam or SKF 38393) were infused into the renal artery of anesthetized normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) (n=5 to 6/drug/strain). Ten or 60 minutes after starting the D1-like agonist infusion, both the infused kidney and the noninfused kidney that served as control were prepared for analysis. The D1-like agonists produced a greater diuresis and natriuresis and inhibited Na+,K+-ATPase activity in proximal tubule (PT) and medullary thick ascending limb (mTAL) to a greater extent in WKY (Delta20+/-1%) than in SHR (Delta7+/-1%, P<0.001). D1-like agonists had no effect on PKC-alpha or PKC-lambda expression in either membrane or cytosol but increased PKC-theta expression in PT in both WKY and SHR at 10 minutes but not at 60 minutes. However, membranous PKC-delta expression in PT and mTAL decreased in WKY but increased in SHR with either 10 or 60 minutes of D1-like agonist infusion. D1-like agonists also decreased membranous PKC-zeta expression in PT and mTAL in WKY but increased it in PT but not in mTAL in SHR. We conclude that there is differential regulation of PKC isoform expression by D1-like agonists that inhibits membranous PKC-delta and PKC-zeta in WKY but stimulates them in SHR; this effect in SHR is similar to the stimulatory effect of norepinephrine and angiotensin II and may be a mechanism for their differential effects on sodium transport.     

Cellular regulation of islet hormone secretion by the incretin hormone glucagon-like peptide 1

Glucagon-like peptide 1 is a gastrointestinally derived hormone with profound effects on nutrient-induced pancreatic hormone release. GLP-1 modulates insulin, glucagon and somatostatin secretion by binding to guanine nucleotide binding protein-coupled receptors resulting in the activation of adenylate cyclase and generation of cyclic adenosine monophosphate (cAMP). In the B-cell, cAMP, via activation of protein kinase A, interacts with a plethora of signal transduction processes including ion channel activity, intracellular Ca2+ handling and exocytosis of the insulin-containing granules. The stimulatory action of GLP-1 on insulin secretion, contrary to that of the currently used hypoglycaemic sulphonylureas, is glucose dependent and requires the presence of normal or elevated concentrations of the sugar. For this reason, GLP-1 attracts much interest as a possible novel principle for the treatment of human type-2 diabetes. Here we review the actions of GLP-1 on islet cell function and attempt to integrate current knowledge into a working model for the control of pancreatic hormone secretion.     

Consideration of conformational transitions and racemization during process development of recombinant glucagon-like peptide-1

This article summarizes the results of a combined analysis from two identical multicenter clinical trials that investigated the efficacy and safety of sertraline versus placebo for treating panic disorder. Patients with panic disorder who were treated with sertraline had a statistically significant reduction in the mean number of panic attacks per week (the primary efficacy measure) as compared with placebo (4.8 vs. 2.5, p < .001). Sertraline-treated patients also showed greater improvement that was statistically significant on several ratings of panic disorder symptomatology and functioning. The design characteristics, clinical rating measures, and outcome measures in these trials included most of the features deemed essential by Shear and Maser (1994) in their summary of the NIMH Consensus Conference for the development of standardized assessments for panic disorder. This suggests that the NIMH Consensus Conference played a key role in developing successful multicenter pharmacological treatment studies, such as this one that ultimately demonstrated that sertraline was an effective treatment for panic disorder.     

Exendin-(9-39) is an inverse agonist of the murine glucagon-like peptide-1 receptor: implications for basal intracellular cyclic adenosine 3',5'-monophosphate levels and beta-cell glucose competence

The effect of exendin-(9-39), a described antagonist of the glucagon-like peptide-1 (GLP-1) receptor, was evaluated on the formation of cAMP- and glucose-stimulated insulin secretion (GSIS) by the conditionally immortalized murine betaTC-Tet cells. These cells have a basal intracellular cAMP level that can be increased by GLP-1 with an EC50 of approximately 1 nM and can be decreased dose dependently by exendin-(9-39). This latter effect was receptor dependent, as a beta-cell line not expressing the GLP-1 receptor was not affected by exendin-(9-39). It was also not due to the endogenous production of GLP-1, because this effect was observed in the absence of detectable preproglucagon messenger RNA levels and radioimmunoassayable GLP-1. Importantly, GSIS was shown to be sensitive to this basal level of cAMP, as perifusion of betaTC-Tet cells in the presence of exendin-(9-39) strongly reduced insulin secretion. This reduction of GSIS, however, was observed only with growth-arrested, not proliferating, betaTC-Tet cells; it was also seen with nontransformed mouse beta-cells perifused in similar conditions. These data therefore demonstrated that 1) exendin-(9-39) is an inverse agonist of the murine GLP-1 receptor; 2) the decreased basal cAMP levels induced by this peptide inhibit the secretory response of betaTC-Tet cells and mouse pancreatic islets to glucose; 3) as this effect was observed only with growth-arrested cells, this indicates that the mechanism by which cAMP leads to potentiation of insulin secretion is different in proliferating and growth-arrested cells; and 4) the presence of the GLP-1 receptor, even in the absence of bound peptide, is important for maintaining elevated intracellular cAMP levels and, therefore, the glucose competence of the beta-cells.     

Distribution of pre-pro-glucagon and glucagon-like peptide-1 receptor messenger RNAs in the rat central nervous system

Glucagon-like peptide-1 (GLP-1) is derived from the peptide precursor pre-pro-glucagon (PPG) by enzymatic cleavage and acts via its receptor, glucagon-like peptide-1 receptor (GLP-1R). By using riboprobes complementary to PPG and GLP-1R, we described the distribution of PPG and GLP-1R messenger RNAs (mRNAs) in the central nervous system of the rat. PPG mRNA-expressing perikarya were restricted to the nucleus of the solitary tact or to the dorsal and ventral medulla and olfactory bulb. GLP-1R mRNA was detected in numerous brain regions, including the mitral cell layer of the olfactory bulb; temporal cortex; caudal hippocampus; lateral septum; amygdala; nucleus accumbens; ventral pallium; nucleus basalis Meynert; bed nucleus of the stria terminalis; preoptic area; paraventricular, supraoptic, arcuate, and dorsomedial nuclei of the hypothalamus; lateral habenula; zona incerta; substantia innominata; posterior thalamic nuclei; ventral tegmental area; dorsal tegmental, posterodorsal tegmental, and interpeduncular nuclei; substantia nigra, central gray; raphe nuclei; parabrachial nuclei; locus ceruleus, nucleus of the solitary tract; area postrema; dorsal nucleus of the vagus; lateral reticular nucleus; and spinal cord. These studies, in addition to describing the sites of GLP-1 and GLP-1R synthesis, suggest that the efferent connections from the nucleus of the solitary tract are more widespread than previously reported. Although the current role of GLP-1 in regulating neuronal physiology is not known, these studies provide detailed information about the sites of GLP-1 synthesis and potential sites of action, an important first step in evaluating the function of GLP-1 in the brain. The widespread distribution of GLP-1R mRNA-containing cells strongly suggests that GLP-1 not only functions as a satiety factor but also acts as a neurotransmitter or neuromodulator in anatomically and functionally distinct areas of the central nervous system.     

Stimulation of G-CSF gene expression in the macrophage cell line by contact with extracellular matrix proteins and a pre-B leukaemia cell line

Freshly prepared dentine specimens of human teeth were perfused with either horse serum or physiologic saline. After application of AllBond2, ART Bond, Syntac or an experimental dentine bonding agent called P-Bond, a composite cylinder was added and cured at the same time. After 1500 thermal cycles with constant imitation of intrapulpal pressure, the shear bond strengths were measured. Resulting shear bond strength values were analysed with Students t-test or Mann-Whitney Test. The values for AllBond2 were not significantly different. The values for ART Bond (P < 0.05) and for Syntac (P < 0.05) were significantly higher if the dentine was perfused with horse serum. For P-Bond (P < 0.001) the values were significantly higher if the dentine was perfused with physiologic saline. According to these results it does not seem to be appropriate to take a clear decision as to which of the two perfusing media investigated might be more suitable to imitate in vivo conditions.     

Oxyntomodulin: a cAMP-dependent stimulus of rat parietal cell function via the receptor for glucagon-like peptide-1 (7-36)NH2

Six patients with hepatocellular carcinoma were subjected to percutaneous microwave coagulation therapy (PMCT) by ultrasonic guiding. The size of the main tumor in the present cases was limited to not more than 2 cm. From 18 to 48 days after PMCT, each patient was subjected to surgery and pathological examination. By macroscopic observation, the PMCT area including both non-tumor and tumor regions looked yellowish white, and the boundary was clearly recognized. In the histological examination, the coagulation area surrounded by fibrous capsule was found, and deletion of nuclei and changes in stainability were observed in the marginal region. These changes indicated obvious coagulation necrosis, but the changes became less intense toward the center in the area, and in some portions, the tissue was indistinguishable from viable cells by light microscopy. In 2 cases out of the 6, part of the tumor remained outside the coagulation area. Since only the area determined by the microwave electrode is coagulated to cause necrosis on PMCT, sufficient safety margin should be required.     

Truncated glucagon-like peptide-1 and oxyntomodulin stimulate somatostatin release from rabbit fundic D-cells in primary culture

OBJECTIVE: To create a neovagina using a combined laparoscopic and ultrasonographic technique in Mayer-Rokitansky-Kuster-Hauser syndrome by modification of Vecchietti's operation. DESIGN: Case report. SETTING: Division of Physiopathology of Reproduction, University of Palermo, Palermo, Italy. MAIN OUTCOME MEASURE(S): The advancement of the needle from the pseudohymen, through the vesicorectal space using a triple contrast ultrasonographic technique. RESULT(S): The ultrasonographic scanning guides the accurate transit from external genitalia to the peritoneal cavity. CONCLUSION(S): This original approach allowed a safe and rapid creation of a neovagina in a case of Mayer-Rokitansky-Kuster-Hauser syndrome.     

Mouse pancreatic beta-cells exhibit preserved glucose competence after disruption of the glucagon-like peptide-1 receptor gene

Previous work suggested that glucagon-like peptide 1 (GLP-1) can acutely regulate insulin secretion in two ways, 1) by acting as an incretin, causing amplification of glucose-induced insulin release when glucose is given orally as opposed to intravenous glucose injection; and 2) by keeping the beta-cell population in a glucose-competent state. The observation that mice with homozygous disruption of the GLP-1 receptor gene are diabetic with a diminished incretin response to glucose underlines the first function in vivo. Isolated islets of Langerhans from GLP-1 receptor -/- mice were studied to assess the second function in vitro. Absence of pancreatic GLP-1 receptor function was observed in GLP-1 receptor -/- mice, as exemplified by loss of [125I]GLP-1 binding to pancreatic islets in situ and by the lack of GLP-1 potentiation of glucose-induced insulin secretion from perifused islets. Acute glucose competence of the beta-cells, assessed by perifusing islets with stepwise increases of the medium glucose concentration, was well preserved in GLP-1 receptor -/- islets in terms of insulin secretion. Furthermore, neither islet nor total pancreatic insulin content was significantly changed in the GLP-1 receptor -/- mice when compared with age-and sex-matched controls. In conclusion, mouse islets exhibit preserved insulin storage capacity and glucose-dependent insulin secretion despite the loss of functional GLP-1 receptors. The results demonstrate that the glucose responsiveness of islet beta-cells is well preserved in the absence of GLP-1 receptor signaling.     

Characterization of the release of cholecystokinin from a murine neuroendocrine tumor cell line, STC-1

Seven fatty acid derivatives containing one or two butenolide moieties along with ancepsenolide [1], a known bis-butenolide of marine origin, were isolated from the Caribbean gorgonian Pterogorgia citrina. The structures of the new metabolites were elucidated on the basis of spectroscopic data and were confirmed upon chemical conversion to either ancepsenolide or to its homolog, homoancepsenolide [4].     

Xanomeline: a novel muscarinic receptor agonist with functional selectivity for M1 receptors

Xanomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1- methylpyridine] has been evaluated as a muscarinic receptor agonist. In vitro, xanomeline had high affinity for muscarinic receptors in brain homogenates, but had substantially less or no affinity for a number of other neurotransmitter receptors and uptake sites. In cells stably expressing genetic m1 receptors, xanomeline increased phospholipid hydrolysis in CHO, BHK and A9 L cells to 100, 72 and 55% of the nonselective agonist carbachol. In isolated tissues, xanomeline had high affinity for M1 receptors in the rabbit vas deferens (IC50 = 0.006 nM), low affinity for M2 receptors in guinea pig atria (EC50 = 3 microM), was a weak partial agonist in guinea pig ileum and was neither an agonist nor antagonist in guinea pig bladder. In vivo, xanomeline increased striatal levels of dopamine metabolites, presumably by acting at M1 heteroreceptors on dopamine neurons to increase dopamine release. In contrast, xanomeline had only a relatively small effect on acetylcholine levels in brain, indicating that it is devoid of actions at muscarinic autoreceptors. In the gastrointestinal tract, xanomeline inhibited small intestinal and colonic motility, but increased small intestinal transmural potential difference. In contrast to the nonselective muscarinic agonist oxotremorine, xanomeline did not produce salivation, tremor nor hypothermia; it did, however, increase heart rate. The present data are consistent with the interpretation that xanomeline is a novel muscarinic receptor agonist with functional selectivity for M1 muscarinic receptors both in vitro and in vivo.     

Protein kinase C-alpha modulates lipopolysaccharide-induced functions in a murine macrophage cell line

Lipopolysaccharide (LPS), a potent modulator of macrophage functional activity, binds to CD14 and triggers the activation of several protein kinases, leading to the secretion of variety of immunomodulatory molecules such as nitric oxide and proinflammatory cytokines. In this study, we have examined the role of the alpha isoenzyme of protein kinase C (PKC) in the regulation of LPS-initiated signal transduction in macrophages. To this end, we have stably overexpressed a dominant-negative (DN) version of PKC-alpha (DN PKC-alpha) in the murine macrophage cell line RAW 264. 7. Clones overexpressing DN PKC-alpha were indistinguishable from the parental line with respect to morphology and growth characteristics. At the functional level, DN PKC-alpha overexpression strongly inhibited LPS-induced interleukin-1alpha mRNA accumulation, and to a lesser extent inducible nitric oxide synthase and tumor necrosis factor-alpha expression. DN-PKC-alpha overexpression did not cause a general unresponsiveness to LPS, as secretion of the matrix metalloproteinase-9 was up-regulated in our DN PKC-alpha-overexpressing clones. Moreover, LPS-induced phosphorylation and degradation of IkappaBalpha, NF-kappaB activation, as well as p38 mitogen-activated protein kinase and Jun N-terminal kinase phosphorylation, were not affected by DN PKC-alpha overexpression. Collectively, these data provide evidence that PKC-alpha regulates selective LPS-induced macrophage functions involved in host defense and inflammation.