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Objective: To evaluate efficacy and safety of switching from twice-daily exenatide to once-daily liraglutide or of 40 weeks of continuous liraglutide therapy., Research Design and Methods: When added to oral antidiabetes drugs in a 26-week randomized trial (Liraglutide Effect and Action in Diabetes [LEAD]-6), liraglutide more effectively improved A1C, fasting plasma glucose, and the homeostasis model of beta-cell function (HOMA-B) than exenatide, with less persistent nausea and hypoglycemia. In this 14-week extension of LEAD-6, patients switched from 10 microg twice-daily exenatide to 1.8 mg once-daily liraglutide or continued liraglutide., Results: Switching from exenatide to liraglutide further and significantly reduced A1C (0.32%), fasting plasma glucose (0.9 mmol/l), body weight (0.9 kg), and systolic blood pressure (3.8 mmHg) with minimal minor hypoglycemia (1.30 episodes/patient-year) or nausea (3.2%). Among patients continuing liraglutide, further significant decreases in body weight (0.4 kg) and systolic blood pressure (2.2 mmHg) occurred with 0.74 episodes/patient-year of minor hypoglycemia and 1.5% experiencing nausea., Conclusions: Conversion from exenatide to liraglutide is well tolerated and provides additional glycemic control and cardiometabolic benefits.

Background: Unlike most antihyperglycaemic drugs, glucagon-like peptide-1 (GLP-1) receptor agonists have a glucose-dependent action and promote weight loss. We compared the efficacy and safety of liraglutide, a human GLP-1 analogue, with exenatide, an exendin-based GLP-1 receptor agonist., Methods: Adults with inadequately controlled type 2 diabetes on maximally tolerated doses of metformin, sulphonylurea, or both, were stratified by previous oral antidiabetic therapy and randomly assigned to receive additional liraglutide 1.8 mg once a day (n=233) or exenatide 10 microg twice a day (n=231) in a 26-week open-label, parallel-group, multinational (15 countries) study. The primary outcome was change in glycosylated haemoglobin (HbA(1c)). Efficacy analyses were by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00518882., Findings: Mean baseline HbA(1c) for the study population was 8.2%. Liraglutide reduced mean HbA(1c) significantly more than did exenatide (-1.12% [SE 0.08] vs -0.79% [0.08]; estimated treatment difference -0.33; 95% CI -0.47 to -0.18; p<0.0001) and more patients achieved a HbA(1c) value of less than 7% (54%vs 43%, respectively; odds ratio 2.02; 95% CI 1.31 to 3.11; p=0.0015). Liraglutide reduced mean fasting plasma glucose more than did exenatide (-1.61 mmol/L [SE 0.20] vs -0.60 mmol/L [0.20]; estimated treatment difference -1.01 mmol/L; 95% CI -1.37 to -0.65; p<0.0001) but postprandial glucose control was less effective after breakfast and dinner. Both drugs promoted similar weight losses (liraglutide -3.24 kg vs exenatide -2.87 kg). Both drugs were well tolerated, but nausea was less persistent (estimated treatment rate ratio 0.448, p<0.0001) and minor hypoglycaemia less frequent with liraglutide than with exenatide (1.93 vs 2.60 events per patient per year; rate ratio 0.55; 95% CI 0.34 to 0.88; p=0.0131; 25.5%vs 33.6% had minor hypoglycaemia). Two patients taking both exenatide and a sulphonylurea had a major hypoglycaemic episode., Interpretation: Liraglutide once a day provided significantly greater improvements in glycaemic control than did exenatide twice a day, and was generally better tolerated. The results suggest that liraglutide might be a treatment option for type 2 diabetes, especially when weight loss and risk of hypoglycaemia are major considerations., Funding: Novo Nordisk A/S.

Glucagon-like peptide 1 (GLP-1) lowers glycemia by modulating gastric emptying and endocrine pancreatic secretion. Rapidly after its secretion, GLP-1-(7-36) amide is degraded to the metabolite GLP-1-(9-36) amide. The effects of GLP-1-(9-36) amide in humans are less well characterized. Fourteen healthy volunteers were studied with intravenous infusion of GLP-1-(7-36) amide, GLP-1-(9-36) amide, or placebo over 390 min. After 30 min, a solid test meal was served, and gastric emptying was assessed. Blood was drawn for GLP-1 (total and intact), glucose, insulin, C-peptide, and glucagon measurements. Administration of GLP-1-(7-36) amide and GLP-1-(9-36) amide significantly raised total GLP-1 plasma levels. Plasma concentrations of intact GLP-1 increased to 21 +/- 5 pmol/l during the infusion of GLP-1-(7-36) amide but remained unchanged during GLP-1-(9-36) amide infusion [5 +/- 3 pmol/l; P < 0.001 vs. GLP-1-(7-36) amide administration]. GLP-1-(7-36) amide reduced fasting and postprandial glucose concentrations (P < 0.001) and delayed gastric emptying (P < 0.001). The GLP-1 metabolite had no influence on insulin or C-peptide concentrations. Glucagon levels were lowered by GLP-1-(7-36) amide but not by GLP-1-(9-36) amide. However, the postprandial rise in glycemia was reduced significantly (by approximately 6 mg/dl) by GLP-1-(9-36) amide (P < 0.05). In contrast, gastric emptying was completely unaffected by the GLP-1 metabolite. The GLP-1 metabolite lowers postprandial glycemia independently of changes in insulin and glucagon secretion or in the rate of gastric emptying. Most likely, this is because of direct effects on glucose disposal. However, the glucose-lowering potential of GLP-1-(9-36) amide appears to be small compared with that of intact GLP-1-(7-36) amide.

Background/aims: In vitro studies suggest that glucagon-like peptide 2 (GLP-2), secreted from enteroendocrine cells in the gastrointestinal tract after food intake, is able to ameliorate mucosal injury in settings of human disease characterized by injury and dysfunction of the intestinal mucosal epithelium. We evaluated this potential of GLP-2 after epithelial trauma by using two in vitro models measuring intestinal epithelial cell proliferation and cell migration., Materials and Methods: Injuries were induced in confluent monolayers of the small intestinal cells lines IEC-6 and IEC-18, as well as in the colonic cell lines Caco-2 and Colo 320. GLP-2 (50-500 nM) or other peptides were added to the media. Wound healing was investigated after 24 h by quantification of the number of cells migrating across the wound edge. Proliferation of cells was assessed by using photometric mitochondrial incorporation measurement of MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide). Monoclonal TGF-beta antibodies were added to wounded monolayers to examine whether the GLP-2-induced wound healing was TGF-beta-mediated., Results: Migration assessments revealed a significant stimulation of GLP-2-induced migration in IEC-6 and IEC-18 monolayers compared to the placebo group. No effect was observed in the colon cancer cell lines Caco-2 and Colo 320. Results of the proliferation assays show a significant inhibition of proliferation by GLP-2 in small intestinal cell lines whereas a dose-dependent stimulation of proliferation in colonic epithelial cells was observed. Addition of neutralizing TGF-beta1 antibodies to wounded IEC-6 and IEC-18 monolayers incubated with GLP-2 significantly reduced the number of migrating cells to the level of the placebo group., Conclusions: In our in vitro model, it was shown that the GLP-2-induced improvement of intestinal wound healing is TGF-beta-mediated. These effects were predominant in the epithelium of the small intestine compared to colonic epithelium. Our findings provide further insight into mechanisms leading to GLP-2-induced mucosal wound healing. These results suggest that GLP-2 or analogues of this peptide may potentially be useful for the treatment of intestinal disorders characterized by injury and ineffective repair of the intestinal mucosa., (Copyright 2004 Elsevier B.V.)

Alpha-factor [WHWLQLKPGQPMY], a secreted tridecapeptide pheromone, is required for mating between the a- and alpha-haploid mating types of Saccharomyces cerevisiae (MATa, MATalpha). New analogues of alpha-factor were synthesized and evaluated by morphogenesis assays and receptor binding studies. The Y(0)Nle(12)F(13) analogue [YWHWLQLKPGQPNleF] (MFN5) caused growth arrest and morphological alteration in MATa cells in a fashion identical to that of the native pheromone. Binding of (125)I-labeled MFN5 was saturable, and reversible as shown by equipotent label displacement by MFN5 and native alpha-mating factor. Scatchard analysis of equilibrium binding data on plasma membranes and intact cells indicated the existence of a single high-affinity binding site (K(d) = 6.4 x 10(-8)). Specific binding of (125)I-labeled MFN5 was significantly reduced by guanosine nucleotides. Affinity cross-linking of (125)I-labeled MFN5 to MATa cell membranes identified a specifically labeled 49-kDa protein. The novel synthetic alpha-factor analogue MFN5 can be easily iodinated and used as a probe for the alpha-factor receptor., (Copyright 1999 Academic Press.)

A stimulatory effect on exocrine pancreas secretion could be demonstrated with high concentrations of the 25-amino-acid peptide xenin in non-anesthetized dogs. This peptide has been isolated from gastric mucosa and it is part of a structural coat protein. It has close structural similarities to neurotensin. The longer C-terminal fragments xenin-(13--25) and xenin-(18--25) are essential for the stimulation of exocrine pancreas secretion in vivo. The smaller peptide fragments xenin-(21--25) and xenin-(22--25) failed to stimulate the pancreas as well as the N-terminal peptide fragment xenin-(1--23). The stimulatory effects of xenin may be mediated via neural neurotensin pathways, because neurotensin receptor blockade abolished the stimulatory effect on pancreatic secretion. Cholinergic pathways are not involved, because atropine had no inhibiting effect.

The action of xenin, a novel 25-residue peptide of the neurotensin (NT)/xenopsin family, was investigated in isolated rat ileal muscle strips and in dispersed longitudinal smooth muscle cells of rat small intestine in vitro. Xenin relaxes KCl-precontracted ileal strips dose dependently (1 nM-3 microM). The order of potency of the investigated peptides was as follows: xenopsin = NT = xenin > neuromedin N. Kinetensin was inactive. Tetrodotoxin, hexamethonium, tetraethylammonium, 4-aminopyridine, and NG-nitro-L-arginine did not influence the relaxant effects of xenin or NT, whereas the K+ channel blocker apamin nearly abolished their effects. Desensitization against one of the peptides or blockade of NT receptors by SR-48692 prevented the effect of xenin and NT. Structure-activity experiments revealed that the COOH-terminal part of the molecules of xenin and NT is essential for biological activity. Experiments with isolated dispersed smooth muscle cells and binding studies on intestinal smooth muscle cell membranes confirmed and extended the results obtained with muscle strips. In conclusion, xenin relaxes rat ileal smooth muscle via a muscular NT-type apamin-sensitive receptor.

Human galanin (hGal) is an important neuro-modulator present in the brain, gastrointestinal system and the hypothalamo-pituitary axis. A specific receptor for hGal has been identified in various areas in human brain. A single class of high affinity binding sites was found on plasma membranes of the amygdala (Kd 0.23 nM, Bmax 44 fmol/mg), the hypothalamus (Kd 0.20 nM, Bmax 25 fmol/mg) and the cortex cerebri (Kd 0.11 nM, Bmax 8.2 fmol/mg). Other brain areas, i.e. cerebellum, thalamus or pons, expressed binding sites of identical high affinity in lower quantities (Bmax < 3 fmol/mg). Specific binding of 125I-labelled hGal was found to be reversible, time- and temperature-dependent and inhibited by Ca2+, Na+ and K+ ions at a concentration of 5 mM. Non-hydrolysable guanosine nucleotides potently reduced specific binding of 125-I-labelled hGal by more than 80%. Synthetic hGal analogues substituted in the N-terminal region exhibited strongly reduced binding affinity for the hGal receptor. Using 3-[(3-cholamidopropyl) dimethylammonio]-2-hydroxy-1-propanesulphonate, hGal receptors were successfully solubilized from human cortical membranes, exhibiting no significant loss of binding affinity. Affinity cross-linking to 125I-labelled hGal revealed a labelled band of approximately 60 kDa sensitive to unlabelled Gal. This putative hGal receptor is glycosylated since its molecular size was reduced after treatment with endoglycosidase F. Receptors bound to 125I-labelled hGal could be specifically adsorbed to wheat germ agglutinin and ricinus communis agglutinin, suggesting that receptor glycosylation involves N-acetyl glucosamine and galactose respectively.

The intraduodenal application of a potent protease inhibitor (camostate, 600 mg) causes a significant increase in basal pancreatic secretion in the manner of a negative-feedback mechanism. The integrated protein secretion during the entire study period was 9.9 +/- 1.8 g in 120 min. The iv application of anti-GRP immunoglobulin caused a significant reduction to 5.0 +/- g in 120 min. No significant changes in the plasma concentrations of GRP and CCK were detectable. The increased secretion occurring after the administration of the protease inhibitor could be mediated by neural GRP-dependent mechanisms. These may also be relevant for CCK-dependent factors, which are only briefly mentioned.

Exocrine pancreatic secretion in conscious dogs is significantly stimulated by the intravenous application of small doses of gastrin-releasing peptide (GRP; 30 pmol x kg-1 x h-1). There is no increase of the GRP concentrations in peripheral blood which is also the case under physiological postprandial conditions. There is also no increase in the peripheral cholecystokinin (CCK) concentrations, in contrast to previous reports. Nevertheless other CCK-related mechanisms may play an important role, since the administration of the highly specific CCK receptor antagonist MK-329 causes a marked reduction of the GRP-induced exocrine pancreatic secretion.

After ingestion of glucose both GIP (gastric inhibitory polypeptide, glucose-dependent insulinotropic polypeptide) and GLP-1(7-36amide) (glucagon-like polypeptide-1, 7-36amide) may play a physiological role in augmenting insulin release. Their insulinotropic effect was compared in isolated rat islets after 24-h maintenance in tissue culture (11 mmol l-1 glucose). Ten islets per vial were then incubated in Krebs-Ringer-Hepes buffer for 30 min; insulin was measured radioimmunologically. Both hormones were always compared in the same experiment. At 16.7 mmol l-1 glucose both GIP and GLP-1(7-36amide) 2 x 10(-10) mol l-1 significantly increased insulin release; 10(-10) mol l-1 of either hormone had no significant effect. The response at 10(-9) and 10(-8) mol l-1 was similar for both; at 4 x 10(-10) mol l-1 GLP-1(7-36amide), however, was clearly more effective than GIP. At low glucose (2.8 or 5.0 mol l-1) no significant differences were found. A concentration of 10(-8) mol l-1 of both hormones was slightly stimulatory. At 8.3 mmol l-1 glucose, 10(-9) mol l-1 GLP-1(7-36amide) was 60% more effective than GIP (4.8 +/- 0.4 vs. 3.0 +/- 0.4, n = 13, P less than 0.005), the response to 10(-8) mol l-1 was similar. These data show comparable effects of high concentrations of GIP and GLP-1(7-36amide) on glucose-induced insulin release; at presumably physiological concentrations, however, GLP-1(7-36amide) was clearly more effective. The combination of the two peptides was not more than additive, suggesting that they act via the same final mechanism.

Galanin (Gal), a 29-amino acid C-terminally amidated neuropeptide, is widely distributed throughout the central and peripheral nervous system. The primary structures of rat and bovine Gals were derived from the cDNA sequences of their precursors. To elucidate the structure of human Gal (hGal), we extracted 280 postmortem pituitaries in trifluoroacetic acid and purified hGal binding activity, by three successive HPLC steps, to homogeneity based on a radioreceptor assay. The primary structure of hGal was determined by automatic Edman degradation to be Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu- Gly-Pro-His-Ala-Val-Gly-Asn-His-Arg-Ser-Phe-Ser-Asp-Lys-Asn-Gly-Leu-Thr- Ser-COOH. The structure was confirmed by plasma desorption time-of-flight mass spectrometry, revealing a mass of 3156.1. Compared to the 29-residue porcine, rat, and bovine Gals, hGal uniquely comprises 30 amino acids possessing an additional nonamidated serine residue as C terminus. The nonamidated carboxylic group at the C terminus was proven by synthesis of amidated and nonamidated hGal and by mass spectrometry after selective methylation of all free carboxylic groups. Synthetic hGal possesses full biological activity on isolated rat fundus muscle strips.

Receptors for galanin, a neuropeptide inhibiting insulin release, have been described on RINm5F insulinoma cells. To characterize structural requirements for binding and biological activity of galanin, we studied binding and inhibition of hormone stimulated intracellular cAMP-production of N-terminal galanin fragments and -analogues in RINm5F cells. Half-maximal binding and potency were the same for all peptides used. Active peptides had the following rank of potency: galanin = galanin(1-22(23)Cys) greater than galanin(1-29(4)NLe) greater than galanin(1-18) greater than galanin(1-29(7)DAla) greater than galanin(1-29(2)DTrp4NLe7DAla) greater than galanin(1-29(2)DTrp). Galanin(3-29) was inactive. Therefore the first two amino acids of the galanin molecule with the indole side chain of the tryptophane residue in the right steric position are crucial for receptor binding.

1. Rat and porcine galanin (rGal and pGal) produced dose-dependent contraction of rat fundus strips in a concentration range of 6 nM-100 nM. 2. The stimulatory effect of rGal on rat fundus strips was not modified in the presence of somatostatin (250 nM), naloxone (1 microM), guanethidine (10 microM), a mixture of propranolol (3 microM) and phentolamine (3 microM), tetrodotoxin (1 microM), indomethacin (10 microM), atropine (1 microM), a mixture of methysergide (2.5 microM) and ketanserine (2.5 microM), a mixture of mepyramine (10 microM) and cimetidine (10 microM), and saralasin (10 microM) or when strips were desensitized to substance P and neurotensin. 3. These results suggest the localization of specific Gal receptors on the surface of smooth muscle cells of rat fundus. 4. The galanin analogues [D-Trp2]-rGal, [Nle4]-rGal, [D-Ala7]-rGal, [D-Trp2-NLe4-D-Ala7]-rGal and fragments [Cys23]-Gal (1-23), Gal (1-18) were fully active. In contrast, rGal (3-29) was completely inactive and showed no antagonistic properties to the contractile effect of intact galanin. 5. The order of potency of the galanin peptides, analogues and fragments to contract rat fundus strips was: pGal greater than rGal greater than [NLe4]-rGal greater than [Cys23]-Gal (1-23) greater than Gal (1-18) greater than [D-Ala7]-rGal greater than [Trp2]-rGal greater than [D-Trp2-NLe4-D-Ala7]-rGal. 6. The data originating from our structure-activity study suggest that the C-terminal portion of Gal contributes mainly to the affinity of Gal receptors whereas the N-terminal portion of Gal is responsible for the full activation of Gal receptors in this tissue. In particular the amino acids in position 1 and 2 of Gal (Gly-Trp) appear to be essential for binding and intrinsic activity.

Glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) is a potent stimulator of insulin secretion. Receptors for this hormone have been found on different insulinoma-derived cell lines, e.g. the RINm5F cell line which is derived from a radiation-induced rat insulinoma. To characterize the part of the GLP-1(7-36)amide molecule that is responsible for binding to its receptor on RINm5F cells, binding studies with synthetic C-terminal (GLP-1(21-36)amide) and synthetic N-terminal (GLP-1(7-25] GLP-1 fragments were carried out. GLP-1(21-36)amide showed dose-dependent binding to the GLP-1(7-36)amide receptor but was approximately 1500 times less potent in inhibiting binding of 125I-labelled GLP-1(7-36)amide than the intact hormone. GLP-1(7-25) at concentrations up to 10 mumol/l did not inhibit binding of label. Neither fragment changed intracellular cyclic AMP concentrations, in contrast to GLP-1(7-36)amide which increased intracellular cyclic AMP. GLP-1(21-36)amide, however, acted as a weak partial antagonist of GLP-1(7-36)amide with respect to GLP-1(7-36)amide-dependent stimulation of cyclic AMP production.

Glucagon-like peptide-1 and glucagon-like peptide-2 are encoded by the m-RNA of pancreatic preproglucagon. They show high conservation in different species and substantial sequence homology to glucagon. Because no definite biological activity of these peptides has been reported, we investigated the effect of synthetic C-terminally amidated glucagon-like peptide-1 [1-36] and synthetic human glucagon-like peptide-2 [1-34] with a free C-terminus on insulin release from isolated precultured rat pancreatic islets in the presence of glucose. Glucagon-like peptide-1 stimulates insulin release at 10 and 16.7 mmol/l glucose in a dose-dependent manner. Significant stimulation starts at 2.5 nmol/l in the presence of 10 mmol/l glucose and near maximal release is observed at 250 nmol/l, with approximately 100% increase over basal at both glucose concentrations. The peptide reaches 63% of the maximal stimulatory effect of glucagon. No stimulation occurs in the presence of 2.8 mmol/l glucose. Glucagon-like peptide-2 has no effect on insulin secretion at any glucose concentration tested. It is concluded that glucagon-like peptide-1, in contrast to glucagon-like peptide-2, exhibits a glucose-dependent insulinotropic action on isolated rat pancreatic islets similar to that of glucagon and gastric inhibitory polypeptide.

A sensitive method for the rapid identification of the C-terminally amidated amino acid in peptides is described. Peptides containing the alpha-amide group at the C-terminus were cleaved with endopeptidases. The fragments released (oligopeptides, amino acids and the C-terminally amidated residue) are coupled to phenylisothiocyanate. The phenylthiocarbamoyl derivative of the amino acid alpha-amide is selectively extracted from the mixture by alkaline butyl acetate and identified by a high-performance liquid chromatography system that enables rapid and complete separation of the derivatives of 17 amino acid amides at a detection limit of 20-50 pmol. The C-terminal alpha-amides of neurokinin-A (Met-NH2), mammalian secretin (Val-NH2), pancreatic polypeptide (Tyr-NH2) and peptide HI (Ile-NH2) are unequivocally determined at a level of 0.5-2 nmol per peptide. This method was used to characterize a crude peptide fraction prepared from porcine brain. Cholecystokinin-58 was identified in this fraction by detection of phenylthiocarbamoyl-phenylalaninamide. The method is suitable for the identification of the C-terminal alpha-amidated residue of purified peptides, but can also be used as a screening strategy to isolate from complex biological extracts novel peptides containing an alpha-amidated amino acid at the C-terminus.

The isolation and primary structure of a novel gastrointestinal peptide, designated valosin, is described. The peptide was purified from porcine upper gut extracts using an HPLC and N-terminal sequence screening strategy which depends on chromatographic and structural characteristics as isolation criterion. The amino acid sequence of this peptide consists of 25 amino acid residues:

Valosin, a novel 25-amino acid gastrointestinal peptide with N-terminal valine and C-terminal tyrosine, has recently been isolated from porcine upper gut extracts. Its physiologic role is unknown and it does not belong to one of the structurally related gut peptide families. Assuming that valosin may influence gastrointestinal functions, we investigated the effect of high-performance liquid chromatography-pure valosin on gastric and exocrine pancreatic secretion and on the intestinal myoelectric activity in conscious dogs. Intravenous injection of valosin (0.125-1 microgram/kg) dose-dependently increased gastric acid secretion 80-fold over basal, corresponding to 18% of the maximal pentagastrin-induced effect. Pepsin output increased 10-fold over basal (30% of the pentagastrin-stimulated secretion). Half-maximal stimulation by pentagastrin could be further increased dose-dependently by simultaneous administration of valosin. Pancreatic bicarbonate secretion was stimulated 11-fold over basal at 1.0 microgram/kg, reaching about 6% of the secretin-induced maximal output, whereas protein secretion increased 12-fold over basal, corresponding to about 55% of the cholecystokinin-induced maximal output. In fasted dogs, spontaneously occurring migrating myoelectric complexes were substantially delayed during infusion of valosin at a dose of 0.2 microgram/kg. These experiments indicate that valosin may represent a novel member of the regulatory gastrointestinal peptides.