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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effects of aging and a high fat diet on body weight and glucose tolerance in glucagon-like peptide-1 receptor -/- mice

Disruption of glucagon-like peptide-1 (GLP-1) receptor signaling in mice results in mild glucose intolerance, principally due to elimination of the incretin effect of GLP-1. Despite the inhibitory effects of GLP-1 on food intake, 6- to 8-week-old GLP-1 receptor -/-(GLP-1R-/-) mice were not obese and did not exhibit disturbances of feeding behavior. As both diabetes and obesity frequently become more phenotypically evident in older rodents, we studied the consequences of aging and a high fat diet on glucose control and body weight in GLP-1R-/- mice. No evidence of obesity or deterioration in glucose control was detected in 11- and 16-month-old GLP-1R-/- mice (mean weight, 34.7 +/- 2.0, 30.5 +/- 1.5, and 34.6 +/- 2.8 g in male and 25.3 +/-1.6, 28.4 +/-1.2, and 31.9 +/- 2.9 g in female GLP-1R+/+, GLP-1R+/-, and GLP-1R-/- mice, respectively; P = NS). After 18 weeks of high fat feeding, GLP-1R-/- mice gained similar (males) or less (females) weight than age- and sex-matched CD1 controls. No significant deterioration in glucose tolerance was observed after high fat feeding in GLP-1R-/- mice. These observations demonstrate that long term disruption of GLP-1 signaling in the central nervous system and peripheral tissues of older mice is not associated with the development of obesity or deterioration in glucose homeostasis.     

Structure-function analysis of the active sites of complement receptor type 1

Two functionally distinct but homologous sites in complement receptor type 1 (CR1) (CD35) were further characterized by homologous substitution mutagenesis of two CR1 derivatives, each containing one site. In both sites, reducing negative and/or increasing positive charge augmented interaction with iC3/C3b and C4b, supporting a role of ionic forces in the binding reaction. In one case, substitution of Asp at the end of complement control protein repeat (CCP) 2 with an Asn transformed the protein, with negligible cofactor activity and iC3 binding, into a mutant with activities similar to native CR1. Consequently, this protein, one-fourth the size of CR1, is a therapeutic candidate for a complement inhibitor. Another important observation is that the residues between two CCPs contribute to activity, probably because they influence positioning of one CCP relative to the next. The initial characterization of the third CCP of an active site led to identification of three peptides necessary for binding. In line with earlier findings for the first two CCPs, interactions with iC3/C3b are similar but not identical to those with C4b, implying overlapping but distinct binding domains. Moreover, changes in cofactor activity usually, but not always, parallel alterations in binding, indicating that these two activities are separable. We also mapped epitopes for a blocking and a function enhancing monoclonal antibody. Their effects can be explained by epitope location. The first antibody binds near functionally important residues. The second may shield inhibitory (negatively charged) residues. These results represent a comprehensive analysis of the active sites of CR1, which is built of modules found in more than 50 mammalian proteins.     

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.     

Peptide YY, glucagon-like peptide-1, and neurotensin responses to luminal factors in the isolated vascularly perfused rat ileum

Exposure of the ileum to nutrients markedly inhibits several upper gastrointestinal functions. Hormonal peptides of the ileal wall, i.e. peptide YY (PYY), glucagon-like peptide-1 (GLP-1), and neurotensin (NT), are thought to play a role in this negative feedback mechanism. The present study was conducted to comparatively assess the secretion of PYY, GLP-1, and NT upon luminal infusion of a variety of individual luminal factors in the isolated vascularly perfused rat ileum preparation. PYY, GLP-1, and NT were measured in the portal effluent with specific RIAs. Glucose (250 mM) induced a pronounced release of the three peptides, whereas a physiological concentration of 5 mM did not induce peptide secretion. Peptone (5%, wt/vol) evoked a sustained release of PYY, GLP-1, and NT. Only NT secretion was increased upon luminal administration of 100 mM sodium oleate. Short chain fatty acids (20 mM) evoked an early and transient release of the three peptides. In contrast, taurocholate (20 mM) induced a sustained release of PYY, GLP-1, and NT, but the threshold concentration for peptide release was lower for NT than for PYY or GLP-1. Cellulose or pectin (0.5%, wt/vol) did not modify peptide secretion. In conclusion, glucose and peptone are potent stimulants of PYY, GLP-1, and NT release. Only NT is released upon oleic acid stimulation. Finally, taurocholate is a potent stimulant of the release of the three peptides. Overall, PYY, GLP-1, and NT may participate cooperatively in the ileal brake. As relatively high concentrations of the various stimulants were required to elicit peptide release, it seems likely that this mechanism operates in cases of maldigestion or malabsorption.     

Mechanisms of the antidiabetic action of subcutaneous glucagon-like peptide-1(7-36)amide in non-insulin dependent diabetes mellitus

Twelve patients with non-insulin dependent diabetes mellitus (NIDDM) under secondary failure to sulfonylureas were studied to evaluate the effects of subcutaneous glucagon-like peptide-1(7-36)amide (GLP-1) on (a) the gastric emptying pattern of a solid meal (250 kcal) and (b) the glycemic and endocrine responses to this solid meal and an oral glucose tolerance test (OGTT, 300 kcal). 0.5 nmol/kg of GLP-1 or placebo were subcutaneously injected 20 min after meal ingestion. GLP-1 modified the pattern of gastric emptying by prolonging the time to reach maximal emptying velocity (lag period) which was followed by an acceleration in the post-lag period. The maximal emptying velocity and the emptying half-time remained unaltered. With both meals, GLP-1 diminished the postprandial glucose peak, and reduced the glycemic response during the first two postprandial hours by 54.5% (solid meal) and 32.7% (OGTT) (P < 0.05). GLP-1 markedly stimulated insulin secretion with an effect lasting for 105 min (solid meal) or 150 min (OGTT). The postprandial increase of plasma glucagon was abolished by GLP-1. GLP-1 diminished the postprandial release of pancreatic polypeptide. The initial and transient delay of gastric emptying, the enhancement of postprandial insulin release, and the inhibition of postprandial glucagon release were independent determinants (P < 0.002) of the postprandial glucose response after subcutaneous GLP-1. An inhibition of efferent vagal activity may contribute to the inhibitory effect of GLP-1 on gastric emptying.     

Inositolphosphoglycans possibly mediate the effects of glucagon-like peptide-1(7-36)amide on rat liver and adipose tissue

Cardiac disorders are increasingly recognised as an important source of cerebral embolism. Atrial fibrillation is the most common cardiac dysrrhythmia that can predispose to stroke. Recent advances have significantly increased the identification of clinical, hematological and echocardiographic risk factors that predict the occurrence of atrial fibrillation related stroke. Also, clinical risk stratification has been used to determine medical therapy (aspirin or warfarin) for prevention of atrial fibrillation related brain embolization. Among the various structural heart diseases causing stroke, the role of patent foramen ovale remains controversial. Strides have been made in the use of ultrasonographic techniques such as transesophageal echocardiography and contrast transcranial doppler to detect patent foramen ovale. Coronary artery bypass grafting is often performed in patients with concomitant aortic atheroma and carotid stenosis that may predispose to stroke in the perioperative period. It is now possible to identify perioperatively significant aortic atherosclerosis (using transesophageal echocardiography and aortic ultrasound) and significant carotid disease (using carotid ultrasound) and make appropriate modifications in surgical technique to reduce the incidence of coronary artery bypass grafting related stroke. Because of shared risk factors it is not surprising that coronary artery disease is frequently found in stroke patients. Recent studies suggest that more than one-third of stroke patients have asymptomatic coronary artery disease. Conversely, the brain damaged by infarction may itself be responsible for the production of cardiac structural and electrical abnormalities. Both these factors may contribute to the finding that cardiac events are the leading cause of death in stroke patients on long term follow-up. Recognition of these correlations has enhanced our ability to treat and prevent stroke related mortality.     

Effects of non-glycated and glycated glucagon-like peptide-1(7-36) amide on glucose metabolism in isolated mouse abdominal muscle

This study investigated the actions of non-glycated and glycated glucagon-like peptide-1(7-36)amide (tGLP-1) on glucose uptake and metabolism in isolated mouse abdominal muscle. Monoglycated tGLP-1 (Mr 3463.8) was prepared under hyperglycemic reducing conditions and purified by HPLC. Non-glycated tGLP-1 (10(-10)-10(-8) mol/l) stimulated both 2-deoxy-D-[1-3H]glucose uptake (1.3-1.5 fold) and 14C-glucose oxidation (1.4-1.7 fold) in muscle compared to controls without tGLP-1. Glycation reduced these stimulatory effects by 27-33% and 25% (at 10(-9) mol/l), respectively. tGLP-1 (10(-10)-10(-8) mol/l) promoted muscle glycogenesis and lactate production, whereas glycated peptide was ineffective below 10(-9) mol/l. This study demonstrates that tGLP-1 has potent glycogenic effects in mouse abdominal muscle in vitro and that glycation impairs its action.     

Comparison of sandwich enzyme-linked immunoadsorbent assay and radioimmunoassay for determination of exogenous glucagon-like peptide-1(7-36)amide in plasma

A sensitive sandwich enzyme-linked immunoadsorbent assay (ELISA) for determination of exogenous glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) in plasma samples from pharmacokinetic studies is described. The assay employs an N-terminally directed antibody and a C-terminally directed antibody. The ELISA has a working range from 10 to 500 pmol l-1, and can be applied to plasma samples from humans, dogs, pigs, minipigs, cats, rabbits, and rats. The assay was compared to a validated radioimmunoassay (RIA), employing an antibody directed against the mid-region of GLP-1. After s.c. administration of GLP-1(7-36)amide, the plasma immunoreactivity of GLP-1 (P-GLP-1-IR) measured by ELISA was markedly lower than P-GLP-1-IR measured by RIA. After HPLC fractionation of plasma samples with subsequent RIA and ELISA analyses of the fractions, this difference was shown to be due to cross reaction with biologically inactive fragments of GLP-1(7-36)amide in the RIA but not in the ELISA.     

Influence of fasting and neuropeptide Y on the suppressive food intake induced by intracerebroventricular injection of glucagon-like peptide-1 in the neonatal chick

Recently, we have reported that central administration of glucagon-like peptide-1 (GLP-1) strongly decreased food intake of chicks. The aim of the present study was to elucidate whether suppressed food intake by central injection of GLP-1 would be modified by an appetite stimulant such as fasting and neuropeptide Y (NPY). Birds (2 days old) were starved for 3 or 6 h and then GLP-1 (0.03 microg/10 microl) or saline was injected by the intracerebroventricular (i.c.v.) route. Birds starved for 6 h ate significantly more food than those starved for 3 h, while irrespective of the time for fasting GLP-1 strongly inhibited food intake as rapidly as 10 min after i.c.v injection. The suppressive effect on food intake continued until 4 h after injection. Central administration of NPY (2.5 microg/10 microl) greatly enhanced food intake, but co-injection of GLP-1 (0.01, 0.02 or 0.03 microg/10 microl) decreased food intake in a dose-dependent fashion. Under GLP-1 (0.03 microg/10 microl) treatment, whether NPY modifies food intake of chicks in a dose-dependent manner was investigated by co-injection of graded levels of NPY (0.4, 1.0 and 2.5 microg/10 microl). GLP-1 completely inhibited the effect of NPY on food intake without a dose response. These results suggest that central GLP-1 may interact with NPY and may be the most potent inhibitor of food intake in the chicken.     

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.