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.     

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.     

Glucagon-like peptide-1-(7-36)amide increases pulmonary surfactant secretion through a cyclic adenosine 3',5'-monophosphate-dependent protein kinase mechanism in rat type II pneumocytes

Glucagon-like peptide-1 (GLP-1) receptor messenger RNA has been identified in cells considered type II pneumocytes that are involved in the synthesis and secretion of the pulmonary surfactant. In an attempt to open new insights into the control of surfactant secretion, we studied the effects of glucagon-related peptides in this process. Accordingly, type II pneumocytes were isolated from Wistar rat lungs and cultured overnight with [methyl-14C]choline, and then the basal and stimulated secretions of [14C]phosphatidylcholine were measured. GLP-1(7-36)amide stimulated phosphatidylcholine secretion in a concentration-dependent manner in the 1-100 nM range; the concentration of the peptide that produced a half-maximal response was 10 nM. Exendin-4 induced similar effects. No changes were observed when GLP-1-(1-37), GLP-2, or exendin-(9-39) was added to the medium. However, the latter reversed the stimulatory effects of GLP-1-(7-36)amide and exendin-4. A study of the mechanism through which GLP-1-(7-36)amide exerts its stimulatory effect was carried out using different agents that are well known stimulants of phosphatidylcholine secretion. GLP-1-(7-36)amide did not produce any change in the stimulatory effect observed with terbutaline or 8-bromo-cAMP, suggesting the involvement of a cAMP-dependent protein kinase in the stimulatory effect of this peptide on phosphatidylcholine secretion. It was further supported by the use of inhibitors of protein kinases and by the stimulation of cAMP production in type II pneumocytes incubated with either GLP-1-(7-36)amide or exendin-4.     

Buspirone: future directions

Glucagon-like peptide 1(7-36) amide (GLP-1) is postulated to be the major physiological incretin in humans, but evidence is indirect. We report the first studies examining the physiological role of GLP-1 in the postprandial state in humans using the GLP-1 antagonist exendin 9-39. Exendin 9-39 completely blocked GLP-1-induced glucose-stimulated insulin release from perifused human islets of Langerhans. In healthy fasted volunteers, intravenous infusion of exendin 9-39 at 500 pmol x kg(-1) x min(-1) in the hyperglycemic state abolished the insulinotropic effect of a physiological dose of GLP-1 and fully reversed the glucose-lowering effect of GLP-1. Nine healthy subjects consumed a 150-g oral glucose tolerance test and were infused with 500 pmol x kg(-1) x min(-1) exendin 9-39 or saline. Exendin 9-39 increased the peak postprandial glucose level (exendin 9-39, 8.67 +/- 0.35 vs. saline, 7.67 +/- 0.35 mmol/l, P < or = 0.005) and increased postprandial plasma glucose incremental area under the curve by 35% (exendin 9-39, 152 +/- 19 vs. saline, 113 +/- 16 mmol x min x l(-1), P < or = 0.05). This could be explained as partly secondary to the blockade of glucose-induced suppression of glucagon and maybe also to an increased rate of gastric emptying. Thus, in humans exendin 9-39 acts as an antagonist of GLP-1 both in vitro and in vivo. When infused alone, exendin 9-39 causes a deterioration in postprandial glycemic control, suggesting that GLP-1 may be important for maintenance of normal postprandial glucose homeostasis in humans.     

Glucagon-like peptide-1 retards gastric emptying and small bowel transit in the rat: effect mediated through central or enteric nervous mechanisms

This study investigated effects of glucagon-like peptide-1(7-36)amide (GLP-1) on gastric emptying, small intestinal transit, and contractility of smooth muscle strips in rats. GLP-1 at doses of 10 and 20 pmol/kg/min administered intravenously dose-dependently retarded transit of the small intestine (P < 0.001), while only the higher dose of 20 pmol/kg/min retarded gastric emptying (P < 0.01). GLP-1 at concentrations up to 10(-4) M did not affect the basal tone or contractility of the gastrointestinal muscle strips that were stimulated with electric field stimulation or acetylcholine. Our results demonstrate that small intestinal transit seems more sensitive than gastric emptying to inhibition by GLP-1 at physiologic levels in plasma. Furthermore, this inhibition appears to be mediated through central mechanisms rather than through peripheral actions. Thus, GLP-1 is suggested to inhibit gastric emptying and small intestinal transit through an indirect effect via central or enteric nervous mechanisms.     

Glucagon-like peptide-1 reduces hepatic glucose production indirectly through insulin and glucagon in humans

The effect of glucagon-like peptide-1 (GLP-1) on hepatic glucose production and peripheral glucose utilization was investigated with or without infusion of somatostatin to inhibit insulin and glucagon secretion in 13 healthy, non-diabetic women aged 59 years. After 120 min 3-(3)H-glucose infusion, GLP-1 was added (4.5 pmol kg(-1) bolus + 1.5 pmol kg(-1) min(-1)). Without somatostatin (n = 6), GLP-1 decreased plasma glucose (from 4.8 +/- 0.2 to 4.2 +/- 0.3 mmol L(-1), P = 0.007). Insulin levels were increased (48 +/- 3 vs. 243 +/- 67 pmol L(-1), P = 0.032), as was the insulin to glucagon ratio (P = 0.044). The rate of glucose appearance (Ra) was decreased (P = 0.003) and the metabolic clearance rate of glucose (MCR) was increased during the GLP-1 infusion (P = 0.024 vs. saline). Also, the rate of glucose disappearance (Rd) was reduced during the GLP-1 infusion (P = 0.004). Since Ra was reduced more than Rd, the net glucose flow was negative, which reduced plasma glucose. Somatostatin infusion (500 microg h(-1), n = 7) abolished the effects of GLP-1 on plasma glucose, serum insulin, insulin to glucagon ratio, Ra, Rd, MCR and net glucose flow. The results suggest that GLP-1 reduces plasma glucose levels mainly by reducing hepatic glucose production and increasing the metabolic clearance rate of glucose through indirectly increasing the insulin to glucagon ratio in healthy subjects.     

Gestational transient hyperthyroxinaemia (GTH): screening for thyroid function in 23,163 pregnant women using dried blood spots

1.Glucagon-like peptide-1 (7-36) amide (GLP-1) is released into the circulation after meals and is the most potent physiological insulinotropic hormone in man. GLP-1 has the advantages over other therapeutic agents for Type 2 diabetes of also suppressing glucagon secretion and delaying gastric emptying. One of the initial abnormalities of Type 2 diabetes is the loss of the first-phase insulin response, leading to postprandial hyperglycaemia.2. To investigate the therapeutic potential of GLP-1 in Type 2 diabetes, six patients were entered into a 6-week, double-blind crossover trial during which each received 3 weeks treatment with subcutaneous GLP-1 or saline, self-administered three times a day immediately before meals. A standard test meal was given at the beginning and end of each treatment period.3.GLP-1 reduced plasma glucose area under the curve (AUC) after the standard test meal by 58% (AUC, 0-240 min: GLP-1 start of treatment, 196+/-141 mmol.min-1.l-1; saline start of treatment, 469+/-124 mmol.min-1.l-1; F=16.4, P<0.05). The plasma insulin excursions were significantly higher with GLP-1 compared with saline over the initial postprandial 30 min, the time period during which the GLP-1 concentration was considerably elevated. The plasma glucagon levels were significantly lower over the 240-min postprandial period with GLP-1 treatment. The beneficial effects of GLP-1 on plasma glucose, insulin and glucagon concentrations were fully maintained for the 3-week treatment period. 4. We have demonstrated a significant improvement in postprandial glycaemic control with subcutaneous GLP-1 treatment. GLP-1 improves glycaemic control partially by restoring the first-phase insulin response and suppressing glucagon and is a potential treatment for Type 2 diabetes.     

Colocalization of glucagon-like peptide-1 (GLP-1) receptors, glucose transporter GLUT-2, and glucokinase mRNAs in rat hypothalamic cells: evidence for a role of GLP-1 receptor agonists as an inhibitory signal for food and water intake

This study was designed to determine the possible role of brain glucagon-like peptide-1 (GLP-1) receptors in feeding behavior. In situ hybridization showed colocalization of the mRNAs for GLP-1 receptors, glucokinase, and GLUT-2 in the third ventricle wall and adjacent arcuate nucleus, median eminence, and supraoptic nucleus. These brain areas are considered to contain glucose-sensitive neurons mediating feeding behavior. Because GLP-1 receptors, GLUT-2, and glucokinase are proteins involved in the multistep process of glucose sensing in pancreatic beta cells, the colocalization of specific GLP-1 receptors and glucose sensing-related proteins in hypothalamic neurons supports a role of this peptide in the hypothalamic regulation of macronutrient and water intake. This hypothesis was confirmed by analyzing the effects of both systemic and central administration of GLP-1 receptor ligands. Acute or subchronic intraperitoneal administration of GLP-1 (7-36) amide did not modify food and water intake, although a dose-dependent loss of body weight gain was observed 24 h after acute administration of the higher dose of the peptide. By contrast, the intracerebroventricular (i.c.v.) administration of GLP-1 (7-36) amide produced a biphasic effect on food intake characterized by an increase in the amount of food intake after acute i.c.v. delivery of 100 ng of the peptide. There was a marked reduction of food ingestion with the 1,000 and 2,000 ng doses of the peptide, which also produced a significant decrease of water intake. These effects seemed to be specific because i.c.v. administration of GLP-1 (1-37), a peptide with lower biological activity than GLP-1 (7-36) amide, did not change feeding behavior in food-deprived animals. Exendin-4, when given by i.c.v. administration in a broad range of doses (0.2, 1, 5, 25, 100, and 500 ng), proved to be a potent agonist of GLP-1 (7-36) amide. It decreased, in a dose-dependent manner, both food and water intake, starting at the dose of 25 ng per injection. Pretreatment with an i.c.v. dose of a GLP-1 receptor antagonist [exendin (9-39); 2,500 ng] reversed the inhibitory effects of GLP-1 (7-36) amide (1,000 ng dose) and exendin-4 (25 ng dose) on food and water ingestion. These findings suggest that GLP-1 (7-36) amide may modulate both food and drink intake in the rat through a central mechanism.     

Glucagon-like peptide 1 improves the ability of the beta-cell to sense and respond to glucose in subjects with impaired glucose tolerance

Impaired glucose tolerance (IGT) and NIDDM are both associated with an impaired ability of the beta-cell to sense and respond to small changes in plasma glucose concentrations. The aim of this study was to establish if glucagon-like peptide 1 (GLP-1), a natural enteric peptide and potent insulin secretagogue, improves this defect. Two weight-matched groups, one with eight subjects having IGT (2-h glucose, 10.1 +/- 0.3 mmol/l) and another with seven subjects with diet-treated NIDDM (2-h glucose, 14.5 +/- 0.9 mmol/l), were studied on two occasions during a 12-h oscillatory glucose infusion, a sensitive test of the ability of the beta-cell to sense and respond to glucose. Glucose was infused with a mean rate of 4 mg x kg(-1) x min(-1), amplitude 33% above and below the mean rate, and periodicity of 144 min, with infusion of saline or GLP-1 at 0.4 pmol x kg(-1) x min(-1) for 12 h. Mean glucose levels were significantly lower in both groups during the GLP-1 infusion compared with during saline infusion: 9.2 +/- 0.4 vs. 6.4 +/- 0.1 mmol/l in the IGT subjects (P < 0.0004) and 14.6 +/- 1.0 vs. 9.3 +/- 0.7 mmol/l in NIDDM subjects (P < 0.0002). Despite this significant reduction in plasma glucose concentration, insulin secretion rates (ISRs) increased significantly in IGT subjects (513.3 +/- 77.6 vs. 583.1 +/- 100.7 pmol/min; P < 0.03), with a trend toward increasing in NIDDM subjects (561.7 +/- 122.16 vs. 642.8 +/- 128 pmol/min; P = 0.1). These results were compatible with enhanced insulin secretion in the presence of GLP-1. Spectral power was used as a measure of the ability of the beta-cell to secrete insulin in response to small changes in the plasma glucose concentration during the oscillatory infusion. Spectral power for ISR increased from 2.1 +/- 0.9 during saline infusion to 7.4 +/- 1.3 during GLP-1 infusion in IGT subjects (P < 0.004), but was unchanged in NIDDM subjects (1.0 +/- 0.4 to 1.5 +/- 0.6; P = 0.3). We concluded that low dosage GLP-1 improves the ability of the beta-cell to secrete insulin in both IGT and NIDDM subjects, but that the ability to sense and respond to subtle changes in plasma glucose is improved in IGT subjects, with only a variable response in NIDDM subjects. Beta-cell dysfunction was improved by GLP-1 infusion, suggesting that early GLP-1 therapy may preserve beta-cell function in subjects with IGT or mild NIDDM.     

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.     

Characterisation of the processing by human neutral endopeptidase 24.11 of GLP-1(7-36) amide and comparison of the substrate specificity of the enzyme for other glucagon-like peptides

The post-secretory processing of the potent insulinotropic peptide hormone, GLP-1(7-36)amide, probably involves one or more of a small group of membrane-bound ectopeptidases. Reported here, is the characterisation of the endoproteolysis of human GLP-1(7-36)amide by the recombinant human form of neutral endopeptidase (NEP) 24.11, which is one of the best characterised and widely-distributed of ectopeptidases and is involved in the processing of other peptide hormones. The products of the limited endoproteolysis were characterised by mass and primary structure following fractionation using high performance liquid chromatography. The rate of this endoproteolysis by NEP 24.11 was estimated and compared to that of GLP-1(7-36)amide-related peptides. GLP-1(7-36)amide appears to be good substrate for NEP 24.11 with most, but not all potential target bonds being cleaved. Also, the structurally-related peptides, secretin and glucagon appear to be good substrates whereas GIP and exendin-4 are very poor substrates. That the GLP-1(7-36)amide super-agonist, exendin-4 is a poor substrate for NEP 24.11 is significant for the possible use of this peptide as a prototype for the development of clinically-useful peptide agonists. Further studies should reveal whether NEP 24.11 is important for the metabolic clearance of GLP-1(7-36)amide and will be highly relevant for the attempts to realise the suggested therapeutic value of GLP-1(7-36)amide.     

Glucagon-like peptide-1 can reverse the age-related decline in glucose tolerance in rats

Wistar rats develop glucose intolerance and have a diminished insulin response to glucose with age. The aim of this study was to investigate if these changes were reversible with glucagon-like peptide-1 (GLP-1), a peptide that we have previously shown could increase insulin mRNA and total insulin content in insulinoma cells. We infused 1.5 pmol/ kg-1.min-1 GLP-1 subcutaneously using ALZET microosmotic pumps into 22-mo-old Wistar rats for 48 h. Rat infused with either GLP-1 or saline were then subjected to an intraperitoneal glucose (1 g/kg body weight) tolerance test, 2 h after removing the pump. 15 min after the intraperitoneal glucose, GLP-1-treated animals had lower plasma glucose levels (9.04+/-0.92 mmol/liter, P < 0.01) than saline-treated animals (11.61+/-0.23 mmol/liter). At 30 min the plasma glucose was still lower in the GLP-1-treated animals (8.61+/-0.39 mmol/liter, P < 0.05) than saline-treated animals (10.36+/-0.43 mmol/liter). This decrease in glucose levels was reflected in the higher insulin levels attained in the GLP-1-treated animals (936+/-163 pmol/liter vs. 395+/-51 pmol/liter, GLP-1 vs. saline, respectively, P < 0.01), detected 15 min after glucose injection. GLP-1 treatment also increased pancreatic insulin, GLUT2, and glucokinase mRNA in the old rats. The effects of GLP-1 were abolished by simultaneous infusion of exendin [9-39], a specific antagonist of GLP-1. GLP-1 is therefore able to reverse some of the known defects that arise in the beta cell of the pancreas of Wistar rats, not only by increasing insulin secretion but also by inducing significant changes at the molecular level.     

Augmentation of lung liquid clearance via adenovirus-mediated transfer of a Na,K-ATPase beta1 subunit gene

The gastrointestinal hormone, glucagon-like peptide-1(7-36)amide (GLP-1) is released after a meal. The potency of synthetic GLP-1 in stimulating insulin secretion and in inhibiting glucagon secretion indicates the putative physiological function of GLP-1. In vitro, the nonmammalian peptide, exendin(9-39)amide [ex(9-39)NH2], is a specific and competitive antagonist of GLP-1. This in vivo study examined the efficacy of ex(9-39)NH2 as an antagonist of exogenous GLP-1 and the physiological role of endogenous GLP-1. Six healthy volunteers underwent 10 experiments in random order. In each experiment, a 30-min period of euglycemia was followed by an intravenous infusion of glucose for 150 min that established a stable hyperglycemia of 8 mmol/liter. There was a concomitant intravenous infusion of one of the following: (1) saline, (2) GLP-1 (for 60 min at 0.3 pmol . kg-1 . min-1 that established physiological postprandial plasma levels, and for another 60 min at 0.9 pmol . kg-1 . min-1 to induce supraphysiological plasma levels), (3-5) ex(9-39)NH2 at 30, 60, or 300 pmol . kg-1 . min-1 + GLP-1, (6-8) ex(9-39)NH2 at 30, 60, or 300 pmol . kg-1 . min-1 + saline, (9 and 10) GIP (glucose-dependent insulinotropic peptide; for 60 min at 0.8 pmol . kg-1 . min-1, with saline or ex(9-39)NH2 at 300 pmol . kg-1 . min-1). Each volunteer received each of these concomitant infusions on separate days. ex(9-39)NH2 dose-dependently reduced the insulinotropic action of GLP-1 with the inhibitory effect declining with increasing doses of GLP-1. ex(9-39)NH2 at 300 pmol . kg-1 . min-1 blocked the insulinotropic effect of physiological doses of GLP-1 and completely antagonized the glucagonostatic effect at both doses of GLP-1. Given alone, this load of ex(9-39)NH2 increased plasma glucagon levels during euglycemia and hyperglycemia. It had no effect on plasma levels of insulin during euglycemia but decreased plasma insulin during hyperglycemia. ex(9-39)NH2 did not alter GIP-stimulated insulin secretion. These data indicate that in humans, ex(9-39)NH2 is a potent GLP-1 antagonist without any agonistic properties. The pancreatic A cell is under a tonic inhibitory control of GLP-1. At hyperglycemia, the B cell is under a tonic stimulatory control of GLP-1.     

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.     

Influence of circulating epinephrine and norepinephrine on insulin-like growth factor binding protein-1 in humans

The aim of the present study was to investigate the influence of circulating epinephrine (Epi) and norepinephrine (Norepi) on serum insulin-like growth factor binding protein-1 (IGFBP-1) concentrations. Healthy men received 0.3 nmol.kg.min Epi iv (n = 6), 0.5 nmol.kg.min Norepi iv (n = 7), or saline (n = 5) during 30 min. Arterial blood samples were obtained before, during, and 120 min after infusion. During the catecholamine infusion arterial Epi and Norepi plasma concentrations reached 6.35 +/- 0.53 and 15.65 +/- 2.71 nmol/L, respectively, which resulted in significant increases in glucose concentrations. When Epi was infused, IGFBP-1 increased from 45 +/- 6 micrograms/L to 76 +/- 10 micrograms/L (P < 0.05) 60 min after the infusion. Epi was also followed by increases in insulin, C-peptide, and glucagon. Norepi resulted in a slight increase in circulating IGFBP-1 (43 +/- 6 to 54 +/- 8 nmol/L, NS). The findings suggest that Epi, at plasma concentrations similar to those reached during physical stress, stimulates the production of IGFBP-1 in humans.     

Release and clearance rates of epinephrine in man: importance of arterial measurements

Previous estimates of catecholamine kinetics in human subjects have been based on the measurement of the catecholamine levels in forearm venous plasma. However, the use of forearm venous measurements may introduce considerable error, since venous catecholamine levels may primarily reflect metabolism in the organ drained rather than in the total body. In this study, arterial levels of epinephrine were found to significantly exceed forearm venous levels, both basally (mean +/- SEM, 71 +/- 13 vs. 50 +/- 7 pg/ml; n = 6; P less than 0.05) and during infusions of epinephrine [0.1 microgram/min (112 +/- 9 vs. 77 +/- 11 pg/ml; P less than 0.005) or 2 micrograms/min (862 +/- 71 vs. 437 +/- 66 pg/ml; P less than 0.001)]. During the 2 micrograms/min epinephrine infusion, arterial plasma norepinephrine rose from 191 +/- 37 to 386 +/- 78 pg/ml (P less than 0.001), while venous norepinephrine levels did not change significantly. Fractional extraction (arterial - venous + arterial X 100) of epinephrine across the forearm was 26 +/- 8% in the basal state and increased to 33 +/- 6% and further to 51 +/- 4% during the epinephrine infusions. The addition of propranolol (5 mg, iv, plus an 80 micrograms/min infusion) reduced fractional extraction from 51 +/- 4% to 35 +/- 5%. Whole body clearance of epinephrine, calculated from arterial measurements, was 33 +/- 3 ml/kg . min during the 0.1 microgram/min infusion and 35 +/- 3 ml/kg . min during the 2 micrograms/min epinephrine infusion, values 50% lower than the clearance rates calculated from venous measurements. Propranolol infusion resulted in a fall in whole body clearance to 20 +/- 2 ml/kg . min (P less than 0.001), suggesting that epinephrine clearance is partly dependent on a beta-adrenergic mechanism. Basal endogenous release rate (clearance X basal epinephrine level) was estimated to be approximately 0.18 microgram/min, a value much less than that reported in studies using venous measurements. We conclude that arterial rather than venous measurements should be used to estimate catecholamine kinetics in vivo.     

Repeated intracerebroventricular administration of glucagon-like peptide-1-(7-36) amide or exendin-(9-39) alters body weight in the rat

Central nervous system glucagon-like peptide-1-(7-36) amide (GLP-1) administration has been reported to acutely reduce food intake in the rat. We here report that repeated intracerebroventricular (i.c.v.) injection of GLP-1 or the GLP-1 receptor antagonist, exendin-(9-39), affects food intake and body weight. Daily i.c.v. injection of 3 nmol GLP-1 to schedule-fed rats for 6 days caused a reduction in food intake and a decrease in body weight of 16 +/- 5 g (P < 0.02 compared with saline-injected controls). Daily i.c.v. administration of 30 nmol exendin-(9-39) to schedule-fed rats for 3 days caused an increase in food intake and increased body weight by 7 +/- 2 g (P < 0.02 compared with saline-injected controls). Twice daily i.c.v. injections of 30 nmol exendin-(9-39) with 2.4 nmol neuropeptide Y to ad libitum-fed rats for 8 days increased food intake and increased body weight by 28 +/- 4 g compared with 14 +/- 3 g in neuropeptide Y-injected controls (P < 0.02). There was no evidence of tachyphylaxis in response to i.c.v. GLP-1 or exendin-(9-39). GLP-1 may thus be involved in the regulation of body weight in the rat.     

Prolactin and beta-endorphin responses to hypoglycemia are reduced in well-controlled insulin-dependent diabetes mellitus

Several pituitary hormones, including corticotropin (ACTH), growth hormone (GH), prolactin, and beta-endorphin (but not thyrotropin, follicle-stimulating hormone, or luteinizing hormone), are released in response to hypoglycemia in normal subjects. In patients with insulin-dependent diabetes mellitus (IDDM), the degree of glycemic control is known to alter ACTH and GH responses to hypoglycemia. The current study was performed to examine the effect of glycemic control on prolactin and beta-endorphin responses to hypoglycemia in subjects with IDDM. We performed 3-hour stopped hypoglycemic-hyperinsulinemic clamp studies (12 pmol/kg/min) during which plasma glucose was decreased from 5.0 mmol/L to 2.2 mmol/L in steps of 0.6 mmol/L every 30 minutes in 20 subjects with uncomplicated IDDM (12 males and eight females; age, 26 +/- 2 years; IDDM duration, 10 +/- 1 years; body mass index, 23.6 +/- 0.6 kg/m2) and 10 healthy subjects (five males and five females aged 30 +/- 1 years). The 10 diabetic subjects in good glycemic control (mean hemoglobin A1 [HbA1], 7.5% +/- 0.3%; normal range, 5.4% to 7.4%) were compared with the 10 poorly controlled patients (mean HbA1, 12.6% +/- 0.5%; P < .001 v well-controlled diabetic group). During hypoglycemia, prolactin levels in the well-controlled diabetic group did not change (7 +/- 1 microgram/L at plasma glucose 5.0 mmol/L to 9 +/- 2 micrograms/L at plasma glucose 2.2 mmol/L), whereas prolactin levels increased markedly in the poorly controlled diabetic group (7 +/- 2 micrograms/L to 44 +/- 17 micrograms/L) and healthy volunteers (12 +/- 2 micrograms/L to 60 +/- 19 micrograms/L, P < .05 between IDDM groups). The plasma glucose threshold required for stimulation of prolactin secretion was 2.2 +/- 0.1 mmol/L in well-controlled IDDM, 3.0 +/- 0.4 mmol/L in poorly controlled IDDM, and 2.4 +/- 0.1 mmol/L in healthy subjects (P < .05 between IDDM groups). Responses in males and females were similar. The increase in beta-endorphin levels was also attenuated in well-controlled IDDM patients (4 +/- 1 pmol/L at plasma glucose 5.0 mmol/L to 11 +/- 4 pmol/L at plasma glucose 2.2 mmol/L) versus poorly controlled IDDM patients (5 +/- 1 pmol/L to 26 +/- 7 pmol/L) and healthy subjects (8 +/- 1 pmol/L to 56 +/- 13 pmol/L). The plasma glucose threshold required for stimulation of beta-endorphin release was again lower in well-controlled IDDM versus poorly controlled IDDM patients (2.2 +/- 0.1 v 3.0 +/- 0.3 mmol/L) and healthy subjects (2.5 +/- 0.4 mmol/L, P < .05 between IDDM groups). In conclusion, prolactin and beta-endorphin responses to a standardized hypoglycemic stimulus (plasma glucose, 2.2 mmol/L) are reduced and plasma glucose levels required to stimulate release of prolactin and beta-endorphin are lower in well-controlled IDDM compared with poorly controlled IDDM and healthy subjects. Thus, stress hormones not previously considered to have a primary role in plasma glucose recovery from hypoglycemia are affected by glycemic control, suggesting a more generalized alteration of hypothalamic-pituitary responses to hypoglycemia in IDDM patients with strict glycemic control.     

Role of adenosine in noradrenergic neurotransmission during hemorrhagic hypotension

Glucagon-like peptide-1-(7-36) amide (GLP-1) and glucose-dependent insulinotropic peptide (GIP) are known incretin hormones, released from enteroendocrine cells in response to food, that enhance insulin secretion, but only in the presence of elevated blood glucose. We used a rat insulinoma cell line, RIN 1046-38, to study the mechanisms underlying the interaction of incretins and glucose. We measured insulin secretion using RIA and the reverse hemolytic plaque assay. GLP-1 stimulates insulin secretion, with a half-maximal concentration of 34 pM. GLP-1 is approximately 2 orders of magnitude more potent than GIP. GLP-1 and GIP have additive effects at submaximal concentrations, but probably not at maximal concentrations, suggesting a common signal transduction pathway. The glucose requirement for GLP-1 action can be replaced by cell membrane depolarization (20 mM KCl in the extracellular medium), suggesting that a rise of intracellular Ca2+ may be an early step required for GLP-1 action. GLP-1 stimulates insulin secretion by significantly increasing the maximum rate of insulin secretion from 10.3 +/- 2.25 to 25.2 +/- 2.94 ng insulin/mg protein.h. GLP-1 acts by recruiting 1.5-fold more cells to secrete insulin as well as enhancing insulin secretion by individual cells. Combinations of stimuli, such as glucose, cell membrane depolarization, and GLP-1, can recruit 90% of RIN 1046-38 cells to secrete insulin.     

Effect of omeprazole and cimetidine on plasma aldosterone response to angiotensin II

The genomic organization of the human gene encoding the receptor for glucagon-like peptide-1 (GLP-1 (7-37)/(7-36) amide) was analyzed to reveal the relationship to other G-protein-coupled receptors. The coding sequence of the GLP-1 receptor is interrupted by 12 introns. These introns are uniformly distributed within the open reading frame. The length of the introns varies between 6.6 kb and 100 bp, in contrast to the relative constant length of 100 bp of the exons. All of the exon/intron splice junctions characterized followed the consensus GT-AG rule. A comparison of the genomic structure with other related receptor genes indicates that the exon/intron organization is well-conserved among the VIP/ glucagon/secretin receptor family.