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Udgivelsesdato: 2009-Sep, AIM: The glucagon-like peptide-1 (GLP-1) receptor agonist, exendin-4, has previously been shown to delay the onset of diabetes when administered to Goto-Kakizaki (GK) rats in the prediabetic period. The present study aimed to evaluate whether long-term administration of exendin-4 to GK rats in the diabetic period would improve their diabetes and how glycaemic control was affected following drug wash-out. METHODS: Glycaemic control was assessed in diabetic GK rats during 12 weeks of exendin-4 or vehicle treatment. Moreover, some animals were followed for an additional 9 weeks without treatment. RESULTS: Glycaemic control was seen to deteriorate in vehicle-treated animals, as assessed by increased glycated haemoglobin A1c (HbA1c), whereas HbA1c improved in exendin-4-treated animals. Following an additional 9 weeks without treatment, glycaemic control in exendin-4-treated animals remained below baseline value and thus remained significantly lower than that of vehicle-treated animals. Following exendin-4 administration, oral glucose tolerance tests revealed greatly reduced glucose and insulin excursions compared with vehicle-treated animals, whereas following overnight drug wash-out, only little difference was seen, suggesting that the improvement in glycaemic control may have been obtained primarily by increased postprandial control. No significant differences were observed in pancreatic islet morphology or islet hormone content. CONCLUSIONS: Exendin-4 treatment improved glycaemic control in diabetic GK rats, independent of changes in beta-cell mass. Additionally, progression of the disease seemed to be delayed because the improvement in HbA1c was still apparent 9 weeks after cessation of treatment.

AIMS/HYPOTHESIS: Glucagon-like peptide-2 (GLP-2) is a gut hormone regulating intestinal growth and nutrient absorption. Recently, GLP-2 has been reported to stimulate glucagon secretion in healthy humans. We sought to clarify the mechanism and physiological significance of this endocrine effect.MATERIALS AND METHODS: The expression of the GLP-2 receptor gene, Glpr2, and the localisation of the protein were evaluated by real-time PCR on cDNA from isolated rat islets and by immunohistochemistry in rat and human pancreas. The glucagon, insulin and somatostatin responses to 0.1, 1 and 10 nmol/l GLP-2 and to GLP-1 and GLP-2 given simultaneously were studied in the isolated perfused rat pancreas.RESULTS: Expression of Glp2r transcript was confirmed by PCR. In both human and rat pancreas, GLP-2r immunoreactivity was colocalised with proglucagon. GLP-2 at 10 nmol/l increased glucagon secretion significantly from a pre-infusion level of 0.314 +/- 0.07 to 0.508 +/- 0.09 pmol/min (p < 0.0005), whereas lower GLP-2 concentrations were ineffective. Neither insulin nor somatostatin output was influenced. During simultaneous administration of GLP-1 and GLP-2, net glucagon release was no longer reduced by 0.1, 1 or 10 nmol/l GLP-1, which, when given alone, inhibited glucagon secretion by 25.0 +/- 9.9, 46.2 +/- 4.8, and 44.1 +/- 2.9%, respectively.CONCLUSIONS/INTERPRETATION: Our results suggest that GLP-2 stimulates glucagon secretion through GLP-2r present on the alpha cell in rats. In the presence of GLP-2, the glucagonostatic effect of GLP-1, normally co-secreted with GLP-2, is markedly inhibited. Based on our analogous immunohistochemical findings in human pancreas, this mechanism also applies in all likelihood to humans. However, further in vivo studies are required to assess the physiological significance of the glucagonotropic action of GLP-2 in humans.

Udgivelsesdato: 2007-Sep, CCR5 and CXCR3 are important molecules in regulating the migration of activated lymphocytes. Thus, the majority of tissue-infiltrating T cells found in the context of autoimmune conditions and viral infections express CCR5 and CXCR3, and the principal chemokine ligands are expressed within inflamed tissues. Accordingly, intervention studies have pointed to nonredundant roles of these receptors in models of allograft rejection, viral infection, and autoimmunity. In spite of this, considerable controversy exists, with many studies failing to support a role for CCR5 or CXCR3 in disease pathogenesis. One possible explanation is that different chemokine receptors may take over in the absence of any individual receptor, thus rendering individual receptors redundant. We have attempted to address this issue by analyzing CCR5(-/-), CXCR3(-/-), and CCR5/CXCR3(-/-) mice with regard to virus-induced liver inflammation, generation and recruitment of effector cells, virus control, and immunopathology. Our results indicate that CCR5 and CXCR3 are largely dispensable for tissue infiltration and virus control. In contrast, the T-cell response is accelerated in CCR5(-/-) and CCR5/CXCR3(-/-) mice and the absence of CCR5 is associated with the induction of CD8(+) T-cell-mediated immunopathology consisting of marked hepatic microvesicular steatosis.

Udgivelsesdato: 2007/8

BACKGROUND: The involvement of nitric oxide (NO) in vagal control and vasoactive intestinal polypeptide (VIP)-induced effects on antral motility was studied using isolated perfused preparations of porcine gastric antrum with intact vagal innervation.METHODS: The presence of NO and VIP-producing neurons was studied using immunohistochemistry and histochemical techniques. Widespread, but not total, co-localization of NO and VIP immunoreactivity was found in the submucosa and in the muscle layers.RESULTS: Electrical stimulation of the vagus nerves for 5 min (8 Hz, 10 mA, 4 msec) increased the motility index from 2.47 = 0.44 to 11.50 +/- 2.02 (n = 5). This effect was not influenced by the two NO synthase inhibitors N-nitro-L-arginine methyl ester (10(-4) M) and NG-nitro-L-arginine (10(-5) M). However, infusion of inhibitors increased the spontaneous motility index from 2.40 +/- 0.08 to 5.36 +/- 1.08 (P < 0.05) and 3.05 +/- 1.10 to 4.14 +/- 1.04 (P < 0.05), respectively. The addition of L-arginine reversed this effect. Infusion of VIP 2 x 10(-9)M decreased the motility index from 2.32 +/- 0.43 to 1.32 +/- 0.27 (P < 0.05), an effect that was preserved during NO synthase inhibition. Electrical vagus stimulation increased the release of VIP to the venous effluent, an effect that persisted during NO synthase inhibitors.CONCLUSION: We conclude that NO-producing nerves seem to have a tonic inhibitory action on the porcine antral motility, but are not involved in the motor effects of vagal stimulation or VIP infusion.

Incretin hormones are insulinotropic hormones from the intestinal mucosa, which after being released in response to ingestion of a meal, enhance insulin secretion in excess of that elicited by the absorbed nutrients (glucose. amino acids etc) themselves. To day it is well established that the most important incretin hormones are glucose-dependent insulinotropic polypeptide (GIP, previously known as gastric inhibitory polypeptide) and glucagon-like peptide-1 (GLP-1) from the upper and lower small intestinal mucosa, respectively. It has been shown that interference with the incretin function causes glucose intolerance and it has also been shown that the incretin function is greatly impaired in type 2 diabetes mellitus. The reason for this seems to be twofold: an impaired secretion of GLP-1 and a severely impaired insulinotropic effect of GIP in these patients. In agreement with this, administration of the active incretin, GLP-1, to patients with type 2 diabetes may nearly normalise their fasting and postprandial hyperglycaemia. In addition to its insulinotropic effects, GLP-1 has been shown to stimulate the formation of new beta cells in rodents, partly by enhanced beta cell proliferation and partly by enhancing differentiation of duct progenitor cells to mature beta cells. GLP-1 also inhibits glucagon secretion, inhibits gastric emptying and reduces appetite and food intake. During the last years, therefore, several most promising attempts have been made to develop GLP-1 into a clinically useful therapeutic agent for the treatment of type 2 diabetes.

Specific mechanisms, providing reuptake of cathecholamine and amino acid neurotransmitters (e.g. serotonin and glutamate) into cells of the central nervous system are well known, whereas neuronal uptake of neuropeptide transmitters have not previously been reported. In the present study we present evidence for uptake of the 37 amino acid neuropeptide, calcitonin gene-related peptide (CGRP) into perivascular terminals of capsaicin sensitive nerve fibres, innervating the guinea-pig basilar artery. Release of CGRP from perivascular nerve terminals was obtained by capsaicin-induced vanilloid receptor-stimulation and detected as CGRP receptor-mediated dilation of isolated segments of the guinea-pig basilar artery. Following three repeated capsaicin challenges, CGRP-depleted segments were incubated with CGRP. This caused significant reappearance of capsaicin-induced vasodilatory responses. These responses were dependent on duration and concentration of the preceding CGRP incubation and were inhibited by the CGRP receptor antagonist, CGRP(8 - 37). The CGRP-re-depletion was significantly reduced when CGRP(8 - 37) was present during the preceding CGRP incubation. Thus, presynaptic CGRP receptors are likely to be involved in neuronal CGRP uptake. Incubating the artery segments with (125)I-CGRP allowed subsequent detection of capsaicin-induced (125)I-release. Immunohistochemical experiments showed that only terminal CGRP is subject to capsaicin-induced depletion in vitro, whereas CGRP-immunoreactivity endures in the nerve fibres.

Glucagon-like peptide 2 (GLP-2), produced by enteroendocrine L-cells, regulates intestinal growth. This study investigates circulating and intestinal GLP-2 levels in conditions with altered L-cell exposure to nutrients. Rats were allocated to the following experimental groups: ileal-jejunal transposition, resection of the proximal or distal half of the small intestine, and appropriate sham-operated controls. After two weeks, ileal-jejunal transposition led to pronounced growth of the transposed segment and also of the remaining intestinal segments. Plasma GLP-2 levels increased twofold, whereas GLP-2 levels in the intestinal segments were unchanged. In resected rats with reduced intestinal capacity, adaptive small bowel growth was more pronounced following proximal resection than distal small bowel resection. Circulating GLP-2 levels increased threefold in proximally resected animals, and twofold in the distally resected group. Tissue GLP-2 levels were unchanged in resected rats. The data indicate that transposition of a distal part of the small intestine, and thereby exposure of L cells to a more nutrient-rich chyme, leads to intestinal growth. The adaptive intestinal growth is associated with increased plasma levels of GLP-2, and GLP-2 seems to act in an endocrine as well as a paracrine manner.

Glucagon-like peptide 2 (GLP-2) is a 33-amino acid (1-33) intestinotrophic peptide. In this study, the distribution and binding of i.v. injected radiolabeled GLP-2 (1-33) were investigated in rats using autoradiography in order to target possible binding sites. The major part of (125)I-GLP-2 (1-33) was distributed to kidneys, liver, and the gastrointestinal tract. In the small intestine, a high density of grains was localized in the epithelium with a predominance in the luminal part of the villus. The saturability of (125)I-GLP-2 (1-33) was investigated by administration of excess amounts of non-radioactive GLP-2 (1-33) or the primary metabolite of GLP-2 degradation, GLP-2 (3-33). In the small intestine, (125)I-GLP-2 was displaced both by non-radioactive GLP-2 (1-33) and (3-33), suggesting that the uptake of GLP-2 (1-33) in the small intestine is receptor-specific and that the metabolite GLP-2 (3-33) may interact with the GLP-2 receptor.

The insulinotropic hormone glucagon-like peptide-1 (GLP-1) is stored in the intestinal L cell in an active form, GLP-1-(7-36)amide, but more than half of the endogenous peptide circulates in an inactive, N-terminally truncated form, GLP-1-(9-36)amide. This study examined the GLP-1 newly secreted from the porcine ileum, in vitro (isolated perfused preparation) and in vivo (anesthetized pig), to determine where this conversion occurs. Although the GLP-1 extractable from the porcine ileum is predominantly the intact peptide (94.6+/-1.7%), a large proportion of the GLP-1 that is secreted has already been degraded to the truncated form both in vitro (53.8+/-0.9% intact) and in vivo (32.9+/-10.8% intact). In the presence of a specific dipeptidyl peptidase IV (DPP IV) inhibitor (valine-pyrrolidide), the proportion of intact GLP-1 released from the perfused ileum was increased under both basal (99% intact; P < 0.05) and stimulated (86-101% intact; P < 0.05) conditions. Immunohistochemical and histochemical studies revealed specific DPP IV staining in the brush border epithelium as well as in the capillary endothelium. Double staining showed juxtapositioning of DPP IV-positive capillaries and GLP-1-containing L cells. From these results, we suggest that GLP-1 is degraded as it enters the DPP IV containing blood vessels draining the intestinal mucosa.

Intravenous GLP-1 [7-36 amide] can normalize fasting hyperglycaemia in Type 2 diabetic patients. Whether GLP-1 [7-37] has similar effects and how quickly plasma glucose concentrations revert to hyperglycaemia after stopping GLP-1 is not known. Therefore, 8 patients with Type 2 diabetes (5 female, 3 male; 65+/-6 years; BMI 34.3+/-7.9 kg m(-2); HbA1c 9.6+/-1.2%; treatment with diet alone (n=2), sulphonylurea (n=5), metformin (n=1)) were examined twice in randomized order. GLP-1 [7-36 amide] or [7-37] (1 pmol kg(-1)min(-1) were infused intravenously over 4 h in fasted subjects. Plasma glucose (glucose-oxidase), insulin and C-peptide (ELISA) was measured during infusion and for 4 h thereafter. Indirect calorimetry was performed. Fasting hyperglycaemia was 11.7+/-0.9 [7-36 amide] and 11.3+/-0.9 mmol l(-1) [7-37]. GLP-1 infusions stimulated insulin secretion approximately 3-fold (insulin peak 168+/-32 and 156+/-47 pmol l(-1), p<0.0001 vs basal; C-peptide peak 2.32+/-0.28 and 2.34+/-0.43 nmol l(-1), p<0.0001, respectively, with GLP-1 [7-36 amide] and [7-37]). Four hours of GLP-1 infusion reduced plasma glucose (4.8+/-0.4 and 4.6+/-0.3 mmol l(-1), p<0.0001 vs basal values), and it remained in the non-diabetic fasting range after a further 4 h (5.1+/-0.4 and 5.3+/-0.4 mmol l(-1), for GLP [7-36 amide] and [7-37], respectively). There were no significant differences between GLP-1 [7-36 amide] and [7-37] (glucose, p=0.99; insulin, p=0.99; C-peptide, p=0.99). Neither glucose oxidation nor lipid oxidation (or any other parameters determined by indirect calorimetry) changed during or after the administration of exogenous GLP-1. In conclusion, GLP-1 [7-36 amide] and [7-37] normalize fasting hyperglycaemia in Type 2 diabetic patients. Diabetes therapy (diet, sulphonyl ureas or metformin) does not appear to influence this effect. In fasting and resting patients, the effect persists during administration of GLP-1 and for at least 4 h thereafter, without rebound. Significan

Glucagon-like peptide 1 (GLP-1) has been shown to inhibit gastric emptying of liquid meals in type 2 diabetic patients. It was the aim of the present study to compare the action of physiological and pharmacological doses of intravenous GLP-1-(7-36) amide and GLP-1-(7-37) on gastric emptying in normal volunteers. Nine healthy subjects participated (26 +/- 3 yr; body mass index 22.9 +/- 1.6 kg/m2; hemoglobin A1C 5.0 +/- 0.2%) in five experiments on separate occasions after an overnight fast. A nasogastric tube was positioned for the determination of gastric volume by use of a dye-dilution technique (phenol red). GLP-1-(7-36) amide (0.4, 0.8, or 1.2 pmol.kg-1.min-1), GLP-1-(7-37) (1.2 pmol.kg-1.min-1), or placebo was infused intravenously from -30 to 240 min. A liquid meal (50 g sucrose, 8% amino acids, 440 ml, 327 kcal) was administered at 0 min. Glucose, insulin, and C-peptide were measured over 240 min. Gastric emptying was dose dependently slowed by GLP-1-(7-36) amide (P < 0.0001). Effects of GLP-1-(7-37) at 1.2 pmol.kg-1.min-1 were virtually identical. GLP.1 dose dependently stimulated fasting insulin secretion (-30 to 0 min) and slightly reduced glucose concentrations. After the meal (0-240 min), integrated incremental glucose (P < 0.0001) and insulin responses (P = 0.01) were reduced (dose dependently) rather than enhanced. In conclusion, 1) GLP-1-(7-36) amide or -(7-37) inhibits gastric emptying also in normal subjects, 2) physiological doses (0.4 pmol.kg-1.min-1) still have a significant effect, 3) despite the known insulinotropic actions of GLP-1-(7-36) amide and -(7-37), the net effect of administering GLP-1 with a meal is no change or a reduction in meal-related insulin responses. These findings suggest a primarily inhibitory function for GLP-1 (ileal brake mechanisms).

Streptozotocin, which induces diabetes mellitus in experimental animals, has been reported to be taken up by beta-cells by means of the glucose transporter 2 (GLUT2) and then reduce the cellular level of NAD+, leading to necrosis of the beta-cells. We investigated the effect of insulin pretreatment on the diabetogenic action of streptozotocin (60 mg/kg). Four groups of rats were studied: 1) a group that received streptozotocin (STZ), 2) a group that received insulin pretreatment and streptozotocin (INS + STZ), 3) a group that received insulin (INS), and 4) a control group (CTRL). Insulin treatment reduced the beta-cell immunoreactivity (IR) of insulin and GLUT2, which, thus, was reduced in INS + STZ rats at the time of streptozotocin injection. In STZ rats, plasma insulin concentrations after 3 weeks as well as insulin concentrations in pancreatic tissue samples were significantly lower than those in CTRL rats [plasma, 274.3 +/- 101.9 vs. 1078.8 +/- 254.9 pmol/liter (P <0.05); tissue, 0.46 +/- 0.02 vs. 117.0 +/- 28.4 nmol/g (P <0.01)]. INS + STZ rats did not become hyperglycemic, and the plasma and tissue levels of insulin were higher than those in STZ rats [plasma, 538.3 +/- 80.1 vs. 274.3 +/- 101.9 pmol/liter (P = 0.08); tissue, 0.46 +/- 0.02 vs. 37.90 +/- 2.13 nmol/g (P <0.05)]. The immunohistochemical findings of insulin IR in the pancreatic tissues were in accordance with the results obtained by RIA. We conclude that exogenous insulin suppresses the expression of GLUT2 and insulin in beta-cells, and this may prevent the diabetogenic effect of streptozotocin.

OBJECTIVE: Glucagon-like peptide I(7-36) amide (GLP-1) is a physiological incretin hormone that, in slightly supraphysiological doses, stimulates insulin secretion, lowers glucagon concentrations, and thereby normalizes elevated fasting plasma glucose concentrations in type II diabetic patients. It is not known whether GLP-1 has effects also in fasting type I diabetic patients.RESEARCH DESIGN AND METHODS: In 11 type I diabetic patients (HbA1c 9.1 +/- 2.1%; normal, 4.2-6.3%), fasting hyperglycemia was provoked by halving their usual evening NPH insulin dose. In random order on two occasions, 1.2 pmol . kg-1 . min-1 GLP-1 or placebo was infused intravenously in the morning (plasma glucose 13.7 +/- 0.9 mmol/l; plasma insulin 26 +/- 4 pmol/l). Glucose (glucose oxidase method), insulin, C-peptide, glucagon, GLP-1, cortisol, growth hormone (immunoassays), triglycerides, cholesterol, and nonesterified fatty acids (enzymatic tests) were measured.RESULTS: Glucagon was reduced from approximately 8 to 4 pmol/l, and plasma glucose was lowered from 13.4 +/- 1.0 to 10.0 +/- 1.2 mmol/l with GLP-1 administration (plasma concentrations approximately 100 pmol, P < 0.0001), but not with placebo (14.2 +/- 0.7 to 13.2 +/- 1.0). Transiently, C-peptide was stimulated from basal 0.09 +/- 0.02 to 0.19 +/- 0.06 nmol/l by GLP-1 (P < 0.0001), but not by placebo (0.07 +/- 0.02 to 0.07 +/- 0.02). There was no significant effect on nonesterified fatty acids (P = 0.34), triglycerides (P = 0.57), cholesterol (P = 0.64), cortisol (P = 0.40), or growth hormone (P = 0.53).CONCLUSIONS: Therefore, exogenous GLP-1 is able to lower fasting glycemia also in type I diabetic patients, mainly by reducing glucagon concentrations. However, this alone is not sufficient to normalize fasting plasma glucose concentrations, as was previously observed in type II diabetic patients, in whom insulin secretion (C-peptide response) was stimulated 20-fold.

BACKGROUND: The insulinotropic hormones gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), secreted from the K-cells of the upper small intestine and from the L-cells of the lower small intestine, respectively, are thought to be responsible for intestinal stimulation of insulin secretion. If true, their plasma concentrations should parallel the meal-related diurnal changes in plasma insulin concentrations.METHODS: Using COOH-terminal assays, thought to reflect accurately their rates of secretion, we measured circulating levels of GIP and GLP-1 in six normal subjects for 15 h of a day, during which they ate three mixed meals.RESULTS: Both GIP and GLP-1 concentrations increased significantly and in parallel with insulin in response to all three meals. The plasma insulin concentrations correlated significantly with both GIP and GLP-1 values throughout the study period (correlation coefficients, 0.49 +/- 0.07 and 0.56 +/- 0.05; p < 0.001).CONCLUSIONS: These results support the notion that GLP-1 and GIP are important incretin hormones.

Previous studies have suggested that glucagon-like peptide-1 (GLP-1) (7-36 amide) may have the direct effect of increasing insulin sensitivity in healthy man. To evaluate this hypothesis we infused GLP-1 in seven lean healthy men during a hyper insulinaemic (0.8 mU.kg-1.min-1), euglycaemic (5 mmol/l) clamp. Somatostatin (450 micrograms/h was infused to suppress endogenous insulin secretion, and growth hormone (3 ng.kg-1.min-1) and glucagon (0.8 ng.kg-1.min-1) were infused to maintain basal levels. GLP-1 (50 pmol.kg-1.h-1) or 154 mmol/l NaCl (placebo) was infused after 3 h of equilibration, i.e. from 180-360 min. GLP-1 infusion resulted in GLP-1 levels of approximately 40 pmol/l. Plasma glucose, insulin, growth hormone, and glucagon levels were similar throughout the clamps. The rate of glucose infusion required to maintain euglycaemia was similar with or without GLP-1 infusion (7.69 +/- 1.17 vs 7.76 +/- 0.95 mg kg-1.min-1 at 150-180 min and 8.56 +/- 1.13 vs 8.55 +/- 0.68 mg.kg-1.min-1 at 330-360 min) and there was no difference in isotopically determined hepatic glucose production rates (-0.30 +/- 0.23 vs -0.16 +/- 0.22 mg.kg-1.min-1 at 330-360 min). Furthermore, arteriovenous glucose differences across the forearm were similar with or without GLP-1 infusion (1.43 +/- 0.23 vs 1.8 +/- 0.29 mmol/l), (ANOVA; p > 0.60, in all instances). In conclusion, GLP-1 (7-36 amide) administered for 3 h, leading to circulating levels within the physiological range, does not affect insulin sensitivity in healthy man.

UNLABELLED: Glucagon-like peptide 1 (GLP-1[7-36 amide]) is an incretin hormone primarily synthesized in the lower gut (ileum, colon/rectum). Nevertheless, there is an early increment in plasma GLP-1 immediately after ingesting glucose or mixed meals, before nutrients have entered GLP-1 rich intestinal regions. The responsible signalling pathway between the upper and lower gut is not clear. It was the aim of this study to see, whether small intestinal resection or colonectomy changes GLP-1[7-36 amide] release after oral glucose. In eight healthy controls, in seven patients with inactive Crohn's disease (no surgery), in nine patients each after primarily jejunal or ileal small intestinal resections, and in six colonectomized patients not different in age (p = 0.10), body-mass-index (p = 0.24), waist-hip-ratio (p = 0.43), and HbA1c (p = 0.22), oral glucose tolerance tests (75 g) were performed in the fasting state. GLP-1[7-36 amide], insulin C-peptide, GIP and glucagon (specific (RIAs) were measured over 240 min.STATISTICS: Repeated measures ANOVA, t-test (significance: p < 0.05). A clear and early (peak: 15-30 min) GLP-1[7-36 amide] response was observed in all subjects, without any significant difference between gut-resected and control groups (p = 0.95). There were no significant differences in oral glucose tolerance (p = 0.21) or in the suppression of pancreatic glucagon (p = 0.36). Colonectomized patients had a higher insulin (p = 0.011) and C-peptide (p = 0.0023) response in comparison to all other groups. GIP responses also were higher in the colonectomized patients (p = 0.0005). Inactive Crohn's disease and resections of the small intestine as well as proctocolectomy did not change overall GLP-1[7-36 amide] responses and especially not the early increment after oral glucose. This may indicate release of GLP-1[7-36 amide] after oral glucose from the small number of GLP-1[7-36 amide] producing L-cells in the upper gut rather than from the main source

Intravenous glucagon-like peptide (GLP)-1 [7-36 amide] can normalize plasma glucose in non-insulin-dependent diabetic (NIDDM) patients. Since this is no form for routine therapeutic administration, effects of subcutaneous GLP-1 at a high dose (1.5 nmol/kg body weight) were examined. Three groups of 8, 9 and 7 patients (61 +/- 7, 61 +/- 9, 50 +/- 11 years; BMI 29.5 +/- 2.5, 26.1 +/- 2.3, 28.0 +/- 4.2 kg/m2; HbA1c 11.3 +/- 1.5, 9.9 +/- 1.0, 10.6 +/- 0.7%) were examined: after a single subcutaneous injection of 1.5 nmol/kg GLP [7-36 amide]; after repeated subcutaneous injections (0 and 120 min) in fasting patients; after a single, subcutaneous injection 30 min before a liquid test meal (amino acids 8%, and sucrose 50 g in 400 ml), all compared with a placebo. Glucose (glucose oxidase), insulin, C-peptide, GLP-1 and glucagon (specific immunoassays) were measured. Gastric emptying was assessed with the indicator-dilution method and phenol red. Repeated measures ANOVA was used for statistical analysis. GLP-1 injection led to a short-lived increment in GLP-1 concentrations (peak at 30-60 min, then return to basal levels after 90-120 min). Each GLP-1 injection stimulated insulin (insulin, C-peptide, p < 0.0001, respectively) and inhibited glucagon secretion (p < 0.0001). In fasting patients the repeated administration of GLP-1 normalized plasma glucose (5.8 +/- 0.4 mmol/l after 240 min vs 8.2 +/- 0.7 mmol/l after a single dose, p = 0.0065). With the meal, subcutaneous GLP-1 led to a complete cessation of gastric emptying for 30-45 min (p < 0.0001 statistically different from placebo) followed by emptying at a normal rate. As a consequence, integrated incremental glucose responses were reduced by 40% (p = 0.051). In conclusion, subcutaneous GLP-1 [7-36 amide] has similar effects in NIDDM patients as an intravenous infusion. Preparations with retarded release of GLP-1 would appear more suitable for therapeutic purposes because elevation of GLP-1 concentrations for

The aim of the study was to investigate whether inhibition of gastric emptying of meals plays a role in the mechanism of the blood glucose-lowering action of glucagon-like peptide-1-(7-36) amide [GLP-1-(7-36) amide] in type 2 diabetes. Eight poorly controlled type 2 diabetic patients (age, 58 +/- 6 yr; body mass index, 30.0 +/- 5.2 kg/m2; hemoglobin A1c, 10.5 +/- 1.2%) were studied in the fasting state (plasma glucose, 11.1 +/- 1.1 mmol/L). A liquid meal of 400 mL containing 8% amino acids and 50 g sucrose (327 Kcal) was administered at time zero by a nasogastric tube. Gastric volume was determined by a dye dilution technique using phenol red. In randomized order, GLP-1-(7-36) amide (1.2 pmol/kg.min; Saxon Biochemicals) or placebo (0.9% NaCl with 1% human serum albumin) was infused between -30 and 240 min. In the control experiment, gastric emptying was completed within 120 min, and plasma glucose, insulin, C-peptide, GLP-1-(7-36) amide, and glucagon concentrations transiently increased. With exogenous GLP-1-(7-36) amide (plasma level, approximately 70 pmol/L), gastric volume remained constant over the period it was measured (120 min; P < 0.0001 vs. placebo), and plasma glucose fell to normal fasting values (5.4 +/- 0.7 mmol/L) within 3-4 h, whereas insulin was stimulated in most, but not all, patients, and glucagon remained at the basal level or was slightly suppressed. In conclusion, GLP-1-(7-36) amide inhibits gastric emptying in type 2 diabetic patients. Together with the stimulation of insulin and the inhibition of glucagon secretion, this effect probably contributes to the blood glucose-lowering action of GLP-1-(7-36) amide in type 2-diabetic patients when studied after meal ingestion. At the degree observed, inhibition of gastric emptying, however, must be overcome by tachyphylaxis, reduction in dose, or pharmacological interventions so as not to interfere with the therapeutic use of GLP-1-(7-36) amide in type 2 diabetic patients.

The intestinal incretin hormone glucagon-like peptide I (GLP-I) inhibits gastric motility and secretion in normal, but not in vagotomized subjects, pointing to a centrally mediated effect. Therefore, our aim was to study the availability of rat brain GLP-I receptors to peripherally injected 125I-labeled GLP-I. The specificity of the binding was tested by co-injection of excess amounts of unlabeled GLP-I. Using light microscopical autoradiography of rat brain sections, we found specific 125I-GLP-I binding exclusively in the subfornical organ and the area postrema. This binding was abolished when an excess amount of unlabeled GLP-I was co-injected with the labeled GLP-I. We conclude that cells in the subfornical organ and the area postrema could be responsive to blood-borne GLP-I. The observed binding of peripherally administered GLP-I to the subfornical organ and the area postrema, which both have close neuroanatomical connections with hypothalamic areas involved in water and appetite homeostasis, is consistent with the potential roles of circulating GLP-I in the central regulation of appetite and autonomic functions.

BACKGROUND: Glucagon-like peptide (GLP-1) 7-36 amide and peptide YY (PYY) from the L-cell of the ileal mucosa are potent inhibitors of gastric acid secretion in man. It is not clear, however, by which mechanism(s) they inhibit acid secretion. In dogs the inhibitory effect of PYY on acid secretion may be mediated mainly through neural pathways. The mechanism of action of GLP-1 might be similar. The aim of the present study was to examine the effects of GLP-1 might be similar. The aim of the present study was to examine the effects of GLP-1 and PYY on the vagally induced gastric acid secretion in man.METHODS: A modified sham feeding technique, chew and spit, was used. Six healthy volunteers were randomly assigned to receive intravenous infusion of saline, GLP-1 (41 pmol/kg/h), or peptide YY (50 pmol/kg/h).RESULTS: The infusion of GLP-1 and PYY resulted in plasma concentrations of 60 +/- 9 pmol/l and 84 +/- 11 pmol/l, respectively. GLP-1 and PYY both significantly inhibited the intergrated acid output by 67 +/- 6% and 68 +/- 9%, respectively, compared with the integrated outputs in a control experiment with saline infusion. Serum gastrin and plasma somatostatin concentrations remained unchanged during saline, GLP-1, and PYY infusions.CONCLUSIONS: GLP-1 and PYY are both potent inhibitors of the cephalic phase of acid secretion, indicating that at least part of the inhibitory effect of GLP-1 and PYY in man is mediated through neural pathways. Furthermore, the inhibitory effect seems to be independent of circulating concentrations of gastrin and somatostatin.

The newly discovered intestinal hormone, glucagon-like peptide-1 (GLP-1) (proglucagon 78-107amide), stimulates insulin secretion and inhibits glucagon secretion in man and may therefore be anticipated to influence hepatic glucose production. To study this, we infused synthetic GLP-1 sequentially at rates of 25 and 75 pmol.kg-1.h-1 into eight healthy volunteers after an overnight fast and measured plasma concentrations of glucose, insulin, and glucagon and glucose turnover by a technique involving infusion of 3-3H-glucose. Plasma levels of GLP-1 increased by 21.3 +/- 3.1 and 75.4 +/- 3.2 pmol/L during the infusion, changes that were within physiologic limits. In a control experiment only saline was infused. During GLP-1 infusion, plasma glucose level decreased significantly (from 5.3 +/- 0.1 to 4.7 +/- 0.1 and 4.3 +/- 0.1 pmol/L at the end of the two infusion periods). Despite this, plasma insulin level increased significantly (from 20.5 +/- 2.9 to a peak value of 33.5 +/- 5.2 pmol/L during the second period), and plasma glucagon level decreased (from 9.3 +/- 1.7 to 7.1 +/- 1.0 pmol/L). Glucose rate of appearance (Ra) decreased significantly to 75% +/- 6% of the preinfusion values during GLP-1 infusion. Glucose disappearance rate (Rd) did not change significantly, but glucose clearance increased significantly compared with saline. All parameters of glucose turnover remained constant during saline infusion. We conclude that GLP-1 may potently control hepatic glucose production and glucose clearance through its effects on the pancreatic glucoregulatory hormones. The effect of GLP-1 on glucose production is consistent with its proposed use in the treatment of type II diabetes.

In the pancreas proglucagon (PG), a peptide precursor of 160 amino acids is cleaved to produce glucagon and a 30-amino acid N-terminal flanking peptide, but the fate of the C-terminal flanking peptide (99 amino acids) is incompletely known. We subjected acid ethanol extracts of human and porcine pancreases to gel filtration and analyzed the fractions with specific radioimmunoassays for the following regions of proglucagon: PG 62-69, PG 72-81, PG 78-87, PG 98-107 amide, PG 126-134, and PG 149-158. Based on these assays and successive purifications by high performance liquid chromatography we isolated and purified to homogeneity three porcine peptides which were subjected to mass spectrometry and sequencing. One peptide was PG 64-69. The second was PG 72-108, as determined by mass spectrometry, N-terminal amino acid sequencing, and specific radioimmunoassays. The third had a molecular size of approximately 10,000, an N-terminal sequence corresponding to PG 72-81, and a C-terminal sequence terminating at PG 158 (specific radioimmunoassay). A similar peptide with an identical N-terminal sequence, a C-terminal sequence corresponding to PG 146-158, and a molecular mass of 9969 (theoretical mass for human PG 72-158 = 9971) was isolated from human pancreas together with small amounts of a peptide corresponding to PG 72-107 amide. Thus, the pancreatic processing of the C-terminal flanking peptide in proglucagon includes the formation of equimolar (to glucagon) amounts of PG 64-69 and PG 72-158 (major proglucagon fragment) and smaller amounts of N-terminally extended glucagon-like peptide-1 (GLP-1) (PG 72-108 in pigs and PG 72-107 amide in humans).

Udgivelsesdato: 1994-Feb-15, Insulin-like growth factor-(IGF) II and IGF-I and IGF-II/mannose 6-phosphate receptors were expressed in a thoracopulmonary malignant small round cell tumor (MSRCT) from a 14-year-old boy. Northern analysis showed that the MSRCT expresses multiple IGF-II mRNA of 6.0, 4.8, 4.2, and 2.2 kilobase from promoters P3 and P4 of the human IGF-II gene. Chromatography and radioimmunoassay revealed two forms of IGF-II with molecular masses of 7.5 kilodalton (kDa) and 10 kDa, corresponding to mature IGF-II and IGF-II with a C-terminal extension, in concentrations of 61 and 41 ng/g/tumor tissue, respectively. By a combined reverse transcription-polymerase chain reaction analysis, the authors also show that the MSRCT expresses IGF-I and IGF-II/mannose 6-phosphate receptor mRNA. The plasma concentration of IGF-II was 600 ng/ml and within the normal range of serum IGF-II. IGF binding proteins (IGFBP) of 41.5, 38.5, 34, 30, and 24 kDa were present in serum. Compared with normal plasma from healthy subjects and an age-matched group of boys, the level of the 41.5, 38.5, and 30 kDa IGFBP were approximately 3-fold elevated. The authors conclude that transcription of the IGF-II gene leads to the production of significant amounts of 10 kDa IGF-II and 7.5 kDa IGF-II. IGF-II may stimulate the proliferation of MSRCT by interaction with IGF-I receptors on the cells.

Gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1-(7-36) amide (GLP-1) are glucose-dependent insulinotropic gut hormones that may explain the greater insulin secretory response with oral compared to i.v. glucose (incretin effect). To study their individual and combined contributions, in eight healthy volunteers, on separate occasions, synthetic human GIP (1 pmol/kg.min) and/or GLP-1 (0.3 pmol/kg.min) or placebo were infused i.v. (-30 to 120 min), while at 0 min, a glucose infusion "isoglycemic" to the profile after an oral glucose load of 50 g/400 mL was started. After the administration of 50 g oral glucose, immunoreactive GIP rose several-fold to 337 +/- 43 pmol/L, while there was only a transient (10-30 min) and moderate increment in immunoreactive GLP-1 (from basal, 25-30, to 41 +/- 4 pmol/L). Isoglycemic i.v. glucose infusions led to smaller B-cell responses (estimated incretin effect, 41 +/- 5%). With single infusions of GIP or GLP-1 (circulating concentrations, 464 +/- 73 and 54 +/- 3 pmol/L, respectively), B-cell responses were significantly augmented compared to i.v. glucose alone and were no longer significantly different from those after oral glucose. The combination of GIP and GLP-1 led to B-cell responses that were significantly higher than those with either hormone alone (additive mode of cooperation). Plasma GIP concentrations were similar after endogenous secretion (oral glucose) and i.v. infusion, while exogenously administered GLP-1 led to plasma levels that were maintained at an elevated level for a longer period during exogenous infusion than after stimulation by oral glucose. When, in seven volunteers, a lower dose (0.15 pmol/kg.min) of GLP-1 was infused during isoglycemic glucose infusion experiments only for the duration of elevated plasma levels in the oral glucose challenges (0-30 min), a significant, but transient, increment in insulin and C-peptide concentrations was observed, which was equivalent to 26 +/- 10% of the est

The biological effects and the metabolism of the intestinal hormone glucagonlike peptide-1 7-36 amide and glucagonlike peptide-1 7-37 were studied in normal healthy subjects. GLP-1 7-36 amide and GLP-1 7-37 equipotently stimulated insulin secretion (integrated hormone response 0-60 min, 631 +/- 211 vs. 483 +/- 177 pmol/h x L-1) and C-peptide secretion (integrated hormone response 9064 +/- 1804 vs. 9954 +/- 2031 pmol/h x L-1) and equipotently lowered plasma glucose (integrated decrease 48.3 +/- 5.7 vs. 46.2 +/- 8.4 mmol/h x L-1) and plasma glucagon (integrated decrease 80.4 +/- 24.3 vs. 156.0 +/- 34.6 pmol/h x L-1). Both GLP-1 7-36 amide and GLP-1 7-37 lowered the plasma concentration of free fatty acids significantly. The plasma half-lives of GLP-1 7-36 amide and GLP-1 7-37 were 5.3 +/- 0.4 vs. 6.1 +/- 0.8 min, and the metabolic clearance rates of the two peptides also were similar (14.6 +/- 2.4 vs. 12.2 +/- 1.0 pmol/kg x min). In conclusion, COOH-terminal amidation is neither important for the metabolism of GLP-1 nor for its effects on the endocrine pancreas.

The post-translational processing of proglucagon in the small intestine gives rise to glucagon-like peptide-1 (PG 78-107 amide) which has profound effects on the endocrine pancreas, and in many species also on the stomach. Glucagon-like peptide-1 (PG 78-107 amide) is secreted in man in response to physiological stimuli e.g. a mixed meal. Glucagon-like peptide-1, in concentrations corresponding to those observed in response to meals, strongly stimulates insulin secretion, in all mammals studied, even more potently than the gastric inhibitory peptide. Thus, glucagon-like peptide-1 fulfills the classic criteria for being a hormone and is likely to be a new incretin. The glucagon inhibitory effect of glucagon-like peptide-1 (PG 78-107 amide) probably further potentiates the effect of glucagon-like peptide-1 on glucose metabolism and distinguished this peptide from other intestinal peptides which have been proposed as incretins. Glucagon-like peptide-1 also inhibits gastric acid secretion and gastric emptying in man. The latter delays nutrient entry to the intestine and thereby diminishes meal-induced glucose excursions. Elevated plasma concentrations of immunoreactive glucagon-like peptide-1 have been reported in Type 2 (noninsulin-dependent) diabetic patients, however, the consequences of the elevation are not yet known. However, elevated levels of glucagon-like peptide-1 in patients with increased gastric emptying rate (post-gastrectomy syndromes) may be responsible for the exaggerated insulin secretion seen in these patients.

Gastric inhibitory polypeptide (GIP) and glucagon-like peptide 1 [7-36 amide] (GLP-1) are glucose-dependent insulinotropic gut hormones. Under experimental conditions, both have been shown to reduce stimulated gastric acid secretion. To study their individual and combined effects on pentagastrin-stimulated (0.1 micrograms/kg/h from -90 to 120 min) gastric volume, acid and chloride output, on separate occasions, synthetic human GIP (1 pmol/kg/min) and/or GLP-1 [7-36 amide] (0.3 pmol/kg/min) or placebo (0.9% NaCl with 1% albumin) were infused intravenously (from -30 to 120 min) in 9 healthy volunteers. At 0 min, a glucose infusion was started that mimicked the glycemic profile after an oral glucose load of 50 g/400 ml and allowed for the glucose-dependent insulinotropic action of GIP and GLP-1 [7-36 amide]. Pentagastrin stimulated acid output significantly, but neither GIP nor GLP-1 [7-36 amide] either alone or in combination, reduced pentagastrin-stimulated gastric acid secretion. The circulating concentrations of GIP and GLP-1 [7-36 amide] obtained at steady state during exogenous administration of synthetic peptides were similar to or higher than those reached after oral glucose (endogenous secretion). In conclusion, (penta)gastrin-stimulated gastric acid secretion is not inhibited by physiological circulating concentrations of GIP or GLP-1 [7-36 amide]. Therefore, the insulinotropic action of these intestinal hormones is physiologically more important than their possible role as enterogastrone.

We studied the secretion of somatostatin and HCl and the release of vasoactive intestinal polypeptide (VIP) and gastrin-releasing peptide (GRP) from isolated, vascularly perfused, porcine non-antral stomach. Electric vagus stimulation increased acid secretion and the release of VIP and GRP and inhibited somatostatin secretion as determined in the venous effluent. Atropine abolished the HCl response and reversed the somatostatin inhibition to a three-fold increase, whereas GRP and VIP responses were unchanged. Both intra-arterial carbachol (10(-6) M) and GRP (10(-8) M) increased acid secretion and inhibited somatostatin secretion. VIP (10(-8) M) increased somatostatin secretion and had no effect on acid secretion. By immunohistochemistry, somatostatin was localized to both open-type and closed-type cells equally spread in the various parts of the gastric glands without particular relation to the parietal cells. Numerous GRP- and VIP-immunoreactive nerve fibers were seen between the glands. It is concluded that the fundic and antral secretion of somatostatin, investigated in a previous study, are differently regulated. The relation of fundic somatostatin release to acid secretion seems to be complex.

We investigated the major products of proglucagon (PG) processing in plasma in the fasting state, after intravenous arginine and after an oral glucose load in noninsulin-dependent diabetics (NIDDM) and in weight matched controls using specific radioimmunoassays and analytical gel filtration. In the fasting state the glucagonlike peptide-1 (GLP-1) immunoreactivity was significantly elevated in the NIDDM group compared with the control group. Both after intravenous arginine and after an oral glucose load a rise in the plasma concentrations of all immunoreactive moieties measured was seen. All integrated incremental responses after intravenous arginine were identical in the two groups. After oral glucose the insulin concentrations in plasma were lower and the concentrations of all proglucagon products were higher in the NIDDM group compared to the control group. The gel filtration analysis showed that arginine stimulated the secretion of pancreatic glucagon (PG 33-61), major proglucagon fragment (PG 72-158) and probably GLP-1 (PG 72-107 amide) in both groups, whereas oral glucose stimulated the secretion of glicentin (PG 1-69) and intestinal GLP-1 (PG 78-107 amide), an insulinotropic hormone. The elevated levels of immunoreactive GLP-1 in diabetics in the fasting state were mainly due to an increased concentration of major proglucagon fragment.

We demonstrated specific binding of the insulin-releasing hormone glucagonlike peptide (GLP)-I-(7-36)-amide, an intestinal product of proglucagon, to pancreatic islet cells by autoradiography using 125I-labeled GLP-I-(7-36)-amide incubated with tissue specimens of extracerebral rat organs. We also found binding of 125I-GLP-I to insulin-, glucagon-, and somatostatin-immunoreactive cells by combined autoradiographic and immunohistochemical analysis of pancreatic specimens using antisera against insulin, glucagon, and somatostatin. An accumulation of radioactivity was also observed in the stomach surface epithelium but could not be prevented by excess unlabeled peptide. No binding was found in other tissues investigated, including the lungs and the small intestinal mucosa. Localization of the binding sites identifies the pancreatic islets as the prime target for GLP-I-(7-36)-amide and suggests that it exerts direct effects on islet cells.