Cathrine Ørskov

Tissue and Plasma Concentrations of Amidated and Glycine-Extended Glucagon-Like Peptide I in Humans

Using specific radioimmunoassays, we studied the occurrence of amidated and glycine-extended glucagon-like peptide I (GLP-I) molecules in the human small intestine and pancreas and in the circulation system in response to a breakfast meal. Through gel permeation chromatography of extracts of the human pancreas (n = 5), we found that 71% of the GLP-I immunoreactivity eluted as a large molecule corresponding to the major proglucagon fragment, 24% corresponded to GLP-I 1–36 amide, and 5% to GLP-I 1–37. By gel permeation chromatography of extracts of human small intestine (n = 6), we found that all immunoreactivity eluted in one peak at the common elution position of the two insulin-releasing peptides, GLP-I 7–36 amide and GLP-I 7–37. Of the GLP-I immunoreactivity, 80% corresponded to GLP-I 7–36 amide and 20% to GLP-I 7–37. The mean concentrations of amidated GLP-I and glycine-extended GLP-I in fasting plasma were 7 ± 1 and 6 ± 1 pM, respectively (n = 6). In response to a breakfast meal, the concentration of amidated GLP-I rose significantly amounting to 41 ± 5 pM 90 min after the meal ingestion, whereas the concentration of glycine-extended GLP-I only rose slightly to a maximum of 10 ± 1 pM. Thus, both amidated and glycine-extended GLP-I molecules are produced in the small intestine and in the pancreas in humans. Both amidated and glycine-extended GLP-I are measurable in fasting plasma. The higher meal response of amidated GLP-I compared with glycine-extended GLP-I probably reflects the larger amount of amidated GLP-I produced in the tissues compared with glycine-extended GLP-I.

Glucagon-like peptide 1 inhibition of gastric emptying outweighs its insulinotropic effects in healthy humans

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-1were 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).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).

Glucagon-Like Peptide-1-(7–36)Amide Is Transformed to Glucagon-Like Peptide-1-(9–36)Amide by Dipeptidyl Peptidase IV in the Capillaries Supplying the L Cells of the Porcine Intestine

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...

Glucagonostatic Actions and Reduction of Fasting Hyperglycemia by Exogenous Glucagon-Like Peptide I(7–36) amide in type I diabetic patients

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 ∼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 ∼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.

Biological Effects and Metabolic Rates of Glucagonlike Peptide-1 7–36 Amide and Glucagonlike Peptide-1 7–37 in Healthy Subjects Are Indistinguishable

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 × L−1) and C-peptide secretion (integrated hormone response 9064 ± 1804 vs. 9954 ± 2031 pmol/h × L−1) and equipotently lowered plasma glucose (integrated decrease 48.3 ± 5.7 vs. 46.2 ± 8.4 mmol/h × L-1) and plasma glucagon (integrated decrease 80.4 ± 24.3 vs. 156.0 ± 34.6 pmol/h × 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 × min). In conclusion, COOH-terminal amidation is neither important for the metabolism of GLP-1 nor for its effects on the endocrine pancreas.

GLP-1 and GIP are colocalized in a subset of endocrine cells in the small intestine.

BACKGROUND The incretin hormones GIP and GLP-1 are thought to be produced in separate endocrine cells located in the proximal and distal ends of the mammalian small intestine, respectively. METHODS AND RESULTS Using double immunohistochemistry and in situ hybridization, we found that GLP-1 was colocalized with either GIP or PYY in endocrine cells of the porcine, rat, and human small intestines, whereas GIP and PYY were rarely colocalized. Thus, of all the cells staining positively for either GLP-1, GIP, or both, 55-75% were GLP-1 and GIP double-stained in the mid-small intestine. Concentrations of extractable GIP and PYY were highest in the midjejunum [154 (95-167) and 141 (67-158) pmol/g, median and range, respectively], whereas GLP-1 concentrations were highest in the ileum [92 (80-207) pmol/l], but GLP-1, GIP, and PYY immunoreactive cells were found throughout the porcine small intestine. CONCLUSIONS Our results provide a morphological basis to suggest simultaneous, rather than sequential, secretion of these hormones by postprandial luminal stimulation.

GLP-1 Receptor Localization in Monkey and Human Tissue: Novel Distribution Revealed With Extensively Validated Monoclonal Antibody

Glucagon-like peptide 1 (GLP-1) analogs are increasingly being used in the treatment of type 2 diabetes. It is clear that these drugs lower blood glucose through an increase in insulin secretion and a lowering of glucagon secretion; in addition, they lower body weight and systolic blood pressure and increase heart rate. Using a new monoclonal antibody for immunohistochemistry, we detected GLP-1 receptor (GLP-1R) in important target organs in humans and monkeys. In the pancreas, GLP-1R was predominantly localized in β-cells with a markedly weaker expression in acinar cells. Pancreatic ductal epithelial cells did not express GLP-1R. In the kidney and lung, GLP-1R was exclusively expressed in smooth muscle cells in the walls of arteries and arterioles. In the heart, GLP-1R was localized in myocytes of the sinoatrial node. In the gastrointestinal tract, the highest GLP-1R expression was seen in the Brunners gland in the duodenum, with lower level expression in parietal cells and smooth muscle cells in the muscularis externa in the stomach and in myenteric plexus neurons throughout the gut. No GLP-1R was seen in primate liver and thyroid. GLP-1R expression seen with immunohistochemistry was confirmed by functional expression using in situ ligand binding with (125)I-GLP-1. In conclusion, these results give important new insight into the molecular mode of action of GLP-1 analogs by identifying the exact cellular localization of GLP-1R.

Structure, measurement, and secretion of human glucagon-like peptide-2

By using radioimmunoassays toward the cDNA-predicted amino acid sequence of human glucagon-like peptide-2, a peptide was isolated from extracts of human ileum. By mass spectrometry and Edman sequencing, this peptide was identified as human proglucagon 126-158. High-performance liquid chromatography analyses indicated that a similar immunoreactive peptide (iGLP-2) was present in human plasma. Human plasma concentrations of iGLP-2 were elevated 3- to 4-fold at 1 to 2 h after ingestion of 800 to 1200 kcal meals.

GLP-2 stimulates colonic growth via KGF, released by subepithelial myofibroblasts with GLP-2 receptors

BACKGROUND Glucagon-like peptide-2 is thought to act as a growth factor for the gut, but the localization of the GLP-2 receptor and mechanism of action on epithelial growth is unclear. METHODS AND RESULTS We found glucagon-like peptide-2 (GLP-2) receptors mainly on subepithelial myofibroblasts in rat, mouse, marmoset and human small and large intestine by immunohistochemistry and in situ hybridisation. By double labelling we found that these GLP-2 receptor immunoreactive cells also produce smooth muscle actin and keratinocyte growth factor (KGF). By subcutaneous infusion of either GLP-2 alone, GLP-2 plus KGF antibody, KGF antibody alone or saline in mice, we found that KGF antibody abolished the growth promoting effect of GLP-2 in the large intestine, but not in the small intestine. CONCLUSIONS Our findings suggest that GLP-2 in the gut acts by activating receptors on the subepithelial myofibroblasts, causing the release of growth factors, which in turn stimulate intestinal growth.

Effect of glucagon-like peptide-1 (proglucagon 78-107amide) on hepatic glucose production in healthy man.

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.