Erythromycin Antagonizes the Deceleration of Gastric Emptying by Glucagon-Like Peptide 1 and Unmasks Its Insulinotropic Effect in Healthy Su
http://www.100md.com
糖尿病学杂志 2005年第7期
1 Department of Medicine, Ruhr University, Bochum, Germany
2 Larry Hillblom Islet Research Center, UCLA School of Medicine, Los Angeles, California
3 Department of Medical Physiology, Panum Institute, University of Copenhagen, Copenhagen, Denmark
4 Diabeteszentrum, Bad Lauterberg im Harz, Germany
ABSTRACT
Glucagon-like peptide 1 (GLP-1) has been proposed to act as an incretin hormone due to its ability to enhance glucose-stimulated insulin secretion. Because GLP-1 also decelerates gastric emptying, it physiologically reduces rather than augments postprandial insulin secretory responses. Therefore, we aimed to antagonize the deceleration of gastric emptying by GLP-1 to study its effects on insulin secretion after a meal. Nine healthy male volunteers (age 25 ± 4 years, BMI 25.0 ± 4.9 kg/m2) were studied with an infusion of GLP-1 (0.8 pmol · kgeC1 · mineC1 from eC30 to 240 min) or placebo. On separate occasions, the prokinetic drugs metoclopramide (10 mg), domperidone (10 mg), cisapride (10 mg, all at eC30 min per oral), or erythromycin (200 mg intravenously from eC30 to eC15 min) were administered in addition to GLP-1. A liquid test meal (50 g sucrose and 8% mixed amino acids in 400 ml) was administered at 0 min. Capillary and venous blood samples were drawn for the determination of glucose (glucose oxidase), insulin, C-peptide, GLP-1, glucagon, gastric inhibitory polypeptide (GIP), and pancreatic polypeptide (specific immunoassays). Gastric emptying was assessed by the phenol red dilution technique. Statistical analyses were performed using repeated-measures ANOVA and Duncan’s post hoc test. GLP-1 significantly decelerated the velocity of gastric emptying (P < 0.001). This was completely counterbalanced by erythromycin, whereas the other prokinetic drugs used had no effect. Postprandial glucose concentrations were lowered by GLP-1 (P < 0.001 vs. placebo), but this effect was partially reversed by erythromycin (P < 0.05). Insulin secretory responses to the meal were lower during GLP-1 administration (P < 0.05 vs. placebo). However, when erythromycin was added to GLP-1, insulin concentrations were similar to those in placebo experiments. The suppression of meal-related increments in glucagon secretion by GLP-1 was reversed by erythromycin (P < 0.001). The time course of GIP secretion was delayed during GLP-1 administration (P < 0.05), but when erythromycin was added, the pattern was similar to placebo experiments. GLP-1 administration led to a reduction in pancreatic polypeptide plasma concentrations (P < 0.05). In contrast, pancreatic polypeptide levels were markedly increased by erythromycin (P < 0.001). Intravenous erythromycin counteracts the deceleration of gastric emptying caused by GLP-1, probably by interacting with the parasympathetic nervous system (pancreatic polypeptide responses). Despite augmented rises in insulin secretion, the glucose-lowering effect of GLP-1 is markedly reduced when the deceleration of gastric emptying is antagonized, illustrating the importance of this facet of the multiple antidiabetic actions of GLP-1.
Insulin secretion after meal ingestion is stimulated not only by the rise in glycemia, but also by the secretion of peptide hormones ("incretins") from the gut (1,2). The postprandial enhancement of insulin secretion by gut-derived factors is called the incretin effect (2,3). The first incretin hormone identified was gastric inhibitory polypeptide (GIP), which is also referred to as glucose-dependent insulinotropic polypeptide (4,5). An incretin role has also been proposed for the proglucagon-derived peptide glucagon-like peptide 1 (GLP-1) (6). This was based on its ability to enhance insulin secretion and to suppress glucagon release during an intravenous glucose infusion in healthy human volunteers (6). Moreover, the GLP-1 receptor antagonist exendin [9eC39] blocked the insulin secretory response after intraduodenal administration of glucose in rats (7). Later, Edwards et al. (8) demonstrated a marked deterioration of oral glucose tolerance in healthy subjects during the administration of exendin [9eC39]. However, when GLP-1 was administered during meal ingestion, a dose-dependent deceleration of gastric emptying as well as a reduction in postprandial insulin secretion was found in healthy volunteers as well as in patients with type 2 diabetes (9,10). Because the velocity of gastric emptying is a major determinant of postprandial glycemia and insulin secretion (11,12), it is difficult to ascertain an insulinotropic effect of GLP-1 after meal ingestion. One way to counterbalance the GLP-1 effects on gastric emptying would be to administer prokinetic drugs before meal ingestion. Therefore, it was the aim of the present experiments to antagonize the decelerating effects of GLP-1 on gastric emptying using a panel of prokinetic drugs with different modes of action (metoclopramide, domperidone, cisapride, and erythromycin) to unmask its insulinotropic effects in the postprandial state.
RESEARCH DESIGN AND METHODS
The study protocol was approved by the ethics committee of the medical faculty of Ruhr University (Bochum, Germany) before the study. Written informed consent was obtained from all participants.
Nine healthy male volunteers were studied. They were 25 ± 4 years old, 181 ± 4 cm tall, and weighed 82 ± 17 kg. Their BMI was 25.0 ± 4.9 kg/m2. All had a normal oral glucose tolerance according to World Health Organization criteria (fasting glucose 5.1 ± 0.4 mmol/l, 120-min value 5.0 ± 1.1 mmol/l). None had a family history of diabetes or a personal history of gastrointestinal disorders. Blood cell counts, serum transaminases, creatinine values, triglyceride, cholesterol, and HDL cholesterol concentrations were in the normal range.
The study was performed in a single-blinded fashion with all participants being studied in random order on six occasions.
1) A liquid mixed meal (50 g sucrose plus amino acids, 400 ml Aminosteril Hepa 8%; Fresenius AG, Bad Homburg, Germany) was instilled intragastrically at time 0. Placebo (0.9% NaCl with 1% human serum albumin (Behring AG, Marburg, Germany) was infused intravenously from eC30 to 240 min.
2) A liquid test meal was administered as described in 1. In addition, a continuous intravenous administration of GLP-1 at a dose of 0.8 pmol · kgeC1 · mineC1 was started 30 min before the meal (at eC30 min) and continued until 240 min.
3) In addition to the administration of the meal and GLP-1 (as described in 1 and 2), metoclopramide (Paspertin; 10 mg/10 ml) was administered orally at eC30 min.
4) In addition to the administration of the meal and GLP-1 (as described in 1 and 2), domperidone (Motilium Saft; 10 mg/10 ml) was administered orally at eC30 min.
5) In addition to the administration of the meal and GLP-1 (as described in 1 and 2), cisapride (Propulsin Saft; 10 mg/10 ml) was administered orally at eC30 min.
6) In addition to the administration of the meal and GLP-1 (as described in 1 and 2), erythromycin (Erycinum; 200 mg/100 ml) was administered intravenously between eC30 and eC15 min.
An interval of at least 10 days was kept between the experiments to exclude carryover effects.
Peptides.
Synthetic GLP-1 [7eC36 amide] was purchased from Saxon Biochemicals (Hannover, Germnay). The lot number of GLP-1 [7eC36 amide] (pharmaceutical grade) was PGAS 242, FGLP7369301 A, net peptide content 88%. The peptide was dissolved in 0.9% NaCl and 1% human serum albumin (HSA Behring, Marburg, Germany), filtered through 0.2 e nitrocellulose filters (Sartorius, Gttingen, Germany), and stored frozen at eC30°C as previously described (9). High-performance liquid chromatography profiles (provided by the manufacturer) showed that the preparation was >99% pure (single peak with appropriate standards). Samples were analyzed for bacterial growth (standard culture techniques) and for pyrogens (limulus amebocyte lysate endo-LAL; Chromogenix AB, Mlndal, Sweden). No bacterial contamination was detected. Endotoxin concentrations in the GLP-1 stock solutions were <0.03 EU/ml.
Experimental procedures.
The tests were performed in the morning after an overnight fast. Two forearm veins were punctured with a Teflon cannula (Moskito 123, 18 gauge; Vygon, Aachen, Germany) and kept patent using 0.9% NaCl (for blood sampling and for GLP-1/placebo administration).
After drawing basal blood specimens, at eC30 min an intravenous infusion of GLP-1 [7eC36 amide] or placebo (0.9% NaCl containing 1% human serum albumin) was started and continued for 270 min. Blood was drawn at the time points eC45, eC30, eC15, 0, 15, 30, 45, 60, 120, 180, and 240 min, and plasma glucose was determined immediately.
Before the study, a nasogastric tube (Freka-Ernhrungssonde, 120 cm, CH12; Fresenius AG, Bad Homburg, Germany) was placed and tape-fixed with the tip 55 cm from the nostrils. Gastric juice was aspirated and an acidic pH was ascertained using pH-sensitive Lackmus paper. The gastric lumen was washed with 100 ml water (37°C). The position of the tube was, if necessary, adjusted to allow near-complete aspiration of instilled fluid. The subjects were in a semirecumbent position with the upper half of the body 45 degrees upright. At 0 min, 400 ml (total volume) of the liquid test meal was instilled into the stomach. It was composed of 50 g sucrose dissolved in 400 ml Aminosteril Hepa 8%. This composition of the meal was chosen because the solution had to be clear for the photometric measurement of phenol red (measurement of gastric emptying, see below) and should be similar in caloric and nutrient content to a normal mixed meal. The meal contained 32 g mixed amino acids (131 kcal = 40%) and 50 g sucrose (196 kcal = 60%), with a total energy content of 327 kcal (energy density 0.82 kcal/ml).
Blood specimens.
Blood was drawn into chilled tubes containing EDTA and aprotinin (Trasylol; 20,000 KIU/ml, 200 e蘬/10 ml blood; Bayer AG, Leverkusen, Germany) and kept on ice. A sample (100 e蘬) was stored in NaF (Microvette CB 300; Sarstedt, Ne筸brecht, Germany) for the measurement of glucose. After centrifugation at 4°C, plasma for hormone analyses was kept frozen at eC30°C.
Gastric emptying.
The velocity of gastric emptying was measured as described (9) by a double-sampling dye dilution technique using phenol red (Merck AG, Darmstadt, Germany) according to George (13), with modifications introduced to reduce measurement error by Hurwitz (14). This technique allows the repeated determination of gastric emptying under different experimental conditions. Briefly, at all time points chosen to measure gastric volume, a known amount of the nonabsorbable dye phenol red was added to the translucent liquid test meal in a volume of 5eC15 ml. After thorough mixing with gastric contents for 2 min, a gastric sample was drawn and the resulting step-up in phenol red concentrations was determined photometrically. The volume of gastric contents was determined from the volume of distribution of phenol red. As the experiments proceeded, increasing amounts of phenol red were used to ensure measurability of optical density increments in the presence of previously instilled phenol red. Gastric contents were determined at 0, 30, 60, 90, 120, 180, and 240 min and were expressed as the percentage of the initial volume of 400 ml at 0 min.
Laboratory determinations.
Glucose was measured using a glucose oxidase method with a Glucose Analyzer 2 (Beckman Instruments, Munich, Germany). Insulin was measured using an insulin microparticle enzyme immunoassay (IMx Insulin; Abbott Laboratories, Wiesbaden, Germany). Intra-assay coefficients of variation were 4%. C-peptide was measured using C-peptide-antibodyeCcoated microtiter wells (C-peptide MTPL EIA; DRG Instruments, Marburg, Germany). Intra-assay coefficients of variation were 6%. Human insulin and C-peptide were used as standards.
Immunoreactive GLP-1 was determined in ethanol-extracted plasma as previously described (15), using antiserum 89390 (final dilution 1:150 000) for the measurement of GLP-1 [7eC36 amide] and synthetic GLP-1 [7eC36 amide] for tracer preparation and as standard. The experimental detection limit (2 SD over samples not containing GLP-1 [7eC36 amide]) was <5 pmol/l. Antiserum 89390 binds to the amidated carboxyl terminus of GLP-1 [7eC36 amide] and therefore measures the sum of intact and degraded GLP-1 [9eC36 amide] in plasma. Intra-assay coefficients of variation were 8%.
Pancreatic glucagon was assayed in ethanol-extracted plasma using antibody 4305 as previously described (16). The detection limit was 1 pmol/l and the intra-assay coefficient of variation was <6% in the working range.
Plasma gastric inhibitory polypeptide (GIP) was determined by radioimmunoassay using antiserum R65 as previously described (17). This assay measures the sum of both intact [1eC42] and degraded GIP [3eC42] in plasma.
Plasma pancreatic polypeptide was determined by radioimmunoassay. A detailed description of the assay is given by Wettergren et al. (18).
Phenol red in gastric contents was assayed photometrically after filtration through filter paper (100 e蘬 in 2 ml Na2HPO4/NaH2PO4 buffer, 0.6 mol/l/l, pH 8.0) at a wavelength of 546 nm and read against a standard curve (phenol red in phosphate buffer) as previously described (9). Each patient’s set of plasma samples was assayed at the same time to avoid errors due to interassay variation.
Statistical analysis.
Results are reported as means ± SE. All statistical calculations were carried out by paired repeated-measures ANOVA using Statistica (Statsoft Europe, Hamburg, Germany). If a significant interaction of treatment and time was documented (P < 0.05), values at single time points were compared by one-way ANOVA and Duncan’s post hoc test (paired analyses). A two-sided P value <0.05 was taken to indicate significant differences.
RESULTS
GLP-1 plasma concentrations.
After the liquid test meal, GLP-1 plasma concentrations increased from 5.3 ± 0.8 pmol/l to peak concentrations of 10.3 ± 1.6 pmol/l at 15 min during placebo infusion (Fig. 1; P = 0.0009). GLP-1 plasma concentrations were similar in all experiments with GLP-1 infusion, reaching steady-state levels at 0 min (Fig. 1). When erythromycin was administered before the test meal, significantly higher GLP-1 plasma concentrations were observed after 30 min, compared with the experiments with the administration of GLP-1 alone (P = 0.0035) (Fig. 1).
Gastric emptying.
After 240 min, the stomach had almost completely emptied in all groups (Fig. 2). During the infusion of GLP-1 alone, the velocity of gastric emptying was significantly decelerated compared with placebo administration (Fig. 2) (P < 0.0001). With erythromycin, the decelerating effect of GLP-1 was completely counterbalanced. Accordingly, no differences occurred in the pattern of gastric emptying between the experiments with the administration of placebo and with the combined administration of GLP-1 and erythromycin (Fig. 2). In contrast, metoclopramide, domperidone, and cisapride had no significant effects on gastric emptying during GLP-1 infusion (Fig. 2).
Plasma glucose.
After the liquid test meal, plasma glucose concentrations increased significantly in the placebo group (P < 0.0001). Intravenous infusion of GLP-1 lowered fasting glucose concentrations even before instillation of the test meal (P < 0.05) and the postprandial rise in glycemia was less pronounced than with placebo. The patterns of glucose concentration were not different in the experiments with the administration of GLP-1 alone or in combination with metoclopramide, domperidone, or cisapride (details not shown). When erythromycin was administered in addition to GLP-1, plasma glucose concentrations at 30 min were higher that with GLP-1 alone but lower than during placebo administration (P < 0.05) (Fig. 3). There were no significant differences in glycemia between the experiments with GLP-1 alone and in combination with erythromycin at any other time point.
Insulin secretory response.
In the fasting state, insulin secretion was not different between the experiments with the administration of placebo and of GLP-1, alone or in combination with erythromycin (Fig. 3). After the liquid test meal, insulin secretory responses were significantly higher during placebo administration than during the infusion of GLP-1 (P < 0.05) (Fig. 3). When erythromycin was administered in addition to GLP-1, the overall pattern of insulin secretion and the integrated incremental plasma concentrations of insulin and C-peptide were more similar to placebo experiments (Fig. 3). However, there were still differences in insulin and C-peptide levels at time point 15 min after the test meal between the experiments with the combined administration of GLP-1 and erythromycin and with placebo. In contrast to the effect of erythromycin, none of the other prokinetic drugs used had an influence on postprandial insulin release during GLP-1 infusion (details not shown).
When the plasma insulin levels immediately after meal ingestion (0eC30 min) were expressed in relation to the corresponding glucose concentrations, a shift of the glucose threshold required for the stimulation of insulin secretion became apparent in the experiments with GLP-1 infusion (Fig. 4). However, the glucose responsiveness of insulin secretion was not affected by erythromycin administration (Fig. 4).
Glucagon secretion.
Plasma glucagon concentrations before the test meal were lowered during GLP-1 infusion (Fig. 1). After the instillation of the liquid meal, plasma glucagon concentrations significantly increased during all experiments (P < 0.0001), reaching higher levels during placebo administration than with GLP-1 (Fig. 1). Again, erythromycin almost completely reversed the changes in glucagon secretion by GLP-1, resulting in a similar pattern of glucagon concentrations as during the placebo experiments (Fig. 1). No changes in glucagon secretion were found after the administration of metoclopramide, domperidone, or cisapride (details not shown).
GIP secretion.
GIP plasma concentrations significantly increased after the liquid test meal during all experiments (Fig. 5) (P < 0.0001). The total amount of GIP secreted after the meal was similar in all experiments (P = 0.38), but the time course of GIP secretion was delayed during GLP-1 infusion (P < 0.05 vs. placebo). These changes were almost completely reversed by erythromycin administration (Fig. 5). Accordingly, peak concentrations of GIP were reached after 60 min during placebo administration, after 180 min during GLP-1 infusion, and after 30 min during the combined administration of erythromycin and GLP-1 (Fig. 5). GIP secretion was not affected by the other prokinetic drugs used (details not shown).
Pancreatic polypeptide secretion.
The secretion of pancreatic polypeptide was significantly suppressed by GLP-1 infusion (Fig. 5). Erythromycin administration led to a marked increase in postprandial pancreatic polypeptide concentrations compared with the placebo experiments (P < 0.0001). The secretion of pancreatic polypeptide was not influenced by metoclopramide, domperidone, or cisapride (details not shown).
DISCUSSION
Because GLP-1 reduces postprandial glycemia not only via endocrine pancreatic secretion (6) but also by decelerating the velocity of gastric emptying (9,10,19,20), it was the aim of this study to assess GLP-1eCinduced insulin secretion after a meal, independent of changes in gastric emptying. Although insulin secretion is typically enhanced by GLP-1 under fasting conditions, a dose-dependent reduction rather than stimulation of postprandial insulin secretion has previously been observed during the administration of GLP-1 in healthy subjects and in patients with type 2 diabetes (9,10). Similar to those studies, a considerably smaller amount of insulin was secreted after the meal during GLP-1 administration in the present experiments. However, when changes in gastric emptying were reversed by erythromycin, insulin secretion was increased to similar levels as in the placebo experiments, although at lower levels of glycemia (Fig. 3). This indicates that the glucose concentration threshold required to stimulate insulin secretion was shifted downward due to the administration of GLP-1. Indeed, when plasma insulin concentrations are expressed in relation to the corresponding glucose levels (Fig. 4), it becomes clear that during GLP-1 administration insulin secretion was increased at lower levels of glycemia compared with placebo. Therefore, in the postprandial state GLP-1 possesses insulinotropic properties independent of changes in the rate of gastric emptying in healthy individuals.
Interestingly, despite the similar patterns of insulin, C-peptide, and glucagon concentrations, postprandial plasma glucose levels were lower in the GLP-1 plus erythromycin experiments than in the placebo experiments. Most likely, this was based on the glucose-lowering effect of GLP-1 before instillation of the test meal. In support of this, plasma glucose concentrations were already lowered by 13 mg/dl at the time of meal administration in the GLP-1 experiments. If these initial differences in glycemia are taken into consideration, the glycemic profiles appear rather similar between the time points of 30 and 60 min in the experiments with GLP-1 plus erythromycin and with placebo. However, there is still a discrepancy regarding the plasma levels of glucose, insulin, C-peptide, and glucagon at the 15-min time point after meal administration between these experiments. Because GIP plasma levels were already increased at that time point in the experiments with GLP-1 and erythromycin, differences in the rate of gastric emptying (which was not determined at this time point) or intestinal nutrient absorption are unlikely to explain this discrepancy. Thus, it is possible that erythromycin had an independent effect on glucose tolerance beyond its effects on gastric emptying.
The present data give rise to a reconsideration of the role of GLP-1 as an incretin hormone. By definition, incretin hormones are released in response to nutrient ingestion and stimulate pancreatic -cells at their typical postprandial concentrations, especially in the presence of elevated plasma glucose concentrations (2,21). Such properties have been ascribed to GIP (22eC24) and cholecystokinin (shown only in dogs and rodents) (25,26). According to this strict definition, GLP-1 would apparently not fulfill the criteria for an incretin hormone in the setting of the present experiments because it reduced rather than stimulated postprandial insulin secretion. In contrast, when the GLP-1 effects on gastric emptying were counterbalanced, the peptide augmented postprandial insulin secretory responses.
However, a word of caution has to be mentioned regarding the GLP-1 doses used for the present experiments. In fact, GLP-1 plasma levels reached with the infusion of 0.8 pmol · kgeC1 · mineC1 exceeded those typically reached after meal ingestion by 100%. Therefore, even though the present data seem to support the notion that, regardless of its gastric emptying effects, GLP-1 possesses typical incretin properties, they do not allow conclusions about the quantitative importance of endogenous GLP-1 for postprandial gastric emptying and glucose tolerance.
The present experiments also underline the importance of gastric emptying for postprandial glucose control. The nutrient supply into the systemic circulation, which is predominantly regulated by the velocity of gastric emptying, has been demonstrated to be a major determinant of postprandial glucose and insulin concentrations (11,12,27). Notably, at least 35% of the variance in postprandial glucose levels can be explained by gastric emptying (11). In addition, there is evidence that the initial rate of duodenal glucose entry not only dictates the subsequent responses in glycemia and insulin secretion, but also impacts on the postprandial rise in incretin hormone secretion (28). This is consistent with the present observation that the pattern of GIP secretion was delayed during the administration of GLP-1 (Fig. 5), which was effectively reversed by erythromycin. Taken together, these findings emphasize the previous observation that the presence of nutrients in the proximal gut and contact with duodenal and ileal K cells are the major stimulus for the secretion of GIP (29). A considerable number of K cells have recently been identified in these portions of the upper gut (30). The close relationship between intestinal nutrient absorption and incretin secretion is further supported by the recent observation that inhibition of intraduodenal free fatty acid generation, using the lipase inhibitor orlistat, attenuates the secretion of GIP and GLP-1 (31). The GLP-1 induced delay in GIP secretion observed in the present study may also be taken as a confirmation that a deceleration of gastric emptying by GLP-1 really occurred and was physiologically relevant.
Stimulation of insulin secretion after 1 week of erythromycin treatment (1,200 mg/day) has been previously reported by Ueno et al. (32) in patients with type 2 diabetes. However, these effects may have been secondary to the improvement in postprandial glycemia, as shown for other prokinetic drugs (12,33,34). In the present study, insulin secretion was not acutely influenced by the administration of 200 mg erythromycin in the fasting state (Fig. 3). However, erythromycin effects on postprandial insulin secretion were not directly addressed. Because cholinergic stimulation represents an important regulator of islet hormone secretion in humans, the increase in pancreatic polypeptide secretion observed after erythromycin administration may be interpreted as an indication of a direct insulinotropic effect of erythromycin.
One potential limitation of the study may be seen in the fact that metoclopramide, cisapride, and domperidone were administered orally, whereas erythromycin was infused intravenously. Moreover, the doses chosen for the administration of metoclopramide, cisapride, and domperidone were comparably low (35eC37). In fact, to avoid overstimulation of gastric emptying and overall gut motility, we aimed for the lower limits of the respective therapeutic ranges used in the treatment of patients with gastric motility disorders (12). Therefore, it is possible that the plasma levels achieved with the oral administration of metoclopramide, cisapride, and domperidone were not sufficient to counterbalance the effects of GLP-1, although in principle these prokinetic drugs would have the potential to decelerate gastric emptying in the presence of GLP-1.
Alternatively, the discrepant efficacy of the prokinetic drugs used in this study may reflect different mechanisms of action. In this way, domperidone and metoclopramide primarily antagonize central and peripheral dompamine receptor activity, whereas cisapride mainly stimulates serotonin 5-hydroxytryptamine-4 receptors (12). In contrast, the action of erythromycin involves activation of motilin receptors as well as direct stimulation of vagal cholinergic nerves (38,39). GLP-1, on the other hand, decelerates gastric emptying by inhibiting the parasympathetic outflow (40eC42). Consistent with these theoretical considerations, in the present experiments erythromycin induced a pronounced rise in pancreatic polypeptide plasma concentrations, whereas GLP-1 suppressed the release of pancreatic polypeptide (Fig. 5). Therefore, it is possible that the effectiveness of erythromycin in this study reflects a direct interaction of GLP-1 and erythromycin at the level of vagal activation.
The potent deceleration of gastric emptying induced by GLP-1 may have substantial consequences for the treatment of patients with type 2 diabetes with incretin hormones. Given the high prevalence of gastric motility disorders in patients with diabetes, further inhibition of gastric emptying in these patients could potentially induce upper gastrointestinal symptoms, such as nausea, vomiting, or reflux. In fact, nausea and vomiting have been observed in some individuals in response to GLP-1 or its derivatives/analogs (43eC45). Antagonizing the GLP-1eCinduced deceleration of gastric emptying using erythromycin may potentially reduce these side effects.
In conclusion, intravenous erythromycin counterbalances the GLP-1eCinduced deceleration of gastric emptying and unmasks its insulinotropic effect in the postprandial period. The unequal effectiveness of the different prokinetic agents tested suggests an involvement of the vagal nervous system in the mediation of GLP-1 effects of the stomach. Although the consequences resulting from the delay in gastric emptying by GLP-1 under physiological conditions require further investigation, the present data demonstrate that, at pharmacological concentrations, the predominant effect of GLP-1 on postprandial glucose homeostasis is mediated by a delay in gastric emptying rather than by a modulation of endocrine pancreatic secretion.
ACKNOWLEDGMENTS
This study was supported by the Deutsche Forschungsgemeinschaft Grant 203/2-2 and the Danish Medical Research Council.
The authors are indebted to S. Richter, Th. Gottschling, K. Hltner, L. Rabenhj, and L. Albk for their excellent technical assistance. The contribution of Dr. Stefanie Grosser is particularly acknowledged.
GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide 1
REFERENCES
Elrick H, Stimmler L, Hlad CJ, Arai Y: Plasma insulin response to oral and intravenous glucose administration. J Clin Endocrinol Metab24 :1076 eC1082,1964
Creutzfeldt W: The incretin concept today. Diabetologia16 :75 eC85,1979
Creutzfeldt W, Nauck M: Gut hormones and diabetes mellitus. Diabetes Metab Rev8 :149 eC177,1992
Brown JC: A astric inhibitory polypeptide. I: the amino acid composition and the tryptic peptides. Can J Biochem49 :255 eC261,1971
Pederson RA, Brown JC: The insulinotropic action of gastric inhibitory polypeptide in the perfused rat pancreas. Endocrinology99 :780 eC785,1976
Kreymann B, Williams G, Ghatei MA, Bloom SR: Glucagon-like peptide-1 [7eC36]: a physiological incretin in man. Lancet2 :1300 eC1304,1987
Kolligs F, Fehmann HC, Gke R, Gke B: Reduction of the incretin effect in rats by the glucagon-like peptide 1 receptor antagonist exendin (9eC39) amide. Diabetes44 :16 eC19,1995
Edwards CM, Todd JF, Mahmoudi M, Wang Z, Wang RM, Ghatei MA, Bloom SR: Glucagon-like peptide 1 has a physiological role in the control of postprandial glucose in humans: studies with the antagonist exendin 9eC39. Diabetes48 :86 eC93,1999
Nauck MA, Niedereichholz U, Ettler R, Holst JJ, Orskov C, Ritzel R, Schmiegel WH: Glucagon-like peptide 1 inhibition of gastric emptying outweighs its insulinotropic effects in healthy humans. Am J Physiol Endocrinol Metab273 :E981 eCE988,1997
Meier JJ, Gallwitz B, Salmen S, Goetze O, Holst JJ, Schmidt WE, Nauck MA: Normalization of glucose concentrations and deceleration of gastric emptying after solid meals during intravenous glucagon-like peptide 1 in patients with type 2 diabetes. J Clin Endocrinol Metab88 :2719 eC2725,2003
Horowitz M, Edelbroek MA, Wishart JM, Straathof JW: Relationship between oral glucose tolerance and gastric emptying in normal healthy subjects. Diabetologia36 :857 eC862,1993
Horowitz M, O’Donovan D, Jones KL, Feinle C, Rayner CK, Samsom M: Gastric emptying in diabetes: clinical significance and treatment. Diabet Med19 :177 eC194,2002
George JD: New clinical method for measuring the rate of gastric emptying; the double sampling test meal. Gut9 :237 eC242,1968
Hurwitz A: Measuring gastric volume by dilution. Gut22 :85 eC93,1981
rskov C, Holst JJ: Radio-immunoassays for glucagon-like peptides 1 and 2 (GLP-1 and GLP-2). Scand J Clin Lab Invest47 :165 eC174,1987
Holst JJ: Evidence that enteroglucagon (II) is identical with the C-terminal sequence (residues 33eC69) of glicentin. Biochem J207 :381 eC388,1982
Deacon CF, Nauck MA, Meier JJ, He筩king K, Holst JJ: Degradation of endogenous and exogenous gastric inhibitory polypeptide (GIP) in healthy and in type 2 diabetic subjects as revealed using a new assay for the intact peptide. J Clin Endocrinol Metab85 :3575 eC3581,2000
Wettergren A, Wojdemann M, Meisner S, Stadil F, Holst JJ: The inhibitory effect of glucagon-like peptide-1 (GLP-1) 7eC36 amide on gastric acid secretion in humans depends on an intact vagal innervation. Gut40 :597 eC601,1997
Wettergren A, Schjoldager B, Mortensen PE, Myhre J, Christiansen J, Holst JJ: Truncated GLP-1 (proglucagon 78eC107-amide) inhibits gastric and pancreatic functions in man. Dig Dis Sci38 :665 eC673,1993
Kieffer TJ, Habener JF: The glucagon-like peptides. Endocr Rev20 :876 eC913,1999
Unger RH, Eisentraut AM: Entero-insular axis. Arch Intern Med123 :261 eC266,1969
Dupree J, Ross SA, Watson D, Brown JC: Stimulation of insulin secretion by gastric inhibitory polypeptide in man. J Clin Endocrinol Metab37 :826 eC828,1973
Nauck MA, Bartels E, rskov C, Ebert R, Creutzfeldt W: Additive insulinotropic effects of exogenous synthetic human gastric inhibitory polypeptide and glucagon-like peptide-1-(7eC36) amide infused at near-physiological insulinotropic hormone and glucose concentrations. J Clin Endocrinol Metab76 :912 eC917,1993
Vilsbll T, Krarup T, Madsbad S, Holst JJ: Both GLP-1 and GIP are insulinotropic at basal and postprandial glucose levels and contribute nearly equally to the incretin effect of a meal in healthy subjects. Regul Pept114 :115 eC121,2003
Ipp E, Dobbs R, Harris V, Arimura A, Vale W, Unger RU: The effect of gastrin, gastric inhibitory polypeptide, secretion, and the octapeptide of cholecystokinin upon immunoreactive somatostatin release by the perfused canine pancreas. J Clin Invest60 :1216 eC1219,1977
Zawalich WS, Diaz VA, Zawalich KC: Stimulatory effects of cholecystokinin on isolated perifused islets inhibited by potent and specific antagonist L 364718 [corrected]. Diabetes37 :1432 eC1437,1988
Schwartz JG, Guan D, Green GM, Phillips WT: Treatment with an oral proteinase inhibitor slows gastric emptying and acutely reduces glucose and insulin levels after a liquid meal in type II diabetic patients. Diabetes Care17 :255 eC262,1994
O’Donovan DG, Doran S, Feinle-Bisset C, Jones KL, Meyer JH, Wishart JM, Morris HA, Horowitz M: Effect of variations in small intestinal glucose delivery on plasma glucose, insulin, and incretin hormones in healthy subjects and type 2 diabetes. J Clin Endocrinol Metab89 :3431 eC3435,2004
Polak JM, Bloom SR, Kuzio M, Brown JC, Pearse AGE: Cellular localization of gastric inhibitory polypeptide in the duodenum and jejunum. Gut14 :284 eC288,1973
Mortensen K, Petersen LL, Holst JJ, rskov C: GLP-1 and GIP are colocalized in a subset of endocrine cells in the small intestine. Regul Pept114 :189 eC196,2003
Pilichiewicz A, O’Donovan D, Feinle C, Lei Y, Wishart JM, Bryant L, Meyer JH, Horowitz M, Jones KL: Effect of lipase inhibition on gastric emptying of, and the glycemic and incretin responses to, an oil/aqueous drink in type 2 diabetes mellitus. J Clin Endocrinol Metab88 :3829 eC3834,2003
Ueno N, Inui A, Asakawa A, Takao F, Tani S, Komatsu Y, Itoh Z, Kasuga M: Erythromycin improves glycaemic control in patients with type II diabetes mellitus. Diabetologia43 :411 eC415,2000
Stacher G, Schernthaner G, Francesconi M, Kopp HP, Bergmann H, Stacher-Janotta G, Weber U: Cisapride versus placebo for 8 weeks on glycemic control and gastric emptying in insulin-dependent diabetes: a double blind cross-over trial. J Clin Endocrinol Metab84 :2357 eC2362,1999
Thompson DG, Wingate DL, Thomas M, Harrison D: Gastric emptying as a determinant of the oral glucose tolerance test. Gastroenterology82 :51 eC55,1982
Schade RR, Dugas MC, Lhotsky DM, Gavaler JS, Van Thiel DH: Effect of metoclopramide on gastric liquid emptying in patients with diabetic gastroparesis. Dig Dis Sci30 :10 eC15,1985
Horowitz M, Harding PE, Chatterton BE, Collins PJ, Shearman DJ: Acute and chronic effects of domperidone on gastric emptying in diabetic autonomic neuropathy. Dig Dis Sci30 :1 eC9,1985
McCallum RW, Prakash C, Campoli-Richards DM, Goa KL: Cisapride. A preliminary review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use as a prokinetic agent in gastrointestinal motility disorders. Drugs36 :652 eC681,1988
Janssens J, Peeters TL, Vantrappen G, Tack J, Urbain JL, De Roo M, Muls E, Bouillon R: Improvement of gastric emptying in diabetic gastroparesis by erythromycin: preliminary studies. N Engl J Med322 :1028 eC1031,1990
Feighner SD, Tan CP, McKee KK, Palyha OC, Hreniuk DL, Pong SS, Austin CP, Figueroa D, MacNeil D, Cascieri MA, Nargund R, Bakshi R, Abramovitz M, Stocco R, Kargman S, O’Neill G, Van Der Ploeg LH, Evans J, Patchett AA, Smith RG, Howard AD: Receptor for motilin identified in the human gastrointestinal system. Science284 :2184 eC2188,1999
Imeryuz N, Yegen BC, Bozkurt A, Coskun T, Villanueva Penacarrillo ML, Ulusoy NB: Glucagon-like peptide-1 inhibits gastric emptying via vagal zfferent-mediated central mechanisms. Am J Physiol Gastroenterol Liver273 :G920 eCG927,1997
Wettergren A, Wojdemann M, Holst JJ: Glucagon-like peptide-1 inhibits gastropancreatic function by inhibiting central parasympathetic outflow. Am J Physiol275 :G984 eCG992,1998
Schirra J, Leicht P, Hildebrand P, Beglinger C, Arnold R, Gke B, Katschinski M: Mechanisms of the antidiabetic action of subcutaneous glucagon-like peptide-1 (7eC36)amide in non-insulin dependent diabetes mellitus. J Endocrinol156 :177 eC186,1998
Ritzel R, rskov C, Holst JJ, Nauck MA: Pharmacokinetic, insulinotropic, and glucagonostatic properties of GLP-1 [7eC36 amide] after subcutaneous injection in healthy volunteers: dose-response-relationships. Diabetologia38 :720 eC725,1995
Fineman MS, Bicsak TA, Shen LZ, Taylor K, Gaines E, Varns A, Kim D, Baron AD: Effect on glycemic control of exenatide (synthetic exendin-4) additive to existing metformin and/or sulfonylurea treatment in patients with type 2 diabetes. Diabetes Care26 :2370 eC2377,2003
Meier JJ, Nauck MA: The potential role of glucagon-like peptide 1 in diabetes. Curr Opin Investig Drugs5 :402 eC410,2004(Juris J. Meier, Guido Kem)
2 Larry Hillblom Islet Research Center, UCLA School of Medicine, Los Angeles, California
3 Department of Medical Physiology, Panum Institute, University of Copenhagen, Copenhagen, Denmark
4 Diabeteszentrum, Bad Lauterberg im Harz, Germany
ABSTRACT
Glucagon-like peptide 1 (GLP-1) has been proposed to act as an incretin hormone due to its ability to enhance glucose-stimulated insulin secretion. Because GLP-1 also decelerates gastric emptying, it physiologically reduces rather than augments postprandial insulin secretory responses. Therefore, we aimed to antagonize the deceleration of gastric emptying by GLP-1 to study its effects on insulin secretion after a meal. Nine healthy male volunteers (age 25 ± 4 years, BMI 25.0 ± 4.9 kg/m2) were studied with an infusion of GLP-1 (0.8 pmol · kgeC1 · mineC1 from eC30 to 240 min) or placebo. On separate occasions, the prokinetic drugs metoclopramide (10 mg), domperidone (10 mg), cisapride (10 mg, all at eC30 min per oral), or erythromycin (200 mg intravenously from eC30 to eC15 min) were administered in addition to GLP-1. A liquid test meal (50 g sucrose and 8% mixed amino acids in 400 ml) was administered at 0 min. Capillary and venous blood samples were drawn for the determination of glucose (glucose oxidase), insulin, C-peptide, GLP-1, glucagon, gastric inhibitory polypeptide (GIP), and pancreatic polypeptide (specific immunoassays). Gastric emptying was assessed by the phenol red dilution technique. Statistical analyses were performed using repeated-measures ANOVA and Duncan’s post hoc test. GLP-1 significantly decelerated the velocity of gastric emptying (P < 0.001). This was completely counterbalanced by erythromycin, whereas the other prokinetic drugs used had no effect. Postprandial glucose concentrations were lowered by GLP-1 (P < 0.001 vs. placebo), but this effect was partially reversed by erythromycin (P < 0.05). Insulin secretory responses to the meal were lower during GLP-1 administration (P < 0.05 vs. placebo). However, when erythromycin was added to GLP-1, insulin concentrations were similar to those in placebo experiments. The suppression of meal-related increments in glucagon secretion by GLP-1 was reversed by erythromycin (P < 0.001). The time course of GIP secretion was delayed during GLP-1 administration (P < 0.05), but when erythromycin was added, the pattern was similar to placebo experiments. GLP-1 administration led to a reduction in pancreatic polypeptide plasma concentrations (P < 0.05). In contrast, pancreatic polypeptide levels were markedly increased by erythromycin (P < 0.001). Intravenous erythromycin counteracts the deceleration of gastric emptying caused by GLP-1, probably by interacting with the parasympathetic nervous system (pancreatic polypeptide responses). Despite augmented rises in insulin secretion, the glucose-lowering effect of GLP-1 is markedly reduced when the deceleration of gastric emptying is antagonized, illustrating the importance of this facet of the multiple antidiabetic actions of GLP-1.
Insulin secretion after meal ingestion is stimulated not only by the rise in glycemia, but also by the secretion of peptide hormones ("incretins") from the gut (1,2). The postprandial enhancement of insulin secretion by gut-derived factors is called the incretin effect (2,3). The first incretin hormone identified was gastric inhibitory polypeptide (GIP), which is also referred to as glucose-dependent insulinotropic polypeptide (4,5). An incretin role has also been proposed for the proglucagon-derived peptide glucagon-like peptide 1 (GLP-1) (6). This was based on its ability to enhance insulin secretion and to suppress glucagon release during an intravenous glucose infusion in healthy human volunteers (6). Moreover, the GLP-1 receptor antagonist exendin [9eC39] blocked the insulin secretory response after intraduodenal administration of glucose in rats (7). Later, Edwards et al. (8) demonstrated a marked deterioration of oral glucose tolerance in healthy subjects during the administration of exendin [9eC39]. However, when GLP-1 was administered during meal ingestion, a dose-dependent deceleration of gastric emptying as well as a reduction in postprandial insulin secretion was found in healthy volunteers as well as in patients with type 2 diabetes (9,10). Because the velocity of gastric emptying is a major determinant of postprandial glycemia and insulin secretion (11,12), it is difficult to ascertain an insulinotropic effect of GLP-1 after meal ingestion. One way to counterbalance the GLP-1 effects on gastric emptying would be to administer prokinetic drugs before meal ingestion. Therefore, it was the aim of the present experiments to antagonize the decelerating effects of GLP-1 on gastric emptying using a panel of prokinetic drugs with different modes of action (metoclopramide, domperidone, cisapride, and erythromycin) to unmask its insulinotropic effects in the postprandial state.
RESEARCH DESIGN AND METHODS
The study protocol was approved by the ethics committee of the medical faculty of Ruhr University (Bochum, Germany) before the study. Written informed consent was obtained from all participants.
Nine healthy male volunteers were studied. They were 25 ± 4 years old, 181 ± 4 cm tall, and weighed 82 ± 17 kg. Their BMI was 25.0 ± 4.9 kg/m2. All had a normal oral glucose tolerance according to World Health Organization criteria (fasting glucose 5.1 ± 0.4 mmol/l, 120-min value 5.0 ± 1.1 mmol/l). None had a family history of diabetes or a personal history of gastrointestinal disorders. Blood cell counts, serum transaminases, creatinine values, triglyceride, cholesterol, and HDL cholesterol concentrations were in the normal range.
The study was performed in a single-blinded fashion with all participants being studied in random order on six occasions.
1) A liquid mixed meal (50 g sucrose plus amino acids, 400 ml Aminosteril Hepa 8%; Fresenius AG, Bad Homburg, Germany) was instilled intragastrically at time 0. Placebo (0.9% NaCl with 1% human serum albumin (Behring AG, Marburg, Germany) was infused intravenously from eC30 to 240 min.
2) A liquid test meal was administered as described in 1. In addition, a continuous intravenous administration of GLP-1 at a dose of 0.8 pmol · kgeC1 · mineC1 was started 30 min before the meal (at eC30 min) and continued until 240 min.
3) In addition to the administration of the meal and GLP-1 (as described in 1 and 2), metoclopramide (Paspertin; 10 mg/10 ml) was administered orally at eC30 min.
4) In addition to the administration of the meal and GLP-1 (as described in 1 and 2), domperidone (Motilium Saft; 10 mg/10 ml) was administered orally at eC30 min.
5) In addition to the administration of the meal and GLP-1 (as described in 1 and 2), cisapride (Propulsin Saft; 10 mg/10 ml) was administered orally at eC30 min.
6) In addition to the administration of the meal and GLP-1 (as described in 1 and 2), erythromycin (Erycinum; 200 mg/100 ml) was administered intravenously between eC30 and eC15 min.
An interval of at least 10 days was kept between the experiments to exclude carryover effects.
Peptides.
Synthetic GLP-1 [7eC36 amide] was purchased from Saxon Biochemicals (Hannover, Germnay). The lot number of GLP-1 [7eC36 amide] (pharmaceutical grade) was PGAS 242, FGLP7369301 A, net peptide content 88%. The peptide was dissolved in 0.9% NaCl and 1% human serum albumin (HSA Behring, Marburg, Germany), filtered through 0.2 e nitrocellulose filters (Sartorius, Gttingen, Germany), and stored frozen at eC30°C as previously described (9). High-performance liquid chromatography profiles (provided by the manufacturer) showed that the preparation was >99% pure (single peak with appropriate standards). Samples were analyzed for bacterial growth (standard culture techniques) and for pyrogens (limulus amebocyte lysate endo-LAL; Chromogenix AB, Mlndal, Sweden). No bacterial contamination was detected. Endotoxin concentrations in the GLP-1 stock solutions were <0.03 EU/ml.
Experimental procedures.
The tests were performed in the morning after an overnight fast. Two forearm veins were punctured with a Teflon cannula (Moskito 123, 18 gauge; Vygon, Aachen, Germany) and kept patent using 0.9% NaCl (for blood sampling and for GLP-1/placebo administration).
After drawing basal blood specimens, at eC30 min an intravenous infusion of GLP-1 [7eC36 amide] or placebo (0.9% NaCl containing 1% human serum albumin) was started and continued for 270 min. Blood was drawn at the time points eC45, eC30, eC15, 0, 15, 30, 45, 60, 120, 180, and 240 min, and plasma glucose was determined immediately.
Before the study, a nasogastric tube (Freka-Ernhrungssonde, 120 cm, CH12; Fresenius AG, Bad Homburg, Germany) was placed and tape-fixed with the tip 55 cm from the nostrils. Gastric juice was aspirated and an acidic pH was ascertained using pH-sensitive Lackmus paper. The gastric lumen was washed with 100 ml water (37°C). The position of the tube was, if necessary, adjusted to allow near-complete aspiration of instilled fluid. The subjects were in a semirecumbent position with the upper half of the body 45 degrees upright. At 0 min, 400 ml (total volume) of the liquid test meal was instilled into the stomach. It was composed of 50 g sucrose dissolved in 400 ml Aminosteril Hepa 8%. This composition of the meal was chosen because the solution had to be clear for the photometric measurement of phenol red (measurement of gastric emptying, see below) and should be similar in caloric and nutrient content to a normal mixed meal. The meal contained 32 g mixed amino acids (131 kcal = 40%) and 50 g sucrose (196 kcal = 60%), with a total energy content of 327 kcal (energy density 0.82 kcal/ml).
Blood specimens.
Blood was drawn into chilled tubes containing EDTA and aprotinin (Trasylol; 20,000 KIU/ml, 200 e蘬/10 ml blood; Bayer AG, Leverkusen, Germany) and kept on ice. A sample (100 e蘬) was stored in NaF (Microvette CB 300; Sarstedt, Ne筸brecht, Germany) for the measurement of glucose. After centrifugation at 4°C, plasma for hormone analyses was kept frozen at eC30°C.
Gastric emptying.
The velocity of gastric emptying was measured as described (9) by a double-sampling dye dilution technique using phenol red (Merck AG, Darmstadt, Germany) according to George (13), with modifications introduced to reduce measurement error by Hurwitz (14). This technique allows the repeated determination of gastric emptying under different experimental conditions. Briefly, at all time points chosen to measure gastric volume, a known amount of the nonabsorbable dye phenol red was added to the translucent liquid test meal in a volume of 5eC15 ml. After thorough mixing with gastric contents for 2 min, a gastric sample was drawn and the resulting step-up in phenol red concentrations was determined photometrically. The volume of gastric contents was determined from the volume of distribution of phenol red. As the experiments proceeded, increasing amounts of phenol red were used to ensure measurability of optical density increments in the presence of previously instilled phenol red. Gastric contents were determined at 0, 30, 60, 90, 120, 180, and 240 min and were expressed as the percentage of the initial volume of 400 ml at 0 min.
Laboratory determinations.
Glucose was measured using a glucose oxidase method with a Glucose Analyzer 2 (Beckman Instruments, Munich, Germany). Insulin was measured using an insulin microparticle enzyme immunoassay (IMx Insulin; Abbott Laboratories, Wiesbaden, Germany). Intra-assay coefficients of variation were 4%. C-peptide was measured using C-peptide-antibodyeCcoated microtiter wells (C-peptide MTPL EIA; DRG Instruments, Marburg, Germany). Intra-assay coefficients of variation were 6%. Human insulin and C-peptide were used as standards.
Immunoreactive GLP-1 was determined in ethanol-extracted plasma as previously described (15), using antiserum 89390 (final dilution 1:150 000) for the measurement of GLP-1 [7eC36 amide] and synthetic GLP-1 [7eC36 amide] for tracer preparation and as standard. The experimental detection limit (2 SD over samples not containing GLP-1 [7eC36 amide]) was <5 pmol/l. Antiserum 89390 binds to the amidated carboxyl terminus of GLP-1 [7eC36 amide] and therefore measures the sum of intact and degraded GLP-1 [9eC36 amide] in plasma. Intra-assay coefficients of variation were 8%.
Pancreatic glucagon was assayed in ethanol-extracted plasma using antibody 4305 as previously described (16). The detection limit was 1 pmol/l and the intra-assay coefficient of variation was <6% in the working range.
Plasma gastric inhibitory polypeptide (GIP) was determined by radioimmunoassay using antiserum R65 as previously described (17). This assay measures the sum of both intact [1eC42] and degraded GIP [3eC42] in plasma.
Plasma pancreatic polypeptide was determined by radioimmunoassay. A detailed description of the assay is given by Wettergren et al. (18).
Phenol red in gastric contents was assayed photometrically after filtration through filter paper (100 e蘬 in 2 ml Na2HPO4/NaH2PO4 buffer, 0.6 mol/l/l, pH 8.0) at a wavelength of 546 nm and read against a standard curve (phenol red in phosphate buffer) as previously described (9). Each patient’s set of plasma samples was assayed at the same time to avoid errors due to interassay variation.
Statistical analysis.
Results are reported as means ± SE. All statistical calculations were carried out by paired repeated-measures ANOVA using Statistica (Statsoft Europe, Hamburg, Germany). If a significant interaction of treatment and time was documented (P < 0.05), values at single time points were compared by one-way ANOVA and Duncan’s post hoc test (paired analyses). A two-sided P value <0.05 was taken to indicate significant differences.
RESULTS
GLP-1 plasma concentrations.
After the liquid test meal, GLP-1 plasma concentrations increased from 5.3 ± 0.8 pmol/l to peak concentrations of 10.3 ± 1.6 pmol/l at 15 min during placebo infusion (Fig. 1; P = 0.0009). GLP-1 plasma concentrations were similar in all experiments with GLP-1 infusion, reaching steady-state levels at 0 min (Fig. 1). When erythromycin was administered before the test meal, significantly higher GLP-1 plasma concentrations were observed after 30 min, compared with the experiments with the administration of GLP-1 alone (P = 0.0035) (Fig. 1).
Gastric emptying.
After 240 min, the stomach had almost completely emptied in all groups (Fig. 2). During the infusion of GLP-1 alone, the velocity of gastric emptying was significantly decelerated compared with placebo administration (Fig. 2) (P < 0.0001). With erythromycin, the decelerating effect of GLP-1 was completely counterbalanced. Accordingly, no differences occurred in the pattern of gastric emptying between the experiments with the administration of placebo and with the combined administration of GLP-1 and erythromycin (Fig. 2). In contrast, metoclopramide, domperidone, and cisapride had no significant effects on gastric emptying during GLP-1 infusion (Fig. 2).
Plasma glucose.
After the liquid test meal, plasma glucose concentrations increased significantly in the placebo group (P < 0.0001). Intravenous infusion of GLP-1 lowered fasting glucose concentrations even before instillation of the test meal (P < 0.05) and the postprandial rise in glycemia was less pronounced than with placebo. The patterns of glucose concentration were not different in the experiments with the administration of GLP-1 alone or in combination with metoclopramide, domperidone, or cisapride (details not shown). When erythromycin was administered in addition to GLP-1, plasma glucose concentrations at 30 min were higher that with GLP-1 alone but lower than during placebo administration (P < 0.05) (Fig. 3). There were no significant differences in glycemia between the experiments with GLP-1 alone and in combination with erythromycin at any other time point.
Insulin secretory response.
In the fasting state, insulin secretion was not different between the experiments with the administration of placebo and of GLP-1, alone or in combination with erythromycin (Fig. 3). After the liquid test meal, insulin secretory responses were significantly higher during placebo administration than during the infusion of GLP-1 (P < 0.05) (Fig. 3). When erythromycin was administered in addition to GLP-1, the overall pattern of insulin secretion and the integrated incremental plasma concentrations of insulin and C-peptide were more similar to placebo experiments (Fig. 3). However, there were still differences in insulin and C-peptide levels at time point 15 min after the test meal between the experiments with the combined administration of GLP-1 and erythromycin and with placebo. In contrast to the effect of erythromycin, none of the other prokinetic drugs used had an influence on postprandial insulin release during GLP-1 infusion (details not shown).
When the plasma insulin levels immediately after meal ingestion (0eC30 min) were expressed in relation to the corresponding glucose concentrations, a shift of the glucose threshold required for the stimulation of insulin secretion became apparent in the experiments with GLP-1 infusion (Fig. 4). However, the glucose responsiveness of insulin secretion was not affected by erythromycin administration (Fig. 4).
Glucagon secretion.
Plasma glucagon concentrations before the test meal were lowered during GLP-1 infusion (Fig. 1). After the instillation of the liquid meal, plasma glucagon concentrations significantly increased during all experiments (P < 0.0001), reaching higher levels during placebo administration than with GLP-1 (Fig. 1). Again, erythromycin almost completely reversed the changes in glucagon secretion by GLP-1, resulting in a similar pattern of glucagon concentrations as during the placebo experiments (Fig. 1). No changes in glucagon secretion were found after the administration of metoclopramide, domperidone, or cisapride (details not shown).
GIP secretion.
GIP plasma concentrations significantly increased after the liquid test meal during all experiments (Fig. 5) (P < 0.0001). The total amount of GIP secreted after the meal was similar in all experiments (P = 0.38), but the time course of GIP secretion was delayed during GLP-1 infusion (P < 0.05 vs. placebo). These changes were almost completely reversed by erythromycin administration (Fig. 5). Accordingly, peak concentrations of GIP were reached after 60 min during placebo administration, after 180 min during GLP-1 infusion, and after 30 min during the combined administration of erythromycin and GLP-1 (Fig. 5). GIP secretion was not affected by the other prokinetic drugs used (details not shown).
Pancreatic polypeptide secretion.
The secretion of pancreatic polypeptide was significantly suppressed by GLP-1 infusion (Fig. 5). Erythromycin administration led to a marked increase in postprandial pancreatic polypeptide concentrations compared with the placebo experiments (P < 0.0001). The secretion of pancreatic polypeptide was not influenced by metoclopramide, domperidone, or cisapride (details not shown).
DISCUSSION
Because GLP-1 reduces postprandial glycemia not only via endocrine pancreatic secretion (6) but also by decelerating the velocity of gastric emptying (9,10,19,20), it was the aim of this study to assess GLP-1eCinduced insulin secretion after a meal, independent of changes in gastric emptying. Although insulin secretion is typically enhanced by GLP-1 under fasting conditions, a dose-dependent reduction rather than stimulation of postprandial insulin secretion has previously been observed during the administration of GLP-1 in healthy subjects and in patients with type 2 diabetes (9,10). Similar to those studies, a considerably smaller amount of insulin was secreted after the meal during GLP-1 administration in the present experiments. However, when changes in gastric emptying were reversed by erythromycin, insulin secretion was increased to similar levels as in the placebo experiments, although at lower levels of glycemia (Fig. 3). This indicates that the glucose concentration threshold required to stimulate insulin secretion was shifted downward due to the administration of GLP-1. Indeed, when plasma insulin concentrations are expressed in relation to the corresponding glucose levels (Fig. 4), it becomes clear that during GLP-1 administration insulin secretion was increased at lower levels of glycemia compared with placebo. Therefore, in the postprandial state GLP-1 possesses insulinotropic properties independent of changes in the rate of gastric emptying in healthy individuals.
Interestingly, despite the similar patterns of insulin, C-peptide, and glucagon concentrations, postprandial plasma glucose levels were lower in the GLP-1 plus erythromycin experiments than in the placebo experiments. Most likely, this was based on the glucose-lowering effect of GLP-1 before instillation of the test meal. In support of this, plasma glucose concentrations were already lowered by 13 mg/dl at the time of meal administration in the GLP-1 experiments. If these initial differences in glycemia are taken into consideration, the glycemic profiles appear rather similar between the time points of 30 and 60 min in the experiments with GLP-1 plus erythromycin and with placebo. However, there is still a discrepancy regarding the plasma levels of glucose, insulin, C-peptide, and glucagon at the 15-min time point after meal administration between these experiments. Because GIP plasma levels were already increased at that time point in the experiments with GLP-1 and erythromycin, differences in the rate of gastric emptying (which was not determined at this time point) or intestinal nutrient absorption are unlikely to explain this discrepancy. Thus, it is possible that erythromycin had an independent effect on glucose tolerance beyond its effects on gastric emptying.
The present data give rise to a reconsideration of the role of GLP-1 as an incretin hormone. By definition, incretin hormones are released in response to nutrient ingestion and stimulate pancreatic -cells at their typical postprandial concentrations, especially in the presence of elevated plasma glucose concentrations (2,21). Such properties have been ascribed to GIP (22eC24) and cholecystokinin (shown only in dogs and rodents) (25,26). According to this strict definition, GLP-1 would apparently not fulfill the criteria for an incretin hormone in the setting of the present experiments because it reduced rather than stimulated postprandial insulin secretion. In contrast, when the GLP-1 effects on gastric emptying were counterbalanced, the peptide augmented postprandial insulin secretory responses.
However, a word of caution has to be mentioned regarding the GLP-1 doses used for the present experiments. In fact, GLP-1 plasma levels reached with the infusion of 0.8 pmol · kgeC1 · mineC1 exceeded those typically reached after meal ingestion by 100%. Therefore, even though the present data seem to support the notion that, regardless of its gastric emptying effects, GLP-1 possesses typical incretin properties, they do not allow conclusions about the quantitative importance of endogenous GLP-1 for postprandial gastric emptying and glucose tolerance.
The present experiments also underline the importance of gastric emptying for postprandial glucose control. The nutrient supply into the systemic circulation, which is predominantly regulated by the velocity of gastric emptying, has been demonstrated to be a major determinant of postprandial glucose and insulin concentrations (11,12,27). Notably, at least 35% of the variance in postprandial glucose levels can be explained by gastric emptying (11). In addition, there is evidence that the initial rate of duodenal glucose entry not only dictates the subsequent responses in glycemia and insulin secretion, but also impacts on the postprandial rise in incretin hormone secretion (28). This is consistent with the present observation that the pattern of GIP secretion was delayed during the administration of GLP-1 (Fig. 5), which was effectively reversed by erythromycin. Taken together, these findings emphasize the previous observation that the presence of nutrients in the proximal gut and contact with duodenal and ileal K cells are the major stimulus for the secretion of GIP (29). A considerable number of K cells have recently been identified in these portions of the upper gut (30). The close relationship between intestinal nutrient absorption and incretin secretion is further supported by the recent observation that inhibition of intraduodenal free fatty acid generation, using the lipase inhibitor orlistat, attenuates the secretion of GIP and GLP-1 (31). The GLP-1 induced delay in GIP secretion observed in the present study may also be taken as a confirmation that a deceleration of gastric emptying by GLP-1 really occurred and was physiologically relevant.
Stimulation of insulin secretion after 1 week of erythromycin treatment (1,200 mg/day) has been previously reported by Ueno et al. (32) in patients with type 2 diabetes. However, these effects may have been secondary to the improvement in postprandial glycemia, as shown for other prokinetic drugs (12,33,34). In the present study, insulin secretion was not acutely influenced by the administration of 200 mg erythromycin in the fasting state (Fig. 3). However, erythromycin effects on postprandial insulin secretion were not directly addressed. Because cholinergic stimulation represents an important regulator of islet hormone secretion in humans, the increase in pancreatic polypeptide secretion observed after erythromycin administration may be interpreted as an indication of a direct insulinotropic effect of erythromycin.
One potential limitation of the study may be seen in the fact that metoclopramide, cisapride, and domperidone were administered orally, whereas erythromycin was infused intravenously. Moreover, the doses chosen for the administration of metoclopramide, cisapride, and domperidone were comparably low (35eC37). In fact, to avoid overstimulation of gastric emptying and overall gut motility, we aimed for the lower limits of the respective therapeutic ranges used in the treatment of patients with gastric motility disorders (12). Therefore, it is possible that the plasma levels achieved with the oral administration of metoclopramide, cisapride, and domperidone were not sufficient to counterbalance the effects of GLP-1, although in principle these prokinetic drugs would have the potential to decelerate gastric emptying in the presence of GLP-1.
Alternatively, the discrepant efficacy of the prokinetic drugs used in this study may reflect different mechanisms of action. In this way, domperidone and metoclopramide primarily antagonize central and peripheral dompamine receptor activity, whereas cisapride mainly stimulates serotonin 5-hydroxytryptamine-4 receptors (12). In contrast, the action of erythromycin involves activation of motilin receptors as well as direct stimulation of vagal cholinergic nerves (38,39). GLP-1, on the other hand, decelerates gastric emptying by inhibiting the parasympathetic outflow (40eC42). Consistent with these theoretical considerations, in the present experiments erythromycin induced a pronounced rise in pancreatic polypeptide plasma concentrations, whereas GLP-1 suppressed the release of pancreatic polypeptide (Fig. 5). Therefore, it is possible that the effectiveness of erythromycin in this study reflects a direct interaction of GLP-1 and erythromycin at the level of vagal activation.
The potent deceleration of gastric emptying induced by GLP-1 may have substantial consequences for the treatment of patients with type 2 diabetes with incretin hormones. Given the high prevalence of gastric motility disorders in patients with diabetes, further inhibition of gastric emptying in these patients could potentially induce upper gastrointestinal symptoms, such as nausea, vomiting, or reflux. In fact, nausea and vomiting have been observed in some individuals in response to GLP-1 or its derivatives/analogs (43eC45). Antagonizing the GLP-1eCinduced deceleration of gastric emptying using erythromycin may potentially reduce these side effects.
In conclusion, intravenous erythromycin counterbalances the GLP-1eCinduced deceleration of gastric emptying and unmasks its insulinotropic effect in the postprandial period. The unequal effectiveness of the different prokinetic agents tested suggests an involvement of the vagal nervous system in the mediation of GLP-1 effects of the stomach. Although the consequences resulting from the delay in gastric emptying by GLP-1 under physiological conditions require further investigation, the present data demonstrate that, at pharmacological concentrations, the predominant effect of GLP-1 on postprandial glucose homeostasis is mediated by a delay in gastric emptying rather than by a modulation of endocrine pancreatic secretion.
ACKNOWLEDGMENTS
This study was supported by the Deutsche Forschungsgemeinschaft Grant 203/2-2 and the Danish Medical Research Council.
The authors are indebted to S. Richter, Th. Gottschling, K. Hltner, L. Rabenhj, and L. Albk for their excellent technical assistance. The contribution of Dr. Stefanie Grosser is particularly acknowledged.
GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide 1
REFERENCES
Elrick H, Stimmler L, Hlad CJ, Arai Y: Plasma insulin response to oral and intravenous glucose administration. J Clin Endocrinol Metab24 :1076 eC1082,1964
Creutzfeldt W: The incretin concept today. Diabetologia16 :75 eC85,1979
Creutzfeldt W, Nauck M: Gut hormones and diabetes mellitus. Diabetes Metab Rev8 :149 eC177,1992
Brown JC: A astric inhibitory polypeptide. I: the amino acid composition and the tryptic peptides. Can J Biochem49 :255 eC261,1971
Pederson RA, Brown JC: The insulinotropic action of gastric inhibitory polypeptide in the perfused rat pancreas. Endocrinology99 :780 eC785,1976
Kreymann B, Williams G, Ghatei MA, Bloom SR: Glucagon-like peptide-1 [7eC36]: a physiological incretin in man. Lancet2 :1300 eC1304,1987
Kolligs F, Fehmann HC, Gke R, Gke B: Reduction of the incretin effect in rats by the glucagon-like peptide 1 receptor antagonist exendin (9eC39) amide. Diabetes44 :16 eC19,1995
Edwards CM, Todd JF, Mahmoudi M, Wang Z, Wang RM, Ghatei MA, Bloom SR: Glucagon-like peptide 1 has a physiological role in the control of postprandial glucose in humans: studies with the antagonist exendin 9eC39. Diabetes48 :86 eC93,1999
Nauck MA, Niedereichholz U, Ettler R, Holst JJ, Orskov C, Ritzel R, Schmiegel WH: Glucagon-like peptide 1 inhibition of gastric emptying outweighs its insulinotropic effects in healthy humans. Am J Physiol Endocrinol Metab273 :E981 eCE988,1997
Meier JJ, Gallwitz B, Salmen S, Goetze O, Holst JJ, Schmidt WE, Nauck MA: Normalization of glucose concentrations and deceleration of gastric emptying after solid meals during intravenous glucagon-like peptide 1 in patients with type 2 diabetes. J Clin Endocrinol Metab88 :2719 eC2725,2003
Horowitz M, Edelbroek MA, Wishart JM, Straathof JW: Relationship between oral glucose tolerance and gastric emptying in normal healthy subjects. Diabetologia36 :857 eC862,1993
Horowitz M, O’Donovan D, Jones KL, Feinle C, Rayner CK, Samsom M: Gastric emptying in diabetes: clinical significance and treatment. Diabet Med19 :177 eC194,2002
George JD: New clinical method for measuring the rate of gastric emptying; the double sampling test meal. Gut9 :237 eC242,1968
Hurwitz A: Measuring gastric volume by dilution. Gut22 :85 eC93,1981
rskov C, Holst JJ: Radio-immunoassays for glucagon-like peptides 1 and 2 (GLP-1 and GLP-2). Scand J Clin Lab Invest47 :165 eC174,1987
Holst JJ: Evidence that enteroglucagon (II) is identical with the C-terminal sequence (residues 33eC69) of glicentin. Biochem J207 :381 eC388,1982
Deacon CF, Nauck MA, Meier JJ, He筩king K, Holst JJ: Degradation of endogenous and exogenous gastric inhibitory polypeptide (GIP) in healthy and in type 2 diabetic subjects as revealed using a new assay for the intact peptide. J Clin Endocrinol Metab85 :3575 eC3581,2000
Wettergren A, Wojdemann M, Meisner S, Stadil F, Holst JJ: The inhibitory effect of glucagon-like peptide-1 (GLP-1) 7eC36 amide on gastric acid secretion in humans depends on an intact vagal innervation. Gut40 :597 eC601,1997
Wettergren A, Schjoldager B, Mortensen PE, Myhre J, Christiansen J, Holst JJ: Truncated GLP-1 (proglucagon 78eC107-amide) inhibits gastric and pancreatic functions in man. Dig Dis Sci38 :665 eC673,1993
Kieffer TJ, Habener JF: The glucagon-like peptides. Endocr Rev20 :876 eC913,1999
Unger RH, Eisentraut AM: Entero-insular axis. Arch Intern Med123 :261 eC266,1969
Dupree J, Ross SA, Watson D, Brown JC: Stimulation of insulin secretion by gastric inhibitory polypeptide in man. J Clin Endocrinol Metab37 :826 eC828,1973
Nauck MA, Bartels E, rskov C, Ebert R, Creutzfeldt W: Additive insulinotropic effects of exogenous synthetic human gastric inhibitory polypeptide and glucagon-like peptide-1-(7eC36) amide infused at near-physiological insulinotropic hormone and glucose concentrations. J Clin Endocrinol Metab76 :912 eC917,1993
Vilsbll T, Krarup T, Madsbad S, Holst JJ: Both GLP-1 and GIP are insulinotropic at basal and postprandial glucose levels and contribute nearly equally to the incretin effect of a meal in healthy subjects. Regul Pept114 :115 eC121,2003
Ipp E, Dobbs R, Harris V, Arimura A, Vale W, Unger RU: The effect of gastrin, gastric inhibitory polypeptide, secretion, and the octapeptide of cholecystokinin upon immunoreactive somatostatin release by the perfused canine pancreas. J Clin Invest60 :1216 eC1219,1977
Zawalich WS, Diaz VA, Zawalich KC: Stimulatory effects of cholecystokinin on isolated perifused islets inhibited by potent and specific antagonist L 364718 [corrected]. Diabetes37 :1432 eC1437,1988
Schwartz JG, Guan D, Green GM, Phillips WT: Treatment with an oral proteinase inhibitor slows gastric emptying and acutely reduces glucose and insulin levels after a liquid meal in type II diabetic patients. Diabetes Care17 :255 eC262,1994
O’Donovan DG, Doran S, Feinle-Bisset C, Jones KL, Meyer JH, Wishart JM, Morris HA, Horowitz M: Effect of variations in small intestinal glucose delivery on plasma glucose, insulin, and incretin hormones in healthy subjects and type 2 diabetes. J Clin Endocrinol Metab89 :3431 eC3435,2004
Polak JM, Bloom SR, Kuzio M, Brown JC, Pearse AGE: Cellular localization of gastric inhibitory polypeptide in the duodenum and jejunum. Gut14 :284 eC288,1973
Mortensen K, Petersen LL, Holst JJ, rskov C: GLP-1 and GIP are colocalized in a subset of endocrine cells in the small intestine. Regul Pept114 :189 eC196,2003
Pilichiewicz A, O’Donovan D, Feinle C, Lei Y, Wishart JM, Bryant L, Meyer JH, Horowitz M, Jones KL: Effect of lipase inhibition on gastric emptying of, and the glycemic and incretin responses to, an oil/aqueous drink in type 2 diabetes mellitus. J Clin Endocrinol Metab88 :3829 eC3834,2003
Ueno N, Inui A, Asakawa A, Takao F, Tani S, Komatsu Y, Itoh Z, Kasuga M: Erythromycin improves glycaemic control in patients with type II diabetes mellitus. Diabetologia43 :411 eC415,2000
Stacher G, Schernthaner G, Francesconi M, Kopp HP, Bergmann H, Stacher-Janotta G, Weber U: Cisapride versus placebo for 8 weeks on glycemic control and gastric emptying in insulin-dependent diabetes: a double blind cross-over trial. J Clin Endocrinol Metab84 :2357 eC2362,1999
Thompson DG, Wingate DL, Thomas M, Harrison D: Gastric emptying as a determinant of the oral glucose tolerance test. Gastroenterology82 :51 eC55,1982
Schade RR, Dugas MC, Lhotsky DM, Gavaler JS, Van Thiel DH: Effect of metoclopramide on gastric liquid emptying in patients with diabetic gastroparesis. Dig Dis Sci30 :10 eC15,1985
Horowitz M, Harding PE, Chatterton BE, Collins PJ, Shearman DJ: Acute and chronic effects of domperidone on gastric emptying in diabetic autonomic neuropathy. Dig Dis Sci30 :1 eC9,1985
McCallum RW, Prakash C, Campoli-Richards DM, Goa KL: Cisapride. A preliminary review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use as a prokinetic agent in gastrointestinal motility disorders. Drugs36 :652 eC681,1988
Janssens J, Peeters TL, Vantrappen G, Tack J, Urbain JL, De Roo M, Muls E, Bouillon R: Improvement of gastric emptying in diabetic gastroparesis by erythromycin: preliminary studies. N Engl J Med322 :1028 eC1031,1990
Feighner SD, Tan CP, McKee KK, Palyha OC, Hreniuk DL, Pong SS, Austin CP, Figueroa D, MacNeil D, Cascieri MA, Nargund R, Bakshi R, Abramovitz M, Stocco R, Kargman S, O’Neill G, Van Der Ploeg LH, Evans J, Patchett AA, Smith RG, Howard AD: Receptor for motilin identified in the human gastrointestinal system. Science284 :2184 eC2188,1999
Imeryuz N, Yegen BC, Bozkurt A, Coskun T, Villanueva Penacarrillo ML, Ulusoy NB: Glucagon-like peptide-1 inhibits gastric emptying via vagal zfferent-mediated central mechanisms. Am J Physiol Gastroenterol Liver273 :G920 eCG927,1997
Wettergren A, Wojdemann M, Holst JJ: Glucagon-like peptide-1 inhibits gastropancreatic function by inhibiting central parasympathetic outflow. Am J Physiol275 :G984 eCG992,1998
Schirra J, Leicht P, Hildebrand P, Beglinger C, Arnold R, Gke B, Katschinski M: Mechanisms of the antidiabetic action of subcutaneous glucagon-like peptide-1 (7eC36)amide in non-insulin dependent diabetes mellitus. J Endocrinol156 :177 eC186,1998
Ritzel R, rskov C, Holst JJ, Nauck MA: Pharmacokinetic, insulinotropic, and glucagonostatic properties of GLP-1 [7eC36 amide] after subcutaneous injection in healthy volunteers: dose-response-relationships. Diabetologia38 :720 eC725,1995
Fineman MS, Bicsak TA, Shen LZ, Taylor K, Gaines E, Varns A, Kim D, Baron AD: Effect on glycemic control of exenatide (synthetic exendin-4) additive to existing metformin and/or sulfonylurea treatment in patients with type 2 diabetes. Diabetes Care26 :2370 eC2377,2003
Meier JJ, Nauck MA: The potential role of glucagon-like peptide 1 in diabetes. Curr Opin Investig Drugs5 :402 eC410,2004(Juris J. Meier, Guido Kem)