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
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ABSTRACT |
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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 [939] 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 [939]. 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.
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RESEARCH DESIGN AND METHODS |
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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 30 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 · kg1 · min1 was started 30 min before the meal (at 30 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 30 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 30 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 30 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 30 and 15 min.
An interval of at least 10 days was kept between the experiments to exclude carryover effects.
Peptides.
Synthetic GLP-1 [736 amide] was purchased from Saxon Biochemicals (Hannover, Germnay). The lot number of GLP-1 [736 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 µm nitrocellulose filters (Sartorius, Göttingen, Germany), and stored frozen at 30°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, Mölndal, 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 30 min an intravenous infusion of GLP-1 [736 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 45, 30, 15, 0, 15, 30, 45, 60, 120, 180, and 240 min, and plasma glucose was determined immediately.
Before the study, a nasogastric tube (Freka-Ernährungssonde, 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 µl/10 ml blood; Bayer AG, Leverkusen, Germany) and kept on ice. A sample (100 µl) was stored in NaF (Microvette CB 300; Sarstedt, Nümbrecht, Germany) for the measurement of glucose. After centrifugation at 4°C, plasma for hormone analyses was kept frozen at 30°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 515 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-antibodycoated 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 [736 amide] and synthetic GLP-1 [736 amide] for tracer preparation and as standard. The experimental detection limit (2 SD over samples not containing GLP-1 [736 amide]) was <5 pmol/l. Antiserum 89390 binds to the amidated carboxyl terminus of GLP-1 [736 amide] and therefore measures the sum of intact and degraded GLP-1 [936 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 [142] and degraded GIP [342] 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 µl 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 patients 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 Duncans post hoc test (paired analyses). A two-sided P value <0.05 was taken to indicate significant differences.
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RESULTS |
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When the plasma insulin levels immediately after meal ingestion (030 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).
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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).
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DISCUSSION |
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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 ß-cells at their typical postprandial concentrations, especially in the presence of elevated plasma glucose concentrations (2,21). Such properties have been ascribed to GIP (2224) 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 · kg1 · min1 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 (3537). 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 (4042). 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 (4345). Antagonizing the GLP-1induced deceleration of gastric emptying using erythromycin may potentially reduce these side effects.
In conclusion, intravenous erythromycin counterbalances the GLP-1induced 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.
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ACKNOWLEDGMENTS |
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The authors are indebted to S. Richter, Th. Gottschling, K. Höltner, L. Rabenhøj, and L. Albæk for their excellent technical assistance. The contribution of Dr. Stefanie Grosser is particularly acknowledged.
Address correspondence and reprint requests to Dr. Michael Nauck, Diabeteszentrum Bad Lauterberg, Kirchberg 21, D-37431 Bad Lauterberg im Harz, Germany. E-mail: m.nauck{at}diabeteszentrum.de
Received for publication July 13, 2004 and accepted in revised form March 23, 2005
GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide 1
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REFERENCES |
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