Inhibition of gastric emptying by acarbose is correlated with GLP-1 response and accompanied by CCK release

Feruze Y. Enç*,1, Nese İmeryüz*,1, Levent Akin2, Turgut Turoglu3, Fuat Dede3, Gonca Haklar4, N&idot;lgün Tekes&idot;n4, Nural Bek&idot;roglu5, Berrak Ç. Yegen6, Jens F. Rehfeld7, Jens J. Holst8, and Nef&idot;se B. Ulusoy1

1 Division of Gastroenterology, Department of Internal Medicine, University of Marmara Medical School, Istanbul 81326; 2 Department of Computer Engineering, Bogaziçi University, Istanbul 80815; Departments of 3 Nuclear Medicine, 4 Biochemistry, 5 Biostatistics, and 6  Physiology, University of Marmara Medical School, Istanbul 81326, Turkey; and 7 Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, DK-2100 Copenhagen; and 8 Department of Medical Physiology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the effect of acarbose, an alpha -glucosidase and pancreatic alpha -amylase inhibitor, on gastric emptying of solid meals of varying nutrient composition and plasma responses of gut hormones. Gastric emptying was determined with scintigraphy in healthy subjects, and all studies were performed with and without 100 mg of acarbose, in random order, at least 1 wk apart. Acarbose did not alter the emptying of a carbohydrate-free meal, but it delayed emptying of a mixed meal and a carbohydrate-free meal given 2 h after sucrose ingestion. In meal groups with carbohydrates, acarbose attenuated responses of plasma insulin and glucose-dependent insulinotropic polypeptide (GIP) while augmenting responses of CCK, glucagon-like peptide-1 (GLP-1), and peptide YY (PYY). With mixed meal + acarbose, area under the curve (AUC) of gastric emptying was positively correlated with integrated plasma response of GLP-1 (r = 0.68, P < 0.02). With the carbohydrate-free meal after sucrose and acarbose ingestion, AUC of gastric emptying was negatively correlated with integrated plasma response of GIP, implying that prior alteration of carbohydrate absorption modifies gastric emptying of a meal. The results demonstrate that acarbose delays gastric emptying of solid meals and augments release of CCK, GLP-1, and PYY mainly by retarding/inhibiting carbohydrate absorption. Augmented GLP-1 release by acarbose appears to play a major role in the inhibition of gastric emptying of a mixed meal, whereas CCK and PYY may have contributory roles.

insulin; diabetes; ileal brake; peptide YY; glucose-dependent insulinotropic polypeptide; blood glucose


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACARBOSE, a complex pseudooligosaccharide of microbial origin with an ability to bind to alpha -glucosidases and reversibly inhibit the activity of these enzymes, possesses 15,000-fold higher affinity to sucrase than its substrate sucrose, and its inhibitory potency on glycoamylase is higher than that of sucrase (6). At the same time, this pseudooligosaccharide inhibits pancreatic alpha -amylase activity in a mixed noncompetitive manner by binding to amylase-substrate complexes or to amylase molecules, resulting in abortive complexes (8). In healthy subjects oral ingestion of acarbose dose-dependently decreases absorption of carbohydrates, increases their ileal delivery, prolongs absorption time, and increases intestinal flow rates (37, 46). Because of its postprandial blood glucose suppressant effect, acarbose is currently used in the treatment of type I and II diabetes mellitus (5, 21). In both healthy and diabetic subjects, acarbose attenuates postprandial insulin and glucose-dependent insulinotropic polypeptide (GIP) concentrations (21, 36, 38, 49) while augmenting plasma glucagon-like peptide-1 (GLP-1) (36), enteroglucagon (gut glucagon-like immunoreactivity by earlier assays) (37, 38, 46) and peptide YY (PYY) (10) levels compared with controls.

On nutrient absorption, GLP-1 is released from the L-type endocrine cells of the intestine and stimulates insulin secretion from pancreatic beta -cells, similar to the actions of GIP (16, 31). Hence, GLP-1 and GIP are considered to be incretin hormones (16). In addition, GLP-1 has gastrointestinal effects, some of which carry potential physiological importance, such as inhibition of gastric motility and acid secretion and induction of satiety (16, 33). PYY is also suggested to play a physiological role in the inhibition of secretory and motor functions of the upper gut (1, 7, 34). Furthermore, GLP-1 and PYY are considered to be among the hormonal mediators of the "ileocolonic brake" phenomenon (7, 16, 34).

In healthy subjects, the effect of acarbose on gastric emptying of solid meals of varying nutrient composition is unknown. Among the determinants of gastric emptying rate are the intestinal nutrient-sensing mechanisms that detect nutrient composition and, accordingly, control food entry to the intestine via an array of complex neurohormonal pathways (12). The emptying rate of carbohydrates is tightly coupled to the energy density and volume of the meal (18, 47). Intraintestinal perfusion of carbohydrates suppresses antral contractions (14, 47), enhances pyloric tone (14), and inhibits gastric emptying via vagal and spinal capsaicin-sensitive afferent pathways (39). Theoretically, acarbose, by attenuating carbohydrate absorption, would suppress the neurohormonal nutrient-sensing mechanisms and hence accelerate gastric emptying. Alternatively, acarbose, by prolonging the absorption time of carbohydrates and increasing their delivery to the distal gut, would augment the neurohormonal sensing mechanisms and thus induce the ileocolonic brake phenomenon, which in turn would delay gastric emptying. Previous studies in healthy subjects documented that acarbose inhibits gastric emptying of a sucrose solution (38) and accelerates mouth-to-cecum transit time (24). On the other hand, in patients with type I diabetes, acarbose was shown to be without effect on the emptying of a mixed meal (21).

The previously documented features of acarbose-induced alteration of carbohydrate absorption and augmentation of GLP-1 and PYY release led us to postulate that in healthy subjects acarbose would delay gastric emptying of a solid mixed meal. A carbohydrate-free meal was included in our study design to determine whether the effect of acarbose on gastric emptying is specifically mediated by alteration of carbohydrate absorption. Postulating that acarbose enhances the intestinal inhibitory neurohormonal signals on gastric emptying and thus mimics the ileal brake effect, we also examined the emptying of a carbohydrate-free meal subsequent to a sucrose premeal. This last condition is also analogous to therapeutic administration of acarbose with meals because the drug is present in the small intestine at least 4 h after its oral ingestion (46). To document whether a relation exists between the rate of gastric emptying and the plasma hormones, which were previously shown to be augmented by acarbose, we measured plasma responses to GLP-1 and PYY. Plasma responses to CCK were also studied because of this peptide's well-established inhibitory role in gastric emptying, including emptying of carbohydrates (9, 30, 39). Carbohydrate absorption was monitored by measuring blood glucose, plasma GIP, and serum insulin concentrations.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Subjects. Thirty-seven healthy male subjects (mean age 23.2 ± 1.5 yr, range 20-26; mean body mass index = 22.9 ± 2.7 kg/m2), who were recruited by advertisement, participated in the study. None of the subjects had chronic diseases or had had abdominal surgery, except appendectomy. Subjects were not on any chronic treatment and did not take any medications within the week before the experiment. Written informed consent was obtained from each subject, and the institutional ethics committee approved the study protocol. Two subjects participated in two groups of studies and one subject participated in three groups of studies.

Study design. All studies were performed in the morning hours after a 12-h fast. Smoking and chewing gum were not allowed 12 h before or during the study. An antecubital vein was cannulated with an indwelling catheter to be used for sampling of blood glucose and hormone responses. Scintigraphic gastric emptying of solid meals was determined using 99mTc-tin colloid-labeled egg as the radioactive component of the meal.

The effect of acarbose on gastric emptying of a mixed-nutrient meal (n = 14), a carbohydrate-free meal (n = 9), or a carbohydrate-free meal after sucrose ingestion (n = 12) was determined (Table 1). In all study groups, each subject randomly underwent two sets of experiments, with or without acarbose administration, at least 1 wk apart. In studies with acarbose, subjects ingested a 100-mg tablet of acarbose (Glucobay; Bayer, Leverkusen, Germany) dissolved in light tea (200 ml, Ceylon Breakfast; Twinings, London, UK) with meals. In studies without acarbose, subjects ingested the same volume of tea (Table 1). In the group with sucrose ingestion, 50 g of sucrose dissolved in 500 ml of water with or without 100 mg of acarbose was ingested 120 min before a carbohydrate-free meal. All solid meals were ingested within 8 min and immediately after scintigraphic acquisitions were obtained. In addition, to determine the effect of acarbose on plasma hormone responses in the absence of nutrients, 500 ml of water were ingested with or without 100 mg of acarbose, and plasma samples were obtained for hormone assays (n = 6).

                              
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Table 1.   Nutrient composition of radiolabeled solid meals

The osmolality of acarbose (100 mg) and sucrose (50 g) in 500 ml of water were ~2 and 299 mosmol/l, respectively, (Osmostat OM-6020; Kagaku, Kyoto, Japan). The osmolality of the sucrose solution with acarbose added was similar to that of the sucrose solution alone. The pH of the solution of sucrose alone was 6.75, and that of sucrose with acarbose was 6.45.

Measurement and analysis of gastric emptying. One millicurie of 99mTc-tin colloid was mixed with the egg component of the meal and cooked with butter until a firm consistency was achieved. Anatomic markers labeled with low-activity 99mTcO4 were attached to the skin at the sternal notch and both anterior superior iliac spines. One-minute anterior and, immediately afterward, posterior scintigraphic acquisitions were obtained in the sitting position using a large field-of-view gamma camera fitted with a low-energy collimator and interfaced with a dedicated computer system (GE XRT; General Electric Medical Systems, Milwaukee, WI). Technetium counts were obtained with a 20% energy window with peak set at 140 keV. The scintigraphic acquisitions were obtained immediately after ingestion of the test meal, every 5 min for the first 30 min and every 10 min thereafter until ~10% of the counts remained in the stomach. Subjects were allowed to ambulate during intervals between image acquisitions. A region of interest was manually outlined corresponding to the stomach for each scintigraphic image. Corrections were made for decay of the radioactivity. Geometric means of the counts obtained in the anterior and posterior projections were calculated for attenuation correction according to the following formula: geometric mean = square root of (anterior × posterior) counts. Data were normalized to 100% based on total gastric counts obtained immediately after ingestion of the radiolabeled meal.

In vivo and in vitro radionuclide labeling stability tests were performed in two subjects as previously described (29). In vitro studies revealed that 93.5% of the radioactivity was in the solid phase 2 h after incubation of the meal with gastric juice. The percentage of the administered 99mTc radioactivity in the blood was 0.75, 1.43, and 1.24 after 1, 2, and 3 h, respectively.

Percent gastric retention radioactivity of each subject was analyzed using the modified power exponential function as described by Siegel et al. (48), according to which y(t) is the fractional meal retention at time t, k is the gastric emptying rate (in min-1), t is the time interval (in min), and beta  is the extrapolated y-intercept from the terminal portion of the curve of the function y(t) = 1 - (1 - e-kt)beta . Using fractional retention y(t) versus t, data as input in a hybrid algorithm were utilized to fit the data to the modified power exponential function. The reason for using a hybrid algorithm was the observation that in nonlinear least-squares applications, the gradient-based optimization algorithms are fast but tend to converge to local minima (2), whereas the evolutionary algorithms are slow but generally converge to the global optimum. Therefore, the global optimum was found by first using a method based on evolutionary strategies (4) followed by the application of the conjugate gradient method. Hence, the unknown parameters k and beta  were determined and a time-activity curve was generated for each subject. Lag phase was calculated by the formula Tlag = ln beta /k (48) representing time t, at which the curve demonstrates an inflection point and after which the slope becomes constant. Gastric half-emptying time (T50) was defined as the time when scintigraphic counts decreased by 50% and was estimated by using data fitted to the modified power exponential function. To determine whether the fitted emptying curve provided a good fit to the data of each subject, an R2 value was obtained based on the formula R2 = 1 - (residual sum of squares/total sum of squares). Total sum of squares was represented by n - 1 times the square of the standard deviation of the observed fractions. The investigators who analyzed fractional gastric emptying were unaware of the nature of the treatment groups.

Blood specimens. Venous blood samples for GIP, CCK, GLP-1, and PYY immunoassays were obtained from an indwelling venous catheter at -15 min and immediately before test meals (0 min) and at appropriate intervals thereafter, as depicted in Figs. 3-5. In the nonnutrient drink group, blood was sampled at 5-min intervals for the first 15 min and then every 15 min for 150 min. Venous blood was collected into chilled tubes containing aprotinin (500 KIU/ml of blood; Trasylol, Leverkusen, Germany) and EDTA (1 mg/ml of blood; Merck, Darmstadt, Germany) and was centrifuged at 4°C, and plasma was immediately stored at -20°C until assayed. In subjects who ingested carbohydrate-containing meals, blood was collected into blank tubes and centrifuged at 4°C, serum was stored at -20°C for insulin determinations, and blood was collected into NaF (Merck)-containing tubes for glucose determination.

Analytical procedures. Glucose was measured with the glucose oxidase method, using an enzymatic colorimetric assay (Glucose GOD-PAP, BM/Hitachi 917 analyzer; Boehringer Mannheim, Mannheim, Germany).

Insulin concentrations were measured using a solid phase, two-site chemiluminescent enzyme-labeled immunometric assay (Immulite Insulin; Diagnostic Products, Los Angeles, CA) in the mixed meal group. The detection limit of the assay was ~2 µIU/ml, and the intra-assay coefficient of variation (CV) was <5%. In the study group with the carbohydrate-free meal after sucrose ingestion, insulin was measured using a solid-phase 125I radioimmunoassay (Coat-A-Count Insulin; Diagnostic Products). The detection limit of the assay was 1.2 µIU/ml, and intra-assay CV was <10%.

Plasma concentrations of CCK, GIP, GLP-1, and PYY were measured as previously described (17, 22, 35, 41). CCK was assayed using an antibody (92128) raised in rabbits against an O-sulfated human CCK-12 analog (41). Antibody 92128 binds all the bioactive forms of CCK with equimolar potency and displays no reactivity with gastrin (41). The tracer used was the Bolton-Hunter labeled sulfated CCK-8 (125I-CCK-8). Separation of antibody-bound and free tracer was achieved by using plasma-coated charcoal. The detection limit of the assay was 0.1 pmol/l. The intra- and interassay variation at different concentrations within the working range of the assay ranged between 5% and 15%. All samples were assayed in duplicate.

Plasma GIP was measured using COOH-terminally directed antibody R65 (22). The sensitivity of the radioimmunoassay was <1 pmol/l. Intra-assay CV was <6% at 20 pmol/l of plasma GIP, and interassay CV was ~15-20%.

Plasma concentrations of GLP-1 were assayed with antibody 89390 at a final dilution of 1:200,000 (35). Antibody 89390 crossreacts 100% with the amidated COOH terminus of GLP-1-(7-36)amide and its immediate metabolite GLP-1-(9-36)amide but crossreacts <0.1% with GLP-1-(7-37) and fragment 1-35. The experimental detection limit was 1 pmol/l, and the intra-assay CV was 6%.

Plasma concentrations of PYY were measured with antiserum 8412-211 (a gift from R. Håkanson, Dept. of Pharmacology, University of Lund, Sweden) by methods previously described (17). The detection limit of the assay was <1 pmol/l, and intra-assay CV was <5%.

Statistical analysis. The data are presented as means ± SE or medians (ranges) according to the distribution of data. Basal plasma hormone concentrations were calculated by taking an average of -15 min and 0 min values. Integrated responses of area under curve (AUC) were calculated according to the trapezoidal rule. To determine whether meal ingestion altered basal plasma determinations, repeated-measures ANOVA with time as a main factor was applied to plasma hormone and glucose concentrations followed by Newman-Keuls test. To determine the effect of acarbose administration on hormonal responses, two-way ANOVA was used, with time and treatment as main factors. Paired data at specific time points were compared by using Wilcoxon signed-rank test or paired t-test, as appropriate. Correlations between gastric emptying data and hormonal responses were investigated by using Pearson's test. Multivariate stepwise regression analysis was used to determine the relationship between dependent and independent variables. Differences were considered statistically significant if P < 0.05.


    RESULTS
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ABSTRACT
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RESULTS
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Acarbose administered with sucrose caused flatulence in three subjects, and two of the three reported diarrhea at ~80 min after the sucrose meal. No symptoms were reported in other study groups.

Blood glucose and serum insulin. The mixed meal alone did not increase blood glucose concentrations except at 40 min (P < 0.001), whereas sucrose ingestion increased glucose levels between 0 and 60 min compared with the basal value (P <0.05-0.001). Acarbose blunted blood glucose concentrations in both groups (Fig. 1). Nevertheless, integrated responses of glucose with acarbose were similar to control values because of the control group's postpeak glucose concentrations, which were below basal values (Fig. 1). Acarbose attenuated responses of serum insulin in both groups (Fig. 1).


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Fig. 1.   Responses of blood glucose (top) and serum insulin (bottom) to a 476-kcal mixed meal (A) and a 228-kcal carbohydrate-free meal (arrows) 120 min after 50 g of sucrose premeal (time 0) (B). Insets, integrated responses. The meals were administered without (open circle , open bars) or with (, solid bars) 100 mg of acarbose at the beginning of each study (means ± SE, n = 12). Acarbose blunted blood glucose responses, but integrated responses were similar because of declining control levels below basal values after glucose peaks. Acarbose attenuated serum insulin responses in both groups. dagger P < 0.05, *P < 0.01, and Dagger P < 0.001 for significant differences between groups without vs. with acarbose intake. AUC, area under curve.

Gastric emptying. In all study groups, the modified power exponential function provided a good fit to each individual gastric emptying curve, and the R2 value was >0.95.

As shown in Table 2 and Fig. 2A, acarbose administration with the mixed meal did not alter the lag phase but prolonged T50 and decreased gastric emptying rate k by ~25%. The median AUC (min-1) for gastric emptying of 54 (40-77) in the mixed meal group was lower than the respective values of 62 (48-72) of the acarbose group (P < 0.05).

                              
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Table 2.   Gastric emptying of a 476-kcal radiolabeled mixed meal ingested with or without 100 mg of acarbose



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Fig. 2.   Percent gastric retention of radiolabeled solid meals without (open circle ) and with ()100 mg of acarbose administered at the beginning of a 476-kcal mixed meal (A) and a 217-kcal carbohydrate-free meal (B). A solution of 50 g of sucrose was ingested without (open circle ) or with () 100 mg of acarbose 120 min before a 228-kcal carbohydrate-free meal (C). Dashed and solid lines represent data without and with acarbose, respectively, fit to the modified power exponential function as described in MATERIALS AND METHODS. Acarbose delayed the emptying of a mixed meal and a carbohydrate-free meal after sucrose ingestion (A and C), whereas emptying of a carbohydrate-free meal without sucrose was unaltered by acarbose (B). See Tables 2 and 3 for details.

Acarbose did not alter any parameter of the gastric emptying of the carbohydrate-free meal (Fig. 2B). The median beta -values of 2.1 (1.1-3.4) and 1.9 (1.2-2.5) for meal alone and meal with acarbose groups, respectively, were consistent with solid gastric emptying (48).

Acarbose delayed the gastric emptying of a carbohydrate-free meal after sucrose ingestion. Both the lag phase and T50 of gastric emptying were prolonged compared with control values (Table 3, Fig. 2C).

                              
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Table 3.   Gastric emptying of a 228-kcal radiolabeled carbohydrate-free meal

Plasma hormone responses. Ingestion of water with or without acarbose did not alter plasma GIP, GLP-1, and PYY levels compared with the respective basal values. Ingestion of water with acarbose significantly increased plasma CCK concentrations compared with the basal value of 0.75 ± 0.13 pmol/l (1.2 ± 0.19 and 1.3 ± 0.17 pmol/l at 5 and 10 min, respectively; P <0.05), whereas water alone did not appreciably alter CCK levels.

Mixed meal group. Plasma samples of only 12 of the 14 study subjects were available for hormone assays for technical reasons. In response to the meal plasma GIP and CCK concentrations were significantly elevated in groups with and without acarbose (P < 0.1-0.001). However, integrated GIP response of the acarbose group was significantly lower and CCK response was significantly higher compared with control values (Fig. 3, A and B, insets).


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Fig. 3.   Responses of plasma immunoreactive glucose-dependent insulinotropic polypeptide (GIP: A), CCK (B), glucagon-like peptide-1 (GLP-1; C), and peptide YY (PYY; D) to ingestion of a 476-kcal radiolabeled mixed meal (61% carbohydrate, 16% protein, and 23% fat) without (open circle ) and with ()100 mg of acarbose in 12 subjects (means ± SE). Insets represent integrated responses between 0 and 180 min without (open bars) and with (filled bars) acarbose. Acarbose attenuated GIP response while augmenting CCK, GLP-1, and PYY responses. dagger P < 0.05, *P < 0.01, and Dagger P < 0.001 for significant differences between groups without and with acarbose intake.

The meal alone caused significant elevation of plasma GLP-1 compared with the basal value only at 20 and 90 min (P <0.05), whereas with acarbose administration, plasma GLP-1 levels were significantly elevated at 40 min and thereafter (P < 0.001). With acarbose administration integrated GLP-1 response was significantly higher than the corresponding control value (P < 0.01; Fig. 3C, inset) and the time × treatment effect for GLP-1 was significant at the level of P < 0.002. Plasma PYY levels remained similar to the basal value in the control group, whereas acarbose caused significantly higher values at 40 min and thereafter (P <0.05 and P < 0.01, respectively). The integrated plasma PYY response was significantly higher in the acarbose group compared with the meal-alone group (Fig. 3D, inset).

Carbohydrate-free meal group. Meal ingestion caused significant elevation of plasma GIP and CCK in groups with and without acarbose (P < 0.001), and integrated responses covering the study period were similar between acarbose and control groups (Fig. 4, A and B, insets). However, plasma GIP showed significant differences between groups with and without acarbose in the inclination and declination portions of the time-response curve (Fig. 4A).


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Fig. 4.   Responses of plasma immunoreactive GIP (A), CCK (B), GLP-1 (C), and PYY (D) to ingestion of a 217-kcal radiolabeled carbohydrate-free meal (35% protein, 65% fat) without (open circle ) and with ()100 mg of acarbose in 9 subjects (means ± SE). Insets, integrated responses between the times shown without (open bars) and with (filled bars) 100 mg of acarbose. Acarbose augmented GIP, GLP-1, and PYY responses ~60 min after meal intake but did not alter CCK response. dagger P < 0.05, *P < 0.01, and Dagger P < 0.001 for significant differences between groups without and with acarbose intake.

Plasma GLP-1 was significantly increased over the basal value at 60 min and thereafter in both groups (P < 0.05-0.01). Integrated GLP-1 responses (0-150 min) were similar between the acarbose and control groups, whereas 90 min after meal ingestion, integrated GLP-1 response in the acarbose group was higher than the corresponding value of the control group (Fig. 4C). Although plasma PYY was modestly elevated after meal ingestion, significantly elevated concentrations compared with basal values were not observed at any time point in groups with and without acarbose, and integrated responses between 0 and 150 min were similar. However, integrated PYY response of the acarbose group between 90 and 150 min was higher compared with control (Fig. 4D, inset).

Carbohydrate-free meal after sucrose ingestion group. As depicted in Fig. 5, acarbose substantially altered all plasma hormone responses to the sucrose premeal and modified GIP and PYY responses to the carbohydrate-free meal. Plasma GIP was abolished and attenuated in response to sucrose and carbohydrate-free meals, respectively (Fig. 5A).


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Fig. 5.   Responses of plasma immunoreactive GIP (A), CCK (B), GLP-1 (C), and PYY (D) to ingestion of a solution of 50 g of sucrose without (open circle ) and with () 100 mg of acarbose at time 0 min followed by a 228-kcal radiolabeled carbohydrate-free meal (21% protein and 79% fat) at 120 min (arrows) in 12 subjects (means ± SE). Insets, integrated responses between the times shown without (open bars) and with (filled bars) 100 mg of acarbose. Acarbose augmented the CCK and GLP-1 responses between 0 and 120 min and did not alter responses between 120 and 240 min. GIP response was attenuated and PYY response was enhanced by acarbose all throughout the study time. dagger P < 0.05, *P < 0.01, and Dagger P < 0.001 for significant differences between groups without and with acarbose intake.

In response to sucrose alone there was a modest but significant increase in plasma CCK only at 15 min compared with the basal value (P < 0.001; Fig. 5B), whereas acarbose caused a significant CCK response during the period of 15-45 min (P < 0.01). Integrated plasma response of CCK to sucrose was significantly higher in the acarbose group compared with the sucrose-alone group (Fig. 5B, inset). Acarbose did not alter integrated CCK response to the carbohydrate-free meal (Fig. 5B, inset).

After sucrose ingestion, plasma GLP-1 concentrations were briefly increased from 15 to 30 min (P < 0.01-0.001 compared with the basal value; Fig. 5C), whereas acarbose administration caused significantly higher concentrations during the period of 15 to 60 min (P <0.01-0.001). Integrated responses of the two groups were significantly different after sucrose ingestion and similar after carbohydrate-free meal ingestion (Fig. 5C, inset).

Neither sucrose nor carbohydrate-free meal ingestion caused significant alteration of plasma PYY concentrations compared with the basal values (Fig. 5D). On the other hand, acarbose administration caused significant elevation of plasma PYY after both meals (P < 0.01), and integrated responses were significantly different after both meals (Fig. 5D, inset).

Correlation of plasma hormone responses with gastric emptying. The delayed gastric emptying time induced by acarbose in the mixed meal group was positively correlated with the integrated GLP-1 response (AUC, r = 0.68, P < 0.02; Fig. 6A). Multivariate stepwise regression analysis demonstrated that integrated plasma GLP-1 response was the only significant determinant of gastric emptying AUC (r2 = 0.46, P < 0.03).


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Fig. 6.   Relationship between integrated plasma immunoreactive GLP-1 (0-180 min) and AUC of gastric emptying of a 476-kcal radiolabeled mixed meal administered with 100 mg of acarbose (A) and integrated immunoreactive GIP (120-240 min) and AUC of gastric emptying of a 228-kcal radiolabeled carbohydrate-free meal ingested at 120 min after 50 g of a sucrose premeal given with 100 mg of acarbose (B) (means ± SE, n = 12). The gastric emptying of the mixed meal administered with acarbose was positively correlated with augmented GLP response (r = 0.68, P < 0.02), and the emptying of the carbohydrate-free meal was negatively correlated with GIP response (r = -0.71, P < 0.01).

Delayed gastric emptying times induced by acarbose in the carbohydrate-free meal after sucrose premeal group were negatively correlated with the integrated GIP response between 120 and 240 min (lag phase, r = -0.73, P < 0.01; T50, r = -0.75, P <0.01; AUC, r = -0.71, P < 0.01) (Fig. 6B). Multivariate stepwise regression analysis revealed that the only significant independent variable for gastric-emptying parameters of lag phase (r2 = -0.68, P <0.01), T50 (r2 = -0.58, P < 0.02), and AUC (r2 = -0.64, P < 0.01) was integrated plasma GIP response between 120 and 240 min. Integrated GIP response between 0 and 240 min was also a significant independent variable for all gastric emptying parameters.

Correlation between hormonal responses. There was a weak correlation between integrated responses of GLP-1 and PYY (r = 0.58, P < 0.5) in the mixed meal acarbose group. A correlation was also observed between integrated responses of GLP-1 and PYY in the acarbose carbohydrate-free meal after sucrose ingestion group (0-240 min) (r = 0.68, P < 0.01). Integrated GLP-1 during the same period was also positively correlated with insulin response (r = 0.75, P < 0.01).


    DISCUSSION
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results demonstrate that acarbose inhibits emptying of carbohydrate-containing meals, whereas it does not alter the emptying of meals composed of fat and protein. In the case of delayed emptying of a mixed meal by acarbose, the duration of the lag phase was similar to that of control, implying that acarbose and carbohydrates had to be emptied from the stomach to initiate the intestinal inhibitory signals that modulate gastric motor activity. On the other hand, acarbose caused prolongation of both the lag phase and gastric half-emptying time of a carbohydrate-free meal subsequent to sucrose ingestion, demonstrating that the intestinal inhibitory signals were already operative while the carbohydrate-free meal was being ingested. The unaltered gastric emptying of a solid mixed meal by acarbose demonstrated in type I diabetic subjects by Juntti-Berggren et al. (21) may be caused by the presence of autonomic neuropathy, which may have blunted the effect of acarbose on intestinal neurohormonal signaling to modify gastric motility. Alternatively, the different results obtained by us and the aforementioned study may be caused by the different experimental designs used.

Carbohydrate absorption is tightly coupled to GIP release (34). Confirming previous findings (36, 46, 49), we observed that acarbose administration causes considerable inhibition of carbohydrate absorption, as reflected by attenuated plasma insulin, GIP, and blood glucose concentrations. It is possible that in healthy and probably in diabetic subjects the postprandial blood glucose-lowering effect of acarbose is partially mediated by its ability to inhibit gastric emptying.

There may be several mechanisms responsible for inhibition of gastric emptying that result from alteration of alpha -glucosidase and alpha -amylase activities by acarbose, and these mechanisms may be particular to the timing of carbohydrate intake. It is conceivable that the inhibitory action of acarbose on gastric emptying is mediated through enhancement of the intestinal neurohormonal inhibitory signals by exposure of a longer length of intestine to the carbohydrate absorptive process, prolongation of carbohydrate absorption time, and increased delivery of unabsorbed carbohydrate residues to the ileum and distally. This assumption is supported by earlier findings demonstrated in humans that gastric emptying of solid nutrients is delayed by slowly absorbed carbohydrates as opposed to rapidly absorbed isocaloric carbohydrates (27). Similarly, a longer length of canine intestine exposed to glucose elicits a higher degree of inhibition of gastric emptying compared with a shorter length of intestinal exposure (26). In addition, unabsorbed carbohydrate residues may have played roles in our findings by increasing osmolality and/or through conversion to short-chain fatty acids (SCFAs), which inhibit gastric motility when perfused into the distal bowel (45).

The absorption of carbohydrates and other nutrients is sensed by intestinal afferent neural transmission, and the information is relayed to other centers to modulate gastric motility (12, 39). Glucosensitive mesenteric and enteric neurons have been identified in the mammalian intestine (13, 23), and, recently, glucose-responsive enteric neurons were demonstrated to express the intestinal brush border Na+-dependent glucose transporter SGLT-1 protein (23). Therefore, in the process of the inhibitory action of acarbose on gastric emptying, it is possible that intestinal afferent neural transmission is altered through modulation of carbohydrate absorption.

The results of the present study demonstrate that acarbose modifies gut peptide release according to the timing and content of nutrient intake. Acarbose augmented release of CCK, GLP-1, and PYY in response to carbohydrate-containing meals (mixed meal and sucrose premeal) compared with controls, whereas it did not modify the hormone responses to a carbohydrate-free meal except for a brief and minor augmentation of plasma GLP-1, PYY, and GIP responses ~90 min after meal ingestion. These later effects of acarbose appear to be unrelated to carbohydrate absorption (see below). In the setting of a sucrose premeal followed by a carbohydrate-free meal, acarbose modified hormone responses to each meal differently. With the sucrose premeal acarbose augmented plasma CCK and GLP-1 compared with control, and the values nearly returned to basal levels before the intake of the carbohydrate-free meal. Acarbose did not modify integrated plasma CCK and GLP-1 in response to the carbohydrate-free meal after the sucrose premeal. On the other hand, acarbose augmented plasma PYY in response to the sucrose premeal and the values remained augmented while the carbohydrate-free meal was being ingested. These findings can be explained on the basis that during the 2 h between meals, unabsorbed carbohydrate residues may have moved from the proximal GLP-1- and CCK-releasing sites distally to sites at which PYY containing endocrine cells are abundant (1). We did not find any correlation between delayed emptying times of the carbohydrate-free meal and augmented PYY response. However, similar to the findings of a study investigating the effect of an amylase inhibitor on gastric emptying (20), attenuated response of plasma GIP by acarbose was negatively correlated with delayed gastric emptying times of the carbohydrate-free meal. These findings support the notion that acarbose-induced inhibition of gastric emptying is mainly related to inhibition/retardation of carbohydrate absorption. At the same time, augmentation of CCK, GLP-1 and PYY release by acarbose appears to be mainly related to its ability to alter carbohydrate absorption.

Augmented plasma GLP-1 response by acarbose in the mixed meal group was positively correlated with gastric emptying AUC. Furthermore, among the other plasma peptides measured, GLP-1 was the only significant determinant of gastric emptying. Augmentation of GLP-1 release by acarbose may be due to absorption of carbohydrates along a longer length of intestine, prolongation of the absorptive process, accelerated intestinal transit time, or increased delivery of carbohydrate residues to distal intestine. In humans, the inhibitory actions of GLP-1 on gastric emptying have been documented by several groups (16, 33). In rats, GLP-1 acts centrally and via capsaicin-sensitive vagal afferent nerves (19), similar to the actions of CCK, to inhibit gastric emptying. Thus, in the inhibition of gastric emptying of mixed nutrients by acarbose, GLP-1 may play a major role. Furthermore, considering our results together with the previous findings, it can be postulated that enhanced GLP-1 response by acarbose participates in the ileocolonic brake phenomenon when carbohydrate absorption is deranged, as occurs in malabsorption syndromes.

The reasons for acarbose-induced augmentation of PYY response may be similar to those operative in augmentation of GLP-1 response. In addition, in our acarbose groups, unabsorbed carbohydrate residues may have converted to SCFAs, which in turn may have caused augmented PYY response (7). CCK stimulates PYY release in dogs (28), but the relevance of this to our findings is not obvious. PYY is implicated as the mediator of the inhibitory action of SCFAs on gastric motility (7) and proposed as one of the hormonal mediators of the ileocolonic brake (34). Although the role played by PYY in our findings is not evident, it appears that inhibition of carbohydrate absorption by acarbose activates neurohormonal mediators, including PYY, that are known to exert inhibitory signals on the upper gut.

Our findings of enhanced CCK release by the acarbose in the mixed meal and sucrose premeal groups were contradictory to the findings of Uttenthal et al. (49), who found an unaltered CCK response. The divergent results are probably caused by different specificities of the assays used in the determination of plasma CCK concentrations (42). The availability of CCK receptor antagonists has facilitated the elucidation of CCK's role in the inhibition of gastric emptying, including emptying of carbohydrates (9, 30, 39). We did not find correlations between augmented CCK response by acarbose and gastric emptying times. However, in response to nutrient intake, plasma levels of CCK and its actions on motor functions of the gut are not necessarily congruent (9, 39). Accumulating evidence suggests that, after its release from the endocrine cells of the upper gut, CCK acts in a paracrine fashion on mucosal chemo- and nutrient-sensitive or mechanosensitive vagal afferents to exert its inhibitory actions on gut motility in addition to its hormonal effects (12, 32, 42a). Therefore, the role of CCK in the delayed gastric emptying times of the acarbose mixed meal group cannot be completely discarded.

The mechanism(s) by which acarbose enhances CCK release in response to carbohydrate intake is not clearly evident. It is not likely that the prolonged absorption time is the sole factor for enhanced peptide release because the presence of acarbose was sensed fairly promptly by CCK-releasing mechanisms (at 15 and 20 min with sucrose ingestion and mixed meal, respectively). In humans the magnitude of carbohydrate-induced CCK release is slightly less than that elicited by isocaloric protein and fat (9, 25). To the best of our knowledge, carbohydrate-stimulated CCK-releasing mechanisms have not been elucidated. Further studies are required to investigate whether inhibition of pancreatic amylase and/or alpha -glucosidase by acarbose results in a feedback signal to augment CCK release.

In our study groups that demonstrated acarbose-induced delay in gastric emptying, there was a correlation between augmented plasma responses of GLP-1 and PYY. As stated above, a longer length of intestinal exposure to carbohydrate absorption may have elicited augmented GLP-1 and PYY release in addition to the other possible mechanisms stated for PYY release. GLP-1 and PYY are costored in a proportion of mucosal L cells, which increase in number distally along the intestine, PYY-containing cells being more abundant in the colon (1). However, the release of GLP-1 and PYY in response to nutrients does not entirely involve similar mechanisms (3). With acarbose administration, the prompt, early rise in plasma GLP-1 after sucrose and mixed meal ingestion as documented in previous studies (36, 47) and in the present study, suggests that there are an adequate number of proximal GLP-1-containing L cells that are capable of sensing the interaction of carbohydrates with absorptive elements. At the same time, in rats, neuroendocrine loops originating in the proximal gut that signal the lower gut to release GLP-1 and PYY were described (11, 44).

Other factors for enhanced release of peptide hormones with acarbose administration should also be considered. Nutrient-induced intestinal neurohormonal signals generally involve the interaction of the nutrient with the absorptive elements. Recent evidence suggests that the nutrients do not necessarily need to be metabolized to elicit their signaling function. Hence, 3-0-methyl glucose, which is transported by the Na+-dependent glucose transporter SGLT-1 but is nonmetabolizable, releases GLP-1 (43) and mimics the action of intestinal glucose to inhibit gastric emptying (40). Therefore, it is intriguing to postulate that acarbose by binding to brush border alpha -glucosidases and/or alpha -amylase initiates signals for peptide release. This assumption is partially supported by our findings that demonstrated that acarbose causes a minor increase in plasma CCK with a nonnutrient water meal and delayed minor increases in plasma GIP, GLP-1, and PYY in response to a carbohydrate-free meal. Further studies are required to investigate the effects of acarbose that are not related to carbohydrate absorption per se on peptide release and gut motility.

In conclusion, our results are compatible with the notion that in healthy subjects acarbose delays gastric emptying by its ability to inhibit/retard carbohydrate absorption. As a result of alteration of carbohydrate absorption, it is likely that acarbose modifies the intestinal afferent neural transmission and the release of gut peptides, which in turn mediate the inhibitory action of acarbose on gastric emptying. Augmented plasma GLP-1 response may be a major mediator of the inhibitory action of acarbose on gastric emptying of a mixed meal, whereas augmented CCK and PYY responses may have contributory roles. It remains to be determined in healthy and diabetic subjects whether the inhibitory action of acarbose on gastric emptying contributes to its postprandial blood glucose blunting effect. Acarbose, in the absence of carbohydrate intake, modifies the release of nutrient-responsive peptide hormones in a minor fashion, which is probably caused by its interaction with carbohydrate absorptive sites or by other unknown factors.


    ACKNOWLEDGEMENTS

The technical assistance of Ayfer Ürün and Lene Albæk is appreciated.


    FOOTNOTES

* F. Y. Enç and N. İmeryüz contributed equally to this work.

This study was supported by grants from the Scientific and Technical Research Council of Turkey (TÜBİTAK, SBAG-1960), University of Marmara Research Fund (SBAG-87), Turkish Government Planning Commission (96-121310), Bayer Türk, and the Danish Medical Research Council.

Address for reprint requests and other correspondence: N. B. Ulusoy, Dept. of Internal Medicine, Univ. of Marmara Medical School, 81326 Haydarpasa, Istanbul, Turkey (E-mail: nefiseulusoy{at}hotmail.com).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 November 2000; accepted in final form 18 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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