We have previously demonstrated that ileal administration of the dietary protein hydrolysate prepared from corn zein (ZeinH) stimulated glucagon-like peptide-1 (GLP-1) secretion and attenuated hyperglycemia in rats. In this study, to examine whether oral administration of ZeinH improves glucose tolerance by stimulating GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) secretion, glucose tolerance tests were performed in normal Sprague-Dawley male rats and diabetic Goto-Kakizaki (GK) male rats. The test solution was gavaged before ip glucose injection in normal rats or gavaged together with glucose in GK rats. Blood samples were collected from the tail vein or by using the jugular catheter to measure glucose, insulin, GLP-1, and GIP levels. In the ip glucose tolerance test, oral administration of ZeinH (2 g/kg) significantly suppressed the glycemic response accompanied by an immediate increase in plasma GLP-1 and GIP levels in normal rats. In contrast, oral administration of another dietary peptide, meat hydrolysate, did not elicit a similar effect. The glucose-lowering effect of ZeinH was attenuated by a GLP-1 receptor antagonist or by a GIP receptor antagonist. Furthermore, oral ZeinH induced GLP-1 secretion and reduced glycemic response in GK rats under the oral glucose tolerance test. These results indicate that the oral administration of the dietary peptide ZeinH improves glucose tolerance in normal and diabetic rats by its incretin-releasing activity, namely, the incretinotropic effect.
Incretins are gut hormones that enhance glucose-dependent insulin secretion, which is known as the incretin effect. Two gut hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are recognized incretins. Based on their stimulative effects on glucose-dependent insulin secretion and pancreatic proliferation (1), incretin systems are a convincing target for treating impaired glucose tolerance and type 2 diabetes. Both incretins are immediately degraded and inactivated by dipeptidyl peptidase-IV (DPP-IV) (2); hence, stable GLP-1 analogs and DPP-IV inhibitors are currently used as clinical drugs.
Secretion of GIP and GLP-1 is increased in response to nutrient ingestion, especially by glucose and free fatty acids. The molecular mechanisms by which these nutrients induce gut hormone secretion were recently elucidated by the discovery of the sweet taste receptor (T1R2/3) (3–5) and free fatty acid receptors (6).
Some dietary proteins and peptides stimulate GLP-1 secretion in animals and humans (7–10). However, the effects of such dietary proteins/peptides on glycemic control and underlying molecular mechanisms are not well studied. We have previously demonstrated that a hydrolysate prepared from zein, a major corn protein, potently stimulated GLP-1 secretion in the murine enteroendocrine cell line GLUTag and in the small intestine of anesthetized rats (11). We also reported that ileal administration of the zein hydrolysate (ZeinH) in conscious rats strongly stimulated GLP-1 secretion, which led to enhanced insulin secretion and attenuation of hyperglycemia (12). Although oral administration of ZeinH attenuated the elevation of glycemia, the GLP-1 response was not investigated.
The purpose of the present study was to examine whether oral administration of ZeinH increases GLP-1 secretion. We also investigated the secretory response of GIP and the involvement of both incretins in the glucose-lowering effect of orally administered ZeinH under an ip glucose tolerance test (IPGTT). It was further examined whether oral administration of ZeinH affects the glycemic response under an oral glucose tolerance test (OGTT) in type 2 diabetic model rats.
Because increasing endogenous incretins, especially GLP-1, has great potential to improve glucose tolerance and pancreatic β-cell function, orally available incretin releasers, including dietary proteins or peptides, are considered promising agents for preventing and treating diabetes and obesity.
Materials and Methods
Materials
ZeinH was prepared as previously described (11). Briefly, zein (50 g; Tokyo Chemical Industry, Tokyo, Japan) was suspended in deionized water (500 mL), and the pH was adjusted to pH 7.2. The suspension was shaken for 60 minutes at 55°C in the presence of papain (250 mg, Papain F; Asahi Food and Health Care, Tokyo, Japan) and then treated in boiling water for 20 minutes to stop the enzyme reaction. After filtration (0.45-μm pore size) and pH adjustment to 7.0, the filtrate was lyophilized as ZeinH. Whey hydrolysate (WheyH) was prepared from whey protein (Optimum Nutrition, Lindesberg, Sweden) with the same procedure as described above. Meat hydrolysate (MHY) was purchased from Sigma-Aldrich Co (St Louis, Missouri). The peptide content of ZeinH was estimated at 77% by total and free amino acid analysis of ZeinH (79.5% and 2.5%, respectively) (12). WheyH and MHY peptide contents were 77.6% and 80.0%, respectively, as determined by the Lowry's protein assay using BSA as a standard protein. ZeinH and MHY had average molecular masses of 1600 to 1700 Da and less than 1200 Da, respectively (13). Additional chemicals were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan) unless otherwise specified.
Animals
Male Sprague-Dawley (SD) rats (7 weeks old), weighing 210 to 230 g, and Goto-Kakizaki (GK) rats (6 weeks old), a type 2 diabetic model, weighing 100 to 160 g were purchased from Japan SLC (Hamamatsu, Japan). All the animals had free access to water and a semipurified diet containing 25% casein based on AIN-93G (14); the rats were housed in individual cages. All animal experiments were performed after an acclimation period (3-7 days) in a temperature-controlled room maintained at 23°C ± 2°C with a 12-hour light, 12-hour dark cycle (8:00 am to 8:00 pm, light period).
This study has been approved by the Hokkaido University Animal Committee, and animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals of Hokkaido University.
Surgical preparation for in vivo experiments
Rats were anesthetized with sodium pentobarbital (50 mg/kg body weight, Somnopentyl Injection; Kyoritsu Seiyaku Co, Tokyo, Japan). The right jugular vein was exposed, and a silicone catheter with a 0.5-mm internal diameter and a 1.0-mm outer diameter (Silascon No. 00; Dow Corning Co, Kanagawa, Japan) was inserted into the vessel and fixed with a thread. The catheter was prefilled with sterilized saline that contained heparin (50 IU/mL; Ajinomoto, Tokyo, Japan).
The free ends of the catheter were dorsally exteriorized, which permitted us to conduct the experiment under nonanesthetized and unrestrained conditions. Rats were used for experiments 2 and 4 after a recovery period of 3 to 4 days. We flushed the jugular catheter with heparinized saline daily to maintain patency.
IPGTT
The glucose solution was administered ip to examine the effect of oral peptides on the GLP-1–mediated glycemic control. All IPGTTs in this study were performed in conscious rats. After a 24-hour fast, a basal (−15 min) blood sample was collected from the tail vein (experiments 1 and 3) or jugular vein (experiments 2 and 4). After basal blood collection, test solutions containing ZeinH, MHY, or deionized water (negative control) were administered into the stomach through the mouth using a feeding tube (5 Fr; Atom Medical Co, Tokyo, Japan). Fifteen minutes after oral administration, the blood sample was collected (0 min) and then the glucose solution was injected (1 g/kg) ip. Blood samples were collected from the tail vein or by using the jugular catheter 15, 30, 60, 90, and 120 minutes after the glucose injection. Plasma was separated by centrifugation at 2500g for 10 minutes at 4°C and frozen at −80°C until glucose, incretin, or insulin measurements. Plasma glucose concentrations were measured using the Glucose CII test kit (Wako).
Experiment 1: effects of oral ZeinH administration on plasma glucose under IPGTT
Peptides (MHY or ZeinH, 2 g/kg) and water (negative control) were administered at −15 minutes to SD rats (8 mL/kg body weight). MHY was selected as a dietary peptide that has GLP-1–releasing activity in vitro (15, 16) and in situ (7). Blood samples (80 μL) were collected from the tail vein and transferred into a 1.5-mL tube containing aprotinin (final concentration, 500 kIU/mL; Wako) and heparin (final concentration, 50 IU/mL) at each time point (−15, 0,15, 30, 60, 90, and 120 min).
Experiment 2: effects of oral ZeinH administration on plasma incretins (GLP-1 and GIP) and insulin under IPGTT
IPGTT was performed in conscious SD rats with the jugular catheter because a large volume of plasma was required to measure glucose and hormone levels. Peptides (MHY or ZeinH at 2 g/kg) and water were administered at −15 minutes, and glucose (1 g/kg) was injected ip at 0 minutes, as previously described. Blood samples (300 μL) were drawn from the jugular catheter into a syringe that contained EDTA (final concentration, 1 mg/mL; Dojindo, Kumamoto, Japan), aprotinin (final concentration, 500 kIU/mL), and DPP-IV inhibitor (final concentration, 50μM; DPP4-010; Millipore Co, Billerica, Massachusetts) at each time point (−15, 0, 15, 30, 60, 90, and 120 min). Between each blood sampling, the catheter was refilled with saline containing heparin (50 IU/mL). Insulin in the plasma (20 μL) was measured using an ELISA kit (AKRIN-010T; Shibayagi Co, Ltd, Gunma, Japan); active GLP-1 and total GIP in the plasma (100 and 20 μL, respectively) were measured by the respective ELISA kits (EGLP-35K and EZRMGIP-55K; Millipore).
Experiment 3: effect of GIP receptor antagonist on ZeinH-attenuated glycemic response (IPGTT)
To investigate the involvement of endogenous GIP in reduced glycemic response after oral ZeinH administration, (Pro3)GIP (Phoenix Pharmaceutical, Inc, Burlingame, California), as a GIP receptor antagonist, was ip injected in SD rats, and IPGTT was performed as described above. (Pro3)GIP (25 nmol/kg, 25 nmol/mL in saline) was injected immediately after oral ZeinH administration (−15 min) under IPGTT. ZeinH (2 g/kg in 8 mL/kg deionized water) and water (negative control) were administered at −15 minutes. Blood samples were collected from the tail vein or the jugular vein at the time indicated in the result, and plasma glucose levels were measured.
Experiment 4: effect of GLP-1 receptor antagonist on plasma insulin after oral ZeinH administration (IPGTT)
A GLP-1 receptor antagonist, exendin(9-39) (Ex9) (synthesized by Thermo Fisher Scientific K.K., Yokohama, Japan) was ip injected in SD rats, and IPGTT was performed. Ex9 (80 nmol/kg) was added in the glucose solution for ip injection. ZeinH (2 g/kg) or water was orally administered at −15 minutes, and then the glucose solution with or without Ex9 was injected ip at 0 minutes. We collected blood samples (300 μL) from the jugular vein from −15 to 60 minutes and measured plasma glucose and insulin levels, as described in experiment 2.
Experiment 5: effect of in vitro digestion on GLP-1–releasing activity of ZeinH in a GLP-1–producing enteroendocrine cell line
ZeinH was digested with pepsin and pancreatin to examine whether the GLP-1–releasing potency of ZeinH remains after luminal protease digestion. ZeinH was dissolved in 0.02N H3PO4 at a concentration of 50 g/L, and the pH was adjusted to 1.85 by using 20N H3PO4. Pepsin (from porcine gastric mucosa; Sigma) was added at 0.5% weight (wt)/substrate wt and incubated for 60 minutes while shaking at 37°C. The pH of the suspension was then adjusted to 8.2 using Ca(OH)2, and pancreatin (4% wt/substrate wt; from porcine pancreas; Sigma) and trypsin (2.5% wt/pancreatin wt, from bovine pancreas; Sigma) were added (17–19). Trypsin was added to sufficiently activate protease zymogens in pancreatin. The mixture was incubated for 120 minutes at 37°C, followed by boiling for 20 minutes to inactivate the enzymes. The mixture was neutralized with H3PO4 and desalted by centrifugation and filtration (0.45-μm pore size). The soluble fraction was lyophilized as the ZeinH-pepsin/pancreatin digest.
To examine the involvement of the peptide fractions of ZeinH in its GLP-1–releasing activity, another in vitro digestion was performed using pronase as the potent protease. Briefly, ZeinH was dissolved in deionized water and the pH was adjusted at 7.0. Pronase (PRONASE Protease, Streptomyces griseus; Calbiochem, Merck KGaA, Darmstadt, Germany) was added to the solution at 0.5% wt/substrate wt and incubated for 60 minutes at 37°C, followed by boiling for 20 minutes to stop the enzymatic reaction. The solution was lyophilized as ZeinH-pronase digest.
The effect of free amino acids on GLP-1 secretion was examined by using the amino acid mixture equivalent to the composition of ZeinH (12). Because Gln and Asn were indistinguishable in the phenyl isothiocyanate amino acid analysis, the concentrations of Gln (21.3 mg/10 mg ZeinH), Glu (0.4 mg/10 mg ZeinH), Asn (5.8 mg/10 mg ZeinH), and Asp (0.5 mg/10 mg ZeinH) were estimated based on the amino acid sequence of zein protein (REFSEQ accession number NM_001112418.1). The total amino acid concentration was finally 62mM in the test solution. To assess the osmotic effect, GLP-1 secretion in response to 31mM NaCl (added to the HEPES buffer described below) was also examined.
GLUTag cells (courtesy of Dr D. J. Drucker, University of Toronto, Toronto, Canada), a murine GLP-1–producing enteroendocrine cell line, were grown in DMEM (Invitrogen, Carlsbad, California; catalog item 12100-038) supplemented with 10% fetal bovine serum, 50 IU/mL penicillin, and 500 μg/mL streptomycin in a humidified 5% CO2 atmosphere at 37°C. Cells were routinely subcultured by trypsinization after reaching 80% to 90% confluency. GLUTag cells were grown in 48-well culture plates at a density of 1.25 × 105 cells per well for 2 days until they reached 80% to 90% confluency. Cells were washed twice with HEPES buffer (140mM NaCl, 4.5mM KCl, 20mM HEPES, 1.2mM CaCl2, 1.2mM MgCl2, and 0.1% BSA [pH 7.4]) to remove the culture media; the cells were then exposed to test agents that were dissolved in the same buffer for 60 minutes at 37°C. Supernatants were collected from the wells, centrifuged at 800g for 5 minutes at 4°C to remove the remaining cells, and then stored at −50°C until the GLP-1 concentration was measured with a commercial enzyme immunoassay kit (Yanaihara Institute Inc, Shizuoka, Japan).
Experiment 6: effects of oral ZeinH on incretin and glycemic response in type 2 diabetic model rats under OGTT
To examine the effect of oral ZeinH on type 2 diabetic models under OGTT, we used GK rats, and blood samples were collected by using the jugular catheter as described above. After basal blood collection (0 min), glucose solution (2 g/kg) as control treatment or the solution containing ZeinH (2 g/kg) or WheyH (2 g/kg) was orally administered. Blood samples (300 μL) were collected from the jugular vein 15, 30, 60, 90, and 120 minutes after the oral administration. Plasma glucose, insulin, active GLP-1, and total GIP levels were measured as described in experiment 2, and total GLP-1 level was measured using an ELISA kit (EZGLP1T-36K; Millipore).
Statistical analysis
Results are expressed as means ± SEM. Statistical analyses were performed by using JMP Pro version 10.0 (SAS Institute Inc, Cary, North Carolina). Statistical significance was assessed by 1-way or 2-way ANOVA. Two-way ANOVA analysis was performed to assess the main effects (treatment and time) and the interaction effect (treatment × time). Significant differences (P < .05) between mean values were determined by Tukey's test or Dunnett's test as appropriate and described in figure legends. The primary endpoints of the present study were the significant increment of plasma incretin levels accompanied by the reduction of plasma glucose levels in ZeinH-treated group compared with the control group.
Results
Experiment 1: changes in plasma glucose concentrations during IPGTT in conscious rats after oral administration of ZeinH
We first examined the dose-response effect of oral ZeinH administration on plasma glucose concentration under IPGTT in conscious rats (experiment 1). To observe the effect of luminal ZeinH on GIP/GLP-1 secretion and to avoid the possible involvement of luminal glucose in the secretion, we used IPGTT rather than OGTT. In the case of OGTT, luminal glucose could stimulate GIP/GLP-1 secretion, and reduced glycemic response might involve delayed gastric emptying and glucose absorption from the intestinal epithelium, apart from the incretin-releasing effect of ZeinH.
Oral administration of test liquids slightly increased plasma glucose concentrations (−15 to 0 min; Figure 1). Increase in the plasma glucose concentration was significantly lower in ZeinH-preloaded rats (2 g/kg) than that in control rats at 30 minutes (Figure 1). Plasma glucose levels similarly decreased in every group after 60 minutes.
Changes in plasma glucose concentrations during IPGTT in conscious rats after oral administration of ZeinH. Water as control (○, 8 mL/kg) and ZeinH at 1 g/kg (□) or 2 g/kg (●) were orally administered 15 minutes before ip glucose injection (1 g/kg). Blood samples were collected from the tail vein before (−15 and 0 minutes) and after (15, 30, 60, 90, and 120 min) glucose injection. Values are displayed as means ± SEM (n = 6). *, Significant differences compared with control treatment at the same time points (Dunnett's test; P < .05).
Experiment 2: changes in plasma glucose, insulin, active GLP-1, and total GIP concentrations during IPGTT after oral administration of ZeinH or MHY
We next examined whether oral ZeinH stimulated incretin and insulin secretion in jugular vein-cannulated rats (experiment 2). Plasma glucose concentrations (Figure 2A) in ZeinH-treated rats, but not in MHY-treated rats, were lower than those in control rats 15 and 30 minutes after ip glucose injection. Plasma insulin concentrations sharply increased at 15 minutes in every group (Figure 2B), and ZeinH-treated rats demonstrated higher insulin concentrations than those in the control group and MHY-treated rats at 15 minutes. After peaking at 15 minutes, insulin concentrations decreased immediately and returned to the basal level at 60 minutes in every group.
Changes in plasma glucose, insulin, active GLP-1, and total GIP concentrations during IPGTT after oral administration of ZeinH or MHY. Water as control (○, 8 mL/kg), ZeinH at 2 g/kg (●), or MHY at 2 g/kg (▵) were administered orally 15 minutes before ip glucose injection (1 g/kg). Blood samples were collected from the jugular vein before and after the glucose injection. Glucose, insulin, active GLP-1, and total GIP concentrations in the plasma were measured. Two-way ANOVA P values were <.01 (treatment), <.01 (time), and <.01 (treatment × time) for insulin (B); <.01 (treatment), .15 (time) and .50 (treatment × time) for active GLP-1 (C); and <.01 (treatment), <.01 (time), <.01 (treatment × time) for total GIP (D). Values are displayed as means ± SEM (n = 7–10). Plots at the same time point that do not share the same letter differ significantly between treatments (Tukey's test; P < .05).
The plasma GLP-1 concentration in the control rats remained at a basal concentration throughout the IPGTT. In contrast, GLP-1 concentrations increased after an oral ZeinH administration; the GLP-1 level at 0 minutes (just before ip glucose injection) was significantly higher than that in control rats. The GLP-1 concentration in the MHY group was at an intermediate level between that in the control group and the ZeinH group; this level was not significantly different compared with that of the control group. The total GIP concentrations changed in a manner similar to GLP-1. Plasma GIP concentrations were significantly higher in ZeinH-treated rats (0–60 minutes) and in MHY-treated rats (0–15 minutes) than in the control rats. The increment of plasma GIP was significantly larger in ZeinH-treated rats than in MHY-treated rats. These results demonstrate that oral ZeinH stimulates the secretion of both incretins, GLP-1 and GIP, independently of luminal glucose.
Experiment 3: glycemic responses during IPGTT after oral preload of ZeinH in rats treated with GIP receptor antagonist
In experiment 3, IPGTT was performed under treatment with the GIP receptor antagonist (Pro3)GIP to examine whether oral ZeinH attenuates plasma glucose increment via the GIP pathway. In this experiment, we collected blood samples until 60 minutes after ip glucose injection because the effect of ZeinH on glycemia was not observed after 60 minutes in the experiments shown in Figures 1 and 2. Plasma glucose levels at 30 minutes in ZeinH/Pro3-treated rats were at intermediate levels between those in control/vehicle-treated rats and ZeinH/vehicle-treated rats, demonstrating partial cancellation of the glucose-lowering effect of ZeinH (Figure 3A). Treatment with (Pro3)GIP had no significant effect on the glycemic response in control rats that received oral water followed by ip glucose injection (Figure 3B).
Glycemic responses during IPGTT after oral preload of ZeinH in rats treated with GIP receptor antagonist. Water as control (8 mL/kg) or ZeinH at 2 g/kg was administered orally 15 minutes before ip glucose injection (1 g/kg). A, Control/vehicle (○) and ZeinH/vehicle (●) groups were ip injected with saline, and the ZeinH/Pro3 (■) group was injected with (Pro3)GIP (25 nmol/kg) at −15 min. Blood samples were collected from the tail vein before and after the glucose injection. B, Plasma glucose concentrations in response to oral water (−15 min) followed by ip glucose injection (1 g/kg, at 0 min) with (□) or without (○) (Pro3)GIP treatment (25 nmol/kg, at −15 min). Blood samples were collected from the jugular vein before and after the glucose injection. Values are displayed as means ± SEM (n = 4–7). Plots at the same time point that do not share the same letter differ significantly between treatments (Tukey's test; P < .05). Abbreviations: Cont, control; Veh, vehicle.
Experiment 4: glycemic and insulin responses during IPGTT after oral administration of ZeinH in rats treated with a GLP-1 receptor antagonist
We examined the effect of the GLP-1 pathway blockage by using the GLP-1 receptor antagonist Ex9 on the glycemic and insulin response in jugular vein-cannulated rats (experiment 4). Consistent with experiment 2, plasma glucose concentrations in ZeinH-treated rats were significantly lower than those in the control rats at 15 and 30 minutes (Figure 4A), and plasma insulin in ZeinH-treated rats was significantly higher than that in the control rats at 0 and 15 minutes (Figure 4B). The treatment with Ex9 had no effect on the glycemic and insulin responses in the control rats (treated with oral water and ip glucose). Treatment with Ex9 attenuated the glucose-lowering effect of ZeinH at 15 and 30 minutes (Figure 4A). As expected, Ex9 treatment reduced the plasma insulin (0–15 min) in response to the oral ZeinH administration (Figure 4B).
Changes in plasma glucose and insulin concentrations during IPGTT after oral administration of ZeinH in rats treated with GLP-1 receptor antagonist. The control groups (○ or □) were orally administered with water (8 mL/kg), and the ZeinH groups (● or ■) were orally administered with ZeinH (2 g/kg) 15 minutes before ip glucose injection (1 g/kg). Rats received ip injection of glucose solution containing Ex9 (□ or ■, 80 nmol/kg) or not containing Ex9 (vehicle treatment; ○ or ●). Blood samples were collected from the jugular vein before and after the glucose injection. Glucose (A) and insulin (B) concentrations in the plasma were measured. Two-way ANOVA P values were <.01 (treatment), <.01 (time), and .05 for (treatment × time) for insulin (B). Values are displayed as means ± SEM (n = 5–9). Plots at the same time point that do not share the same letter differ significantly between treatments (Tukey's test; P < .05). Abbreviations: Cont, control; Veh, vehicle.
Experiment 5: effects of in vitro digestion and amino acids of ZeinH on its GLP-1–releasing activity in enteroendocrine GLUTag cells
We examined the influence of luminal protease (pepsin and pancreatin) digestion on the GLP-1–releasing potency of ZeinH in a GLP-1–producing enteroendocrine cell line. Consistent with the findings of our previous study (11), ZeinH significantly increased GLP-1 release into the supernatant of GLUTag cells after 60 minutes incubation and indicated that ZeinH directly activated GLP-1 secretion in enteroendocrine cells. Pepsin/pancreatin-treated ZeinH also induced a significant increase in GLP-1 secretion compared with the blank (control), with slightly lower potency than untreated ZeinH (Figure 5A). The degree of hydrolysis (DH), determined by using the trinitrobenzenesulfonic method (20, 21), was 8.6% for pepsin/pancreatin-treated ZeinH. In the case of casein, DH was 60.6% after the same treatment; this finding confirmed that a sufficient digestive condition for general protein was used in the present study, and it suggests that ZeinH has a luminal protease-resistant property compared with casein.
Effects of in vitro digestion and the amino acid mixture of ZeinH on its GLP-1–releasing activity in enteroendocrine GLUTag cells. GLUTag cells were exposed to intact or pepsin/pancreatin-treated ZeinH (A), pronase-treated ZeinH (B) at 10 mg/mL, or the amino acid mixture (C) equivalent to 10 mg/mL of ZeinH for 60 minutes. KCl (70mM NaCl was replaced with 70mM KCl in the HEPES buffer) solution was used as positive control that induces GLP-1 secretion via depolarization. The amino acid mixture (AA mix) was prepared to reconstitute the total amino acid composition of 10 mg/mL ZeinH. NaCl (31mM) was added to the control buffer to assess the osmotic effect of the amino acid mixture (C). The supernatant was collected, and the GLP-1 concentration was measured. Values are expressed as means ± SEM (n = 4). Plots at the same time point that do not share the same letter differ significantly between treatments (Tukey's test; P < .05).
We used another strong protease (pronase) (22) to examine the involvement of peptide fractions of ZeinH in its GLP-1–releasing activity. DH of ZeinH-pronase digest was 14.5%, suggesting that ZeinH is more sensitive to pronase than pepsin/pancreatin. GLP-1 release in response to pronase-treated ZeinH was largely reduced compared with intact ZeinH (Figure 5B). The enzymes (pepsin, pancreatin, and pronase) without ZeinH had no effect on GLP-1 secretion in GLUTag cells in the preliminary experiments. The amino acid mixture equivalent to the amino acid composition of ZeinH (12) induced significant increment of GLP-1 concentration in the supernatant (Figure 5C). The increment was around half of the ZeinH-induced increment. The high osmotic control (HEPES buffer added with 31mM NaCl) had only a slight effect on GLP-1 secretion.
Experiment 6: effect of oral ZeinH on glycemic and incretin responses in type 2 diabetic model rats
GK rats are well recognized as one of the best available model of nonobese type 2 diabetes (23, 24). To examine the effect of oral ZeinH under a diabetic condition and under the presence of enteral glucose, OGTT was carried out. The hydrolysate of whey protein (WheyH) was included as another dietary peptide because whey had been reported to increase GLP-1 secretion in vivo (7–10). Fasting glucose levels were approximately 120 mg/dL, and glucose levels in control rats were drastically increased to more than 300 mg/dL after oral glucose load, indicating the typical glucose intolerance condition of GK rats. Oral administration of ZeinH at 2 g/kg significantly reduced glycemic response in GK rats (Figure 6A), similar to the administration of WheyH. Although the experimental conditions differed, the glucose-lowering effect seemed more apparent than that in normal rats as described in the results above. Insulin levels were immediately increased in all groups, with the highest elevation observed in the ZeinH group (Figure 6B). Total GLP-1 level was significantly increased 15 minutes after oral administration of ZeinH (Figure 6C). The increment of total GLP-1 in the WheyH group was not statistically significant, whereas the control group (oral glucose) showed significant reduction of total GLP-1 after 30 minutes. Only slight increments of active GLP-1 were observed after oral administration of test solutions, and significant differences were not detected (Figure 6D). Total GIP levels were immediately increased after the oral glucose load (Figure 6E). Increments in the total GIP levels were similar in the control and ZeinH groups, whereas the WheyH group showed a smaller increase at 15 minutes compared with the other groups.
Glycemic and incretin responses to oral ZeinH or WheyH under OGTT in GK rats. Glucose solution (8 mL/kg) as control (○, 2 g/kg), glucose solution containing ZeinH at 2 g/kg (●), or glucose solution containing WheyH at 2 g/kg (▵) were administered orally. Blood samples were collected from the jugular vein before and after the oral administration, and glucose (A), insulin (B), total GLP-1 (C), active GLP-1 (D), and total GIP (E) concentrations in the plasma were measured. Two-way ANOVA P values were .02 (treatment), <.01 (time), and .02 (treatment × time) for insulin (B); <.01 (treatment), <.01 (time) and .07 (treatment × time) for total GLP-1 (C); .60 (treatment), .08 (time), .96 (treatment × time) for active GLP-1 (D); and .38 (treatment), <.01 (time), <.01 (treatment × time) for total GIP (E). Values are displayed as means ± SEM (n = 6–7). *, Significant differences compared with basal (0 min) values within each treatment (Dunnett's test; P < .05). Plots at the same time point that do not share the same letter differ significantly between treatments (Tukey's test; P < .05).
Discussion
The objective of this study was to investigate whether oral administration of a dietary peptide, ZeinH, stimulates GLP-1 or GIP secretion and improves glucose tolerance in normal rats. We found that oral administration of ZeinH, but not that of MHY, attenuated the glycemia under IPGTT, associated with enhanced secretions of GLP-1 and GIP. The glucose-lowering effect of ZeinH was reversed by GLP-1/GIP receptor antagonists. Oral ZeinH also induced GLP-1 secretion and reduced glycemic response in diabetic model rats. The results of this study demonstrate that the oral administration of the dietary peptide ZeinH can attenuate hyperglycemia by stimulating incretin secretion in normal and diabetic conditions.
Oral ZeinH suppresses plasma glucose elevation
It has been reported that oleoylethanolamide (25) and α-linolenic acid (26) stimulate GLP-1 secretion via GPR119 and GPR120, respectively. With regard to dietary proteins, oral administration of whey protein with carbohydrates decreased postprandial glucose level in individuals with type 2 diabetes mellitus (27), and whey protein increased GLP-1 secretion under OGTT (9). In contrast, by using IPGTT, we demonstrated a dose-response effect of oral ZeinH in attenuating glycemia, and the effective dose was confirmed at 2 g/kg (Figure 1). This result clearly indicates that ZeinH improves glucose tolerance regardless of the modification of intestinal glucose absorption. To clarify whether oral administration of ZeinH has the potential to induce incretin secretion independently of luminal glucose, we used IPGTT in this study.
Oral ZeinH stimulates incretins (GLP-1 and GIP) secretion
Oral administration of ZeinH stimulated GLP-1 secretion (Figure 2C) in conscious rats, as has occurred previously with ileal ZeinH (12). Additionally, we found that GIP secretion was also increased by oral ZeinH (Figure 2D). These incretin secretions were accompanied by enhanced insulin secretion (Figure 2B). The incretin-releasing activity had been proposed as an incretinotropic effect in previous papers (28, 29); thus, ZeinH can be designated as a potent incretinotropic dietary peptide.
Plasma GIP and GLP-1 levels peaked 15 minutes (at 0 min in Figure 2) after oral administration of ZeinH, indicating that luminal ZeinH, not ip glucose, stimulated the secretion of incretins. Enhanced insulin secretion after the glucose injection in ZeinH-treated rats could be responsible for the attenuation of hyperglycemia.
Because GIP-producing K cells are primarily located in the upper small intestine, ZeinH flown into the upper small intestine within 15 minutes might directly stimulate GIP secretion. In contrast, GLP-1–producing L cells are abundant in the distal small intestine and the large intestine. Therefore, oral administration of ZeinH might not reach these regions to directly stimulate L cells within such a short time period. However, we observed that the jejunal lumen was filled with liquid 15 minutes after oral administration of ZeinH solution in another study, and L cells also exist in the jejunum (11, 30). These observations suggest that GLP-1 was initially released from the jejunal L cells by luminal ZeinH, rather than from the ileal L cells. Other reports documented that GIP activates L cells to release GLP-1 via the vagal nerve pathway (31). Such an indirect pathway might also be responsible for GLP-1 secretion after the administration of oral ZeinH.
High GLP-1 levels after 15 minutes might reflect the release of GLP-1 from ileal L cells. In vitro digestion of ZeinH with pepsin and pancreatin failed to attenuate the GLP-1–releasing ability of ZeinH in GLUTag cells (Figure 5A). This finding supports the hypothesis that ZeinH or its partially digested products can stimulate L cells directly even after luminal digestion.
In contrast to pepsin/pancreatin treatment that was carried out to mimic luminal protease digestion, treatment with pronase, a mixture of potent proteases, which was used for nonspecific digestion largely diminished (∼50%) the GLP-1–releasing activity of ZeinH (Figure 5B). This result suggests that peptide fractions in ZeinH are responsible for its GLP-1–releasing activity. Further investigations to identify the active peptides in ZeinH are underway in our laboratory. The residual GLP-1 secretion after pronase treatment may be caused by remaining peptide fragments and liberated amino acids. Although the potency was much lower than intact ZeinH, the amino acid mixture reconstituting the total amino acid composition of ZeinH induced GLP-1 secretion (Figure 5C). Previous reports have documented that amino acids, particularly glutamine, stimulated GLP-1 secretion in vitro (32) and increased plasma GLP-1 level in humans (33). Such amino acids liberated from ZeinH might partially contribute to the incretin-releasing effect of ZeinH in the lumen. Even if glucose and fats were included in ZeinH as minor components, such components would require doses similar to ZeinH to trigger significant GLP-1 secretion. Previous studies demonstrated that GLP-1 secretion was induced by >300 mg glucose (7, 34) or by a 2.2-kcal (244 mg) dose of lipid emulsion (35) in rats that have similar body weights to those in the present study.
Involvement of incretins
The glucose-lowering effect of ZeinH was attenuated by treatment with a GIP receptor antagonist, (Pro3)GIP (Figure 3A), and by a GLP-1 receptor antagonist, Ex9 (Figure 4A). Furthermore, treatment with Ex9 attenuated ZeinH-induced insulin secretion (Figure 4B). These results demonstrated that GLP-1 secretion induced by the administration of oral ZeinH enhanced insulin secretion and resulted in the prevention of hyperglycemia.
GIP is also responsible for the prevention of hyperglycemia by enhancing insulin secretion. As previous reports have documented that GIP is involved in the indirect stimulation of GLP-1 secretion via the vagus nerve (31), GIP released from K cells by luminal ZeinH might activate a vagal pathway to trigger GLP-1 secretion from L cells located at the distal small intestine. This hypothesis is supported by the results that GLP-1 secretion peaked at 0 minutes and at the same time point with GIP. Incretins have the effect of enhancing insulin secretion and protecting islet β-cells, but they also have multiple effects on cardiovascular (36), liver (37), and adipose (38) functions and other systems (39). Stable GLP-1 analogs and DPP-IV inhibitors are already used to treat type 2 diabetes. However, these drugs have potential side effects such as nausea, anorexia, and diarrhea (40). Incretinotropic dietary peptides such as ZeinH prepared from corn could have a lower risk of these side effects than incretin mimetics and incretin enhancers.
Previous studies have demonstrated enhanced incretin secretion by dietary protein/peptides such as whey protein (7–10), casein (41, 42), and MHY (7, 15, 16). In some of these studies, meal tolerance tests or OGTTs were employed. Although these experimental methods are relatively physiological compared with the IPGTT used in our study, the effects on incretin secretion and glycemia could involve a combined effect of oral meal or oral glucose. In addition, modified gastric emptying by other gut hormone secretion, such as cholecystokinin or serotonin, could affect glycemic responses (43, 44). To our knowledge, it has not been reported that oral dietary peptides induce incretin secretion under IPGTT or iv glucose tolerance test. In this study, we demonstrated that single oral administration of dietary peptide ZeinH increased GLP-1 and GIP secretion, which resulted in an improved glucose tolerance without other luminal factors that could enhance incretin secretion.
Oral ZeinH induces GLP-1 secretion and lowers glycemic response in type 2 diabetic model rats
The potent incretin-releasing and glucose-lowering effects of ZeinH led us to apply this peptide to diabetic model rats (GK rats). The glycemic response after an oral glucose load was diminished by coadministration of ZeinH (Figure 6A). Although WheyH had a similar glucose-lowering effect, the insulin response was larger in ZeinH-treated rats than WheyH-treated and control rats (Figure 6B). This could be explained by the significant increment of total GLP-1 only by ZeinH administration. The reason significant increment was not observed in control and WheyH-treated rats might be the defect of nutrient-sensitive incretin responses in GK rats (45). GIP secretion was significantly lower in the WheyH group than in the control and ZeinH groups. These results indicate that WheyH had less potency to stimulate incretin secretion than ZeinH and that the glucose-lowering effect of WheyH was independent of an incretin or insulinotropic effect. Possibly, gastric emptying was strongly inhibited by WheyH, which limited the delivery of glucose into the small intestine. Such an effect might be mediated by cholecystokinin or serotonin released from the upper small intestine (43, 44). A recent paper demonstrated that incretin secretory responses to luminal glucose were impaired in GK rats compared with Wistar rats, but responses to lipids were maintained (45). It is interesting to know whether incretin secretory responses to dietary proteins/peptides were impaired in diabetic models. The present result revealed potent GLP-1–releasing potency of ZeinH even in diabetic model rats.
In summary, our study demonstrated that the oral administration of a dietary peptide, ZeinH, attenuated hyperglycemia by stimulating GLP-1 and GIP secretion in normal rats. The involvement of increased GLP-1/GIP secretion was determined using GLP-1/GIP receptor antagonists. The GLP-1–releasing activity of ZeinH was maintained in the enteroendocrine cell line even after in vitro digestion with pepsin and pancreatin. Additionally, oral ZeinH effectively reduced the glycemic response under OGTT in type 2 diabetic model rats accompanied by increased GLP-1 and insulin secretion. Our data demonstrate the possibility that the oral administration of dietary peptides such as ZeinH potently stimulates incretin secretion and attenuates postprandial hyperglycemia in normal and diabetic conditions.
Acknowledgments
H.H. received grant support (2010-2013) from the Japan Society for the Promotion of Science (KAKENHI 22380070).
Disclosure Summary: The authors have nothing to disclose.
Abbreviations
- DH
degree of hydrolysis
- DPP-IV
dipeptidyl peptidase-IV
- Ex9
exendin(9-39)
- GIP
glucose-dependent insulinotropic polypeptide
- GK
Goto-Kakizaki
- GLP-1
glucagon-like peptide-1
- IPGTT
ip glucose tolerance test
- MHY
meat hydrolysate
- OGTT
oral glucose tolerance test
- SD
Sprague-Dawley
- WheyH
whey hydrolysate
- wt
weight
- ZeinH
zein hydrolysate.
References
