Protein ingestion after injection of the glucagon-like peptide-1 receptor agonist Exendin-4 (Ex-4) causes hyperglycemia in rats. The objectives of this study were to determine the components of protein digestion responsible for this effect and to associate it with changes in the concentrations of other metabolites and hormones. Two experiments were conducted. In the first experiment, food-deprived rats were gavaged with intact whey (WP) or albumin protein, their hydrolysates, amino acid mixtures (1 g/2.5 ml), or water 5 min after injection of either PBS or Ex-4 (0.5 μg/rat). Tail vein blood was analyzed for glucose over 2 h. In the second experiment, food-deprived rats were gavaged with WP with or without Ex-4. Groups of conscious rats were killed by decapitation either before, or at selected times after gavage. Plasma concentrations of glucose, amino acids, free fatty acids (FFA), glycerol, insulin, glucagon, and leptin were measured. In experiment 1, blood glucose was higher when intact proteins and protein hydrolysates, but not amino acid mixtures, were given with than without Ex-4 (P < 0.05). In experiment 2, concentrations of glucose, FFA, and the ratio of tyrosine to branched-chain amino acid were higher (P < 0.01), but leptin and essential amino acid concentrations were lower (P < 0.05), and insulin, glucagon, and glycerol were similar when WP was given with or without Ex-4. We conclude that the hyperglycemia caused by the administration of Ex-4 concurrently with dietary protein arises from the action of peptides released during digestion and their interaction with Ex-4 in the regulation of glucose, fatty acid, and amino acid metabolism.
- amino acids
- glucagon-like peptide-1
glucagon-like peptide-1 (GLP-1) is a hormone secreted by the L cells of the distal gut in response to nutrient ingestion (17). It exerts a myriad of physiological functions through activation of the GLP-1 receptor (9). The most documented role for GLP-1 is the maintenance of glucose homeostasis by stimulating insulin secretion and biosynthesis; suppressing glucagon secretion, gastric emptying, and food intake; increasing proliferation and differentiation while inhibiting apoptosis of pancreatic β-cells; and possibly by enhancing peripheral glucose disposal (9). For this reason, GLP-1 has been of interest as a potential treatment for the management of blood glucose in diabetes, but, unfortunately, its rapid degradation by the enzyme dipeptidyl peptidase IV (DPP IV) in vivo results in a very short half-life (19). As a result, the GLP-1 receptor agonist Exendin-4 (Ex-4) has been widely used to study the biological actions of GLP-1.
Ex-4 possesses structural and pharmacokinetic properties that make it a potent GLP-1 receptor agonist suitable for therapeutic applications. Isolated from the salivary secretion of the Gila monster lizard, Ex-4 shares 53% amino acid sequence homology with GLP-1 (15). In vitro and in vivo studies demonstrated that it binds with high affinity to the GLP-1 receptor (15, 36). The presence of glycine at position 2 of the amino terminal of the peptide makes it resistant to the action of DPP IV, and thus Ex-4 has a much longer half-life in vivo than native GLP-1 (28). Ex-4 exerts powerful antidiabetic actions in animals (42) and in humans (10, 11, 21) in the presence of hyperglycemia, such as that occurring in diabetes or after ingestion of mixed meals or a glucose load. In the absence of elevated blood glucose concentrations, GLP-1 and Ex-4 do not stimulate insulin secretion, a property that protects against the insulin-induced hypoglycemia characteristic of alternative insulin secretagogues (9).
In previous experiments in which we examined the relationship between Ex-4-induced reductions in food intake and changes in blood glucose concentrations, we found that Ex-4 caused hyperglycemia in normal, nondiabetic male Wistar rats only when given before WP but not glucose or corn oil (4). The observation that the glucoregulatory action of Ex-4 is macronutrient dependent is of crucial importance because a synthetic Ex-4 has successfully undergone phase III clinical trials for the management of type 2 diabetes (11, 21). Therefore, the objectives of this study were to determine the components of protein digestion responsible for the hyperglycemic effect of Ex-4 when taken with dietary protein and to associate this effect with selected markers of metabolic regulation.
MATERIALS AND METHODS
Animals and Diets
Male Wistar rats (Charles River, Quebec, Canada) were housed individually in hanging wire-mesh stainless steel cages in a room with a temperature of 22 ± 1°C and a 12:12-h light-dark cycle (lights on at 0600) and had access to the AIN-93G diet (33). Food was available from 1800 to 0800, but water was provided for 24 h a day (5). The University of Toronto Animal Care Committee approved the protocol, and care and maintenance of the animals conformed to the guidelines of the Canadian Council on Animal Care.
The composition (in g/kg) for the AIN-93G diet (33) was casein (203), cornstarch (529.4), sucrose (100.1), soybean oil (70), cellulose (50), vitamin mixture (10), mineral mixture (35), choline bitartarate (2.5), and tertbutyl hydroquinone (0.014), as used previously (5). Corn starch, high-protein casein (87%), and cellulose were purchased from Harland Teklad (Madison, WI). The vitamin mixture, mineral mixture, choline bitartarate, and tertbutyl hydroquinone were purchased from Dyets (Bethlehem, PA), whereas sucrose and soybean oil were purchased from local suppliers in Toronto, Canada (Allied Food Service and Loblaws, respectively).
One gram of whey protein (WP, experiments 1 and 2; Sportpharma, Concord, CA; see Refs. 4 and 5), albumin from chicken egg white (AP; Sigma, St. Louis, MO; experiment 1), whey hydrolysate (WH; experiment 1; Designer Pro, Carlsbad, CA; see Ref. (5), albumin hydrolysate from chicken egg white (AH; Sigma; experiment 1), and an amino acid mixture patterned after WP (AAWP) or AP (AAAP; experiment 1; see Ref. 5), or glucose (GL; experiment 2; see Ref. 4) was dissolved in deionized water to a total volume of 2.5 ml. The nitrogen content for the proteins as determined by the manufacturers was 12.9, 13.2, 12.7, and 12.3% for WP, AP, WH, and AH, respectively. The proteins and their hydrolysates contained small amounts of carbohydrates and ash (≤7 and 4% wt/wt, respectively), were dissolved or suspended (certain amino acids) in water, and were drawn in the gavage syringe while being stirred on a magnetic stirrer to ensure homogeneity.
Ex-4 (American Peptide, Sunnyvale, CA) was prepared as described previously (4). It was diluted in sterile deionized water and divided into aliquots before being quickly frozen on dry ice. The aliquots were lyophilized in a freeze dryer, and the resulting dried peptide was stored at −20°C until used. When needed, freeze-dried peptide was allowed to come to room temperature and reconstituted using a PBS (Sigma) at pH 7.4. The reconstituted peptide was used within 1 h of preparation. All injections were given intraperitoneally in a volume of 0.5 ml. Ex-4 was given at a dose of 0.5 μg/rat (peptide content).
This study consisted of two experiments. Experiment 1 tested the effect of Ex-4 on blood glucose after intact proteins (WP and AP), protein hydrolysates (WH and AH), or amino acid mixtures (AAWP and AAAP). Different rats of similar average body weight of 350 g received oral loads of WP and AP, WH and AH, or AAWP and AAAP 5 min after the injection of either Ex-4 or PBS. Ex-4 alone was also a treatment before water gavage.
Experiment 2 tested the effect of Ex-4 on metabolic responses to WP or GL. WP was given by gavage 5 min after either PBS or Ex-4 was injected intraperitoneally, and plasma glucose, insulin, glucagon, FFA, glycerol, leptin, and amino acid concentrations were measured. GL was given by gavage 5 min after either PBS or Ex-4, and plasma glucose, insulin, and amino acid concentrations were measured. The effect of Ex-4 given before GL on these selected metabolic parameters was investigated to determine whether the actions of Ex-4 were macronutrient specific.
All animals were adapted to injection and gavage before the treatments.
In experiment 1, rats deprived of food for 9 h were divided into two or three groups of five to seven of similar average body weights and received either Ex-4 (0.5 μg) or the vehicle (0.5 ml PBS) 5 min before administration of the treatment (1 g/2.5 ml) or water (2.5 ml) load by gavage. A drop of blood for glucose measurement was obtained from the tail vein before and 15, 30, 60, and 120 min after gavage on conscious rats. Blood glucose was determined by a Precision Q.I.D glucometer using the Precision strips (Medisense, Bedford, MA; see Ref. 4).
In experiment 2, rats deprived of food for at least 6 h were killed by decapitation to obtain blood samples. Decapitation was performed on conscious rats because glucose metabolism is affected by common anesthetics, such as ketamine and pentobarbitone (29, 34). Rats of similar body weights were orally challenged with either WP or GL loads except for those used to obtain the baseline values. Rats were killed 5 min after injection with either Ex-4 or PBS or at 15, 30, 60, or 120 min after receiving the WP or GL loads with or without Ex-4. Blood was collected in tubes containing either 25 IU dried heparin for plasma glucose and amino acid determination or 0.5 ml of TED prepared from Trasylol: 50 ml 10,000 kallikrein inhibitory units/ml (Bayer, Mississauga, ON), 50 ml H2O, 1.2 g EDTA (BDH Chemicals, Toronto, ON), and 3.4 mg the DPP IV inhibitor Diprotin A (Cedarlane, Hornby, ON), for all other assays. Blood was immediately centrifuged at ≈8,000 g for 3 min. Plasma was separated into aliquots, put on ice, and frozen at −70°C.
Glucose was measured by the glucose oxidase method (Beckman 2 Glucose Analyzer; Beckman Coulter, Fullerton, CA). Total free fatty acid (FFA) was determined colorimetrically (WAKO Chemicals, Neuss, Germany). Free glycerol was determined by the glycerol kinase method using an Analox instrument (GMRD-177). Plasma insulin concentrations were determined by an ELISA method specific for rat insulin (Crystal Chemicals, Chicago, IL). Plasma glucagon and leptin concentrations were determined using RIA for rats (LINCO, St. Charles, MO).
Analysis of plasma amino acid concentrations was as previously described (8), with modifications as follows: 10 μl of plasma was added to 127.5 μl of the derivatization mixture composed of 20 μl borate buffer (Fisher Scientific, Toronto, ON, Canada), pH 9.0, 12.5 μl o-phthalic-carboxaldehyde (OPA; Aldrich, Milwaukee, WI), 80 μl methanol (Caledon, Georgetown, ON, Canada), and 5 μl of the internal standard mixture including hydroxylysine (Aldrich). Precisely 2 min after mixing plasma with the derivatization mixture, 50 μl were injected manually in a System Gold (Beckman, Mississauga, ON, Canada) reverse-phase HPLC system utilizing a 5-μm spherical C18 column, 3.9 × 150 mm (Waters, Mississauga, ON, Canada). The mobile phase, with a flow rate of 1.0 ml/min, consisted of two buffers. The initial condition at pH 7.2 was 92% buffer A [10 mM phosphate buffer and tetrahydrofuran (98.5:1.5)] and 8% buffer B [10 mM phosphate buffer and acetonitrile (50:50)], adjusted as required to optimize separation of amino acids. A linear gradient was established such that at 60 min the composition of the mobile phase was 100% buffer B. The latter condition was maintained for 7 min before returning to the initial condition. The eluted amino acids were detected fluorometrically (157 Fluorescence Detector; Beckman). Lysine was not included in the calculations because of poor elution conditions of the assay at the end of the chromatogram.
The standard solution of physiological amino acids (Beckman) was combined with asparagine, γ-aminobutyric acid, glutamine, phosphoserine, taurine, tryptophan, and ornithine. The amount injected on the column was 36.36 pmol for all amino acids except for tryptophan (18.18 pmol). Amino acids were obtained from Sigma Chemicals.
For experiment 1, the blood glucose data collected by tail prick are reported as changes from baseline concentrations (mmol/l) at 15, 30, 60, and 120 min and as incremental areas under the glucose curve (iAUC, min·mmol·l−1; see Ref. 38). Treatment effects at each sampling time were determined by either Student's t-test or one-way ANOVA followed by post hoc Duncan's multiple-range test.
Different groups of rats were killed at the selected time points in experiment 2. Therefore, to determine the effect of treatment and time as main factors and their interaction, the data were analyzed by two-way ANOVA. Student's t-test was used to compare the parameters measured at each of the blood sampling times. The statistical analyses were performed using the SAS system (SAS Institute, Cary, NC). Significance was declared at P < 0.05. All data are expressed as means ± SE.
Experiment 1: Effect of Ex-4 on Blood Glucose After Intact Proteins, Protein Hydrolysates, and Their Amino Acid Mixtures
WP and AP.
Baseline blood glucose concentrations were not different between treatment groups. The increase from baseline in blood glucose concentrations was greater after WP ± Ex-4 than after WP alone at 30 (P ≤ 0.01), 60, and 120 (P ≤ 0.02) min, as was the iAUC (181 ± 31.6 vs. 93 ± 40.4 vs. 13 ± 6.7 min·mmol·l−1, respectively; P ≤ 0.01; Fig. 1A). Similarly, a greater increase in blood glucose concentrations occurred after AP when given with Ex-4 than when given alone at 30 (P ≤ 0.01) and 60 (P ≤ 0.02) min (Fig. 1B). Again, the iAUC was larger after AP ± Ex-4 treatment than after AP treatment (148 ± 31.4 vs. 40 ± 19.9 min·mmol·l−1, respectively; P ≤ 0.05; Fig. 1B)
Ex-4 alone resulted in a blood glucose response that was intermediate between WP and WP ± Ex-4 treatments, as shown by the higher blood glucose after Ex-4 alone than after WP alone at 30 min (P = 0.01), and the intermediate iAUC (93 ± 40.4 min·mmol·l−1).
WH and AH.
Baseline blood glucose concentrations were not different between treatments. The increase from baseline in blood glucose concentrations was greater after WH ± Ex-4 than after WH alone at 60 min (P < 0.02), as was the iAUC (139 ± 36.5 vs. 45 ± 19.2 min·mmol·l−1, respectively; P ≤ 0.05; Fig. 2A). Similarly, a greater change in blood glucose concentrations occurred after AH when given with Ex-4 than when given alone at all times (P ≤ 0.05; Fig. 2B). Similarly, the iAUC was higher after AH ± Ex-4 treatment than after AH treatment (165 ± 43.6 vs. 40 ± 19.9 min·mmol·l−1, respectively; P ≤ 0.05; Fig. 2B).
AAWP and AAAP.
Baseline blood glucose concentrations were not different among treatment groups, and they were not increased by either of the amino acid mixtures given with Ex-4. However, after AAWP alone, blood glucose was lower than after AAWP ± Ex-4 but only at 60 min (P ≤ 0.05; Fig. 3A). Blood glucose was higher after AAAP than after AAAP ± Ex-4 at 15 min (P ≤ 0.05). However, the iAUC was not different between the treatments [39 ± 10.5 vs. 17 ± 6.9 min·mmol·l−1 for AAWP ± Ex-4 and AAWP, respectively (Fig. 3A), and 40 ± 19.2 vs. 35 ± 15.9 min·mmol·l−1 for AAAP ± Ex-4 vs. AAAP, respectively (Fig. 3B)].
Experiment 2: Effect of Ex-4 on Metabolic Responses to WP and GL
Plasma glucose, insulin, and glucagon.
Plasma glucose was higher (P ≤ 0.01) when Ex-4 was given with either WP or GL preloads than after WP or GL alone by two-way ANOVA (Tables 1 and 2). However, there was a significant treatment by time interaction (P ≤ 0.05) only after the WP preloads, indicating that the increase in plasma glucose with time was primarily in rats also receiving Ex-4. When the two groups were compared at specified sampling times, plasma glucose concentrations were higher after WP + Ex-4 than after WP alone at 30 min (P < 0.01), 60 min (P < 0.01), and 120 min (P < 0.05; Table 1), as observed in experiment 1. However, there were no significant differences between GL + Ex-4 and GL alone at any sampling time (Table 2).
There was no effect of either treatment or time, based on the two-way ANOVA, on plasma insulin or glucagon after gavage with either WP or GL. Mean plasma concentrations of insulin and glucagon were not different at any sampling time between the WP + Ex-4 and WP groups (Table 1) and of insulin between GL + Ex-4 and GL groups (Table 2).
Plasma FFA, free glycerol, and leptin.
Plasma FFA concentrations were higher after Ex-4 ± WP treatment than after WP (P ≤ 0.01) but increased with time (P ≤ 0.01) in both groups (Table 3) by the two-way ANOVA. Conversely, leptin was lower (P ≤ 0.01) after the Ex-4 ± WP treatment but also increased with time (P ≤ 0.01) with no interaction between the main factors (Table 3). Plasma glycerol concentrations were lower with time (P ≤ 0.05) but were not affected by treatment (Table 3). When group means were compared at each of the times that the rats were killed, plasma FFA concentrations were higher after the WP + Ex-4 treatment than after the WP treatment at 30 and 60 min (P < 0.05). Glycerol tended to be higher in the WP + Ex-4 treatment, but it was not statistically significantly different from the WP treatments. In contrast to its effect on FFA, the WP + Ex-4 treatment resulted in lower plasma leptin concentrations than the WP treatment at 30 min (P = 0.06) and 60 min (P < 0.01).
Plasma Amino Acid Concentrations and Ratios
Plasma leucine, isoleucine, valine, threonine, glutamine, alanine, and serine and total branched-chain amino acid (BCAA) and essential amino acid concentrations were affected by treatment (P ≤ 0.05 or 0.01), as shown by the two-way ANOVA. A treatment by time interaction (P ≤ 0.05) indicated that plasma concentrations of individual and total BCAAincreased with time after the WP loads but tended to decrease with time after Ex-4 was given with WP (Table 4). At 60 min, many of the amino acids in plasma of the rats receiving WP with Ex-4 were 50% of those receiving WP alone. There were no significant differences in plasma amino acids concentrations between the GL + Ex-4 and GL groups at any sampling time (data not shown).
These results corroborate our previous finding of a hyperglycemic effect of Ex-4 after WP ingestion (4). Moreover, they show that Ex-4, a GLP-1 receptor agonist, caused hyperglycemia after both whey and albumin, and that peptides, but not amino acids, arising from their digestion contribute to this response. Furthermore, the Ex-4-induced hyperglycemia after WP was associated with increased FFA and decreased plasma amino acid concentrations, suggesting that GLP-1 and peptides arising from protein digestion interact in metabolic regulation.
Hyperglycemia arose from an interaction between Ex-4 and both the WP and WH, suggesting that it is peptides arising from digestion that account for the hyperglycemia. Although the mechanism remains to be determined, the action of the peptides is probably in the gastrointestinal tract. Biologically active peptides arising from digestion of food proteins are known to exert their effects by stimulating the release of peptide hormones, which subsequently affect several physiological and metabolic functions (20). For example, caseinomacropeptide, a breakdown product of casein, induces enzymatic secretion from the pancreas, possibly through its stimulation of CCK (30), and β-conglycinin hyrdolysates from soy protein suppresses food intake by stimulating CCK secretion (27). Furthermore, peptides arising from the digestion of soy, casein, and WP suppress food intake in rats through CCK-A, opioid, and/or GLP-1 receptors (13, 31, 5). Also, opioid-like peptides derived from milk and other proteins have many biological actions (20, 24), such as enhancing the peripheral effects of catecholamines, as do endogenous opioid peptides (3, 37).
The increase in plasma glucose concentrations could arise from either decreased glucose uptake by peripheral tissues, increased hepatic glucose production, or both. Despite the higher plasma glucose concentrations in rats given WP + Ex-4 compared with those given WP alone, plasma insulin concentrations were not different between the two groups (Table 1). The higher plasma FFA concentrations found in rats receiving WP + Ex-4 compared with those receiving WP only (Table 3) are consistent with the hyperglycemia because FFA enhance hepatic glucose production and inhibit glucose uptake (6, 22). Consistent with the increase in plasma FFA indicating mobilization of stored triglycerides was a small, but not statistically significant, increase in plasma free glycerol (Table 3).
The metabolic effects of Ex-4 are consistent with a catecholamine-induced stress response (1, 7, 25, 26, 32, 37, 39) and with one report in which a hyperglycemic effect of Ex-4 given to fasted, nondiabetic female Wistar rats (23) was attributed to an increase in plasma catecholamine concentrations. Both GLP-1 and Ex-4 stimulate catecholamine synthesis and secretion (23, 40, 41). Our results suggest that dietary peptides might have enhanced this effect of Ex-4. Unfortunately, plasma catecholamine concentrations were not measured in this study because blood was not collected in a way that would prevent oxidation of catecholamines (18). Even if they had been, the large increase in catecholamines during decapitation may have masked treatment effects (16).
The inhibitory effect of Ex-4 on the increase in plasma amino acid concentrations after WP ingestion is also a novel but difficult to explain observation. Ex-4 with WP resulted in lower plasma concentrations of most amino acids, including alanine, glutamate, isoleucine, and threonine, which serve as substrates for gluconeogenesis (Table 4). Although plasma amino acids concentrations were not measured after Ex-4 given alone, the observations that plasma amino acids were not different between rats given GL alone and in combination with Ex-4 suggest that the reduction in plasma amino acids was the result of an interaction between Ex-4 and WP. Ex-4 slows gastric emptying (12), which would be expected to delay protein digestion and absorption of amino acids, but it is also possible that there was an anti-proteolytic effect of catecholamines leading to the reduction of plasma amino acid concentrations (26).
These results further support a role for GLP-1 in metabolic responses to protein. We previously reported that GLP-1 receptor activation is involved in protein-induced suppression of food intake (4, 5). Because Ex-4 is being considered as a pharmacological agent for the management of type 2 diabetes, there is some urgency to understand the mechanisms and the circumstances under which Ex-4 contributes to, rather than reduces, hyperglycemia. Many individuals with type 2 diabetes are following one of the popular high-protein/low-carbohydrate diets for weight loss (2, 12, 35), which may be a contraindication for use of Ex-4 in this population. Investigation of the effects of Ex-4 and GLP-1 after protein ingestion in human subjects should be undertaken.
In conclusion, the hyperglycemia caused by the administration of Ex-4 concurrently with dietary proteins arises from the action of peptides released during digestion and their interaction with Ex-4 in the regulation of glucose, fatty acid, and amino acid metabolism.
This work was supported by the Natural Sciences and Engineering Research Council of Canada.
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