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American Journal of Clinical Nutrition, Vol. 82, No. 1, 76-83, July 2005
© 2005 American Society for Clinical Nutrition

ORIGINAL RESEARCH COMMUNICATION

Co-ingestion of a protein hydrolysate and amino acid mixture with carbohydrate improves plasma glucose disposal in patients with type 2 diabetes1,2,3

Ralph JF Manders, Anton JM Wagenmakers, René Koopman, Antoine HG Zorenc, Paul PCA Menheere, Nicolaas C Schaper, Wim HM Saris and Luc JC van Loon

1 From the Departments of Human Biology (RJFM, RK, AHGZ, WHMS, and LJCvL) and Movement Sciences (LJCvL), the Nutrition and Toxicology Research Institute Maastricht (NUTRIM), the Maastricht University, Maastricht, Netherlands; the School of Sport and Exercise Sciences, University of Birmingham, Edgbaston, United Kingdom (AJMW); and the Departments of Clinical Chemistry (PPCAM) and Internal Medicine (NCS), Maastricht University Hospital, Maastricht, Netherlands

2 Supported by a grant from DSM Food Specialties (Delft, Netherlands).

3 Address reprint requests to RJF Manders Department of Human Biology, Maastricht University, PO Box 616, 6200 MD Maastricht, Netherlands. E-mail: r.manders{at}hb.unimaas.nl.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background:Although insulin secretion after carbohydrate ingestion is severely impaired in patients with type 2 diabetes, amino acid and protein co-ingestion can substantially increase plasma insulin responses.

Objective:We investigated insulin responses and the subsequent plasma glucose disposal rates after the ingestion of carbohydrate alone (CHO) or with a protein hydrolysate and amino acid mixture (CHO+PRO) in patients with a long-term diagnosis of type 2 diabetes.

Design:Ten type 2 diabetic patients [mean (±SEM) age: 62 ± 2 y; body mass index (kg/m2): 27 ± 1] and 9 healthy control subjects (age: 58 ± 1 y; body mass index: 27 ± 1) participated in 2 trials in which the plasma insulin response was measured after the ingestion of 0.7 g carbohydrate · kg–1 · h–1 with or without 0.35 g · kg–1 · h–1 of a mixture that contained a protein hydrolysate, leucine, and phenylalanine. Continuous infusions with [6,6-2H2]glucose were then given to investigate plasma glucose disposal.

Results:Plasma insulin responses were higher by 299 ± 64% and 132 ± 63% in the CHO+PRO trial than in the CHO trial in the diabetic patients and the matched control subjects, respectively (P < 0.001). The subsequent plasma glucose responses were reduced by 28 ± 6% and 33 ± 3% in the CHO+PRO trial than in the CHO trial in the diabetic patients and the matched control subjects, respectively (P < 0.001). The reduced plasma glucose response in the diabetic patients was attributed to a 13 ± 3% increase in glucose disposal (P < 0.01).

Conclusions:The combined ingestion of carbohydrate with a protein hydrolysate and amino acid mixture significantly increases de novo insulin production in patients with a long-term diagnosis of type 2 diabetes. The increased insulin response stimulates plasma glucose disposal and reduces postprandial glucose concentrations.

Key Words: Glucose disposal • protein hydrolysate • leucine • phenylalanine • metabolism • type 2 diabetes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The stimulating effect of the combined intake of carbohydrate and protein on plasma insulin release was reported in the 1960s (1, 2) and has since been confirmed in healthy subjects (3) and in patients with type 2 diabetes (4-6). Furthermore, intravenous infusion of free amino acids was reported to increase insulin secretion (7-9). In agreement with these findings, various in vitro studies with incubated ß cells have attributed strong insulinotropic properties to arginine, leucine, and phenylalanine (10-17). We have performed various in vivo studies in which we defined an optimal insulinotropic amino acid and protein mixture containing leucine, phenylalanine, and a protein hydrolysate that has repeatedly been shown to augment the insulin response by an additional 100% in healthy subjects (18, 19). Nutritional interventions that effectively stimulate endogenous insulin secretion could be of particular significance in patients with type 2 diabetes. An increase in endogenous insulin secretion could increase blood glucose disposal and thus improve glucose homeostasis. Moreover, preventing or reducing the postprandial rise in blood glucose concentration that follows carbohydrate intake could reduce the risk of developing diabetic and cardiovascular complications (20, 21). Furthermore, the combined administration of amino acids and protein with carbohydrate, which leads to a state of hyperinsulinemia and hyperaminoacidemia, may represent an effective strategy to inhibit proteolysis and to stimulate protein synthesis (22, 23). This outcome would be of particular interest, because muscle protein breakdown rates are markedly elevated in uncontrolled diabetes (24).

In patients with a long-term diagnosis of type 2 diabetes, hyperglycemia is not accompanied by a compensatory hyperinsulinemia. As such, it is generally assumed that the capacity of the ß cell to secrete insulin is severely impaired as the result of several defects (25). These defects, which are all indicative of a progressive insensitivity of the ß cell to glucose, include a reduced early-insulin secretory response to oral glucose, a reduced ability of the ß cell to compensate for the degree of insulin resistance, a decline in the glucose-sensing ability of the ß cell, and a shift to the right in the dose-response curve relating glucose and insulin secretion (26). All of these defects involve glucose-sensing and -signaling pathways in the ß cell. Although insulin secretion in response to carbohydrate intake is impaired in patients with type 2 diabetes, we recently showed that co-ingestion of a protein and amino acid mixture can increase the plasma insulin response 2–3-fold (27). Although such nutritional interventions can effectively stimulate endogenous insulin secretion in patients with a long-term diagnosis of type 2 diabetes, the clinical significance of these interventions in regard to blood glucose homeostasis remains to be established.

In the present study, we investigated the insulinotropic properties of a combination of a mixture of protein hydrolysate, leucine, and phenylalanine with carbohydrate and the glucose disposal rate after its ingestion in patients with a long-term diagnosis of type 2 diabetes and in healthy, matched control subjects.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Ten male patients with a long-term diagnosis of type 2 diabetes and 10 healthy, matched control subjects were selected to participate in the present study. The baseline characteristics of the subjects are shown in Table 1Go. Exclusion criteria were impaired renal or liver function, obesity [body mass index (in kg/m2) >35], cardiac disease, hypertension, diabetic complications, and exogenous insulin therapy. All except one of the type 2 diabetic patients (n = 9) were using oral antidiabetic agents (metformin alone or in combination with sulfonylureas). One control subject withdrew from the experiment for personal reasons. In the type 2 diabetic patients, any medication being used was withheld for 2 d before the screening process. The subjects were screened for glucose intolerance and type 2 diabetes by use of a standard oral-glucose-tolerance test according to the World Health Organization criteria of 1999 (28). All subjects were informed about the nature and the risks of the experimental procedures before their written informed consent was obtained. All clinical trials were approved by the local medical ethical committee.


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  		TABLE 1. Subject characteristics1

 
Screening
Before selection into the study, all subjects were given an oral-glucose-tolerance test. The subjects arrived at the laboratory at 0800 by car or public transportation after having fasted overnight. A blood sample was collected from the fasting subjects, after which a bolus of 75 g glucose (dissolved in 250 mL water) was ingested (t = 0 min). After 120 min, a second blood sample was obtained. Plasma glucose concentrations were measured to determine glucose intolerance and type 2 diabetes according to the World Health Organization criteria of 1999 (28). In addition, basal fasting plasma glucose and insulin concentrations were used to assess whole-body insulin resistance with the homeostasis model assessment insulin resistance index (29), which was calculated as the product of basal fasting plasma glucose (mmol/L) and insulin (mU/L) concentrations divided by 22.5.

Medication, diet, and activity before testing
Medication that stimulates insulin production or secretion (sulfonylurea derivatives) was withheld for 2 d before each test to prevent confounding effects on amino acid–induced insulin secretion. The use of insulin sensitizers (metformin) was continued to support the benefits of increasing endogenous insulin secretion on glucose homeostasis. All subjects maintained normal dietary and physical activity patterns throughout the entire experimental period. In addition, the subjects refrained from heavy physical labor and exercise for ≥3 d before each trial and filled out a food-intake diary for 2 d before the first trial to keep their dietary intake as identical as possible before the second trial. The evening before each trial, the subjects received a standardized meal (43.80 kJ/kg body wt that consisted of 60% of energy as carbohydrate, 28% of energy as fat, and 12% of energy as protein).

Design
Each subject participated in 2 trials, separated by a 2-wk period, in which the plasma insulin response and subsequent plasma glucose disposal rate were measured after the ingestion of 2 different beverage compositions (CHO, carbohydrate only; or CHO+PRO, carbohydrate and a mixture that contained a protein hydrolysate and the free amino acids leucine and phenylalanine). The subjects were placed in a supine position and remained inactive for 3 h. Drinks were provided in a randomized order and a double-blind fashion. The beverages were flavored to make the taste comparable in both trials (see below).

Protocol
The subjects reported to the laboratory at 0800 after an overnight fast. A catheter (Baxter BV, Utrecht, Netherlands) was inserted into an antecubital vein for the isotope infusion. Another catheter was inserted into a dorsal vein on the contralateral hand and was placed in a hot-box (60°C) for arterialized blood sampling. After 10 min, a blood sample was collected from the resting subjects (t = 0 min). After the administration of an intravenous bolus of 13.5 µmol [6,6-2H2]glucose/kg, a continuous infusion of 277 ± 3 nmol · kg–1 · min–1 of [6,6-2H2]glucose was started via a calibrated IVAC 560 pump (IVAC Corp, San Diego, CA) and continued until t = 180 min. At t = 0 min, the subjects drank an initial bolus (2 mL/kg) of the test drink (CHO or CHO+PRO). Repeated boluses (2 mL/kg) were ingested every 15 min until t = 165 min. Blood samples were drawn every 15 min during the first hour and then every 30 min until t = 180 min for the measurement of plasma glucose, glucose enrichment, and insulin. In addition, proinsulin and C-peptide concentrations were measured in the blood samples that were collected at t = 0, 60, 120, and 180 min.

Beverages
The subjects received repeated boluses of 2 mL/kg to ensure a given dose of 0.7 g carbohydrate · kg–1 · h–1 (50% as glucose and 50% as maltodextrin) with or without 0.35 g · kg–1 · h–1 of a protein hydrolysate and amino acid mixture (50% as casein hydrolysate, 25% as free leucine, and 25% as free phenylalanine) every 15 min until t = 165 min. Glucose and maltodextrin were obtained from AVEBE (Veendam, Netherlands), crystalline amino acids were from BUFA (Uitgeest, Netherlands), and the casein protein hydrolysate was prepared by DSM Food Specialties (Delft, Netherlands). The casein hydrolysate (Insuvital; DSM Food Specialties) was obtained by enzymatic hydrolysis of sodium caseinate with the use of a neutral protease and a prolyl-specific endoproteinase. Both drinks were uniformly flavored by the addition of 0.2 g sodiumsaccharinate, 1.8 g citric acid, and 5 g of a cream vanilla flavor (Quest International, Naarden, Netherlands) for each 1 L of beverage.

Isotope tracer calculations
The glucose tracer (99% enriched; Cambridge Isotope laboratories, Andover, MA) was first dissolved in 0.9% saline. The glucose tracer concentration in the infusates averaged 22 ± 0.4 mmol/L. The [6,6-2H2]glucose infusion rate averaged 277 ± 3 nmol · kg–1 · min–1. Plasma glucose enrichments are expressed as tracer/tracee ratios. The rate of appearance (Ra) and rate of disappearance (Rd) of glucose were calculated with the use of the single-pool non–steady state Steele equations (30) adapted for stable-isotope studies as described elsewhere (31).

(1)

(2)
where F is the infusion rate (in µmol · kg–1 · min–1); V is the distribution volume for glucose (160 mL/kg); C1 and C2 are the glucose concentrations (in mmol/L) at time 1 (t1) and 2 (t2), respectively; and E1 and E2 are the plasma glucose enrichments (tracer/tracee ratios) at time 1 and 2, respectively.

Blood sample analysis
Blood (10 mL) was collected in EDTA-containing tubes and centrifuged at 1000 x g for 10 min at 4°C. Aliquots of plasma were immediately frozen in liquid nitrogen and stored at –80°C until analyzed. Glucose concentrations (Uni Kit III; Roche, Basel, Switzerland) were analyzed with the COBAS FARA semiautomatic analyzer (Roche). Plasma insulin, proinsulin, and C-peptide concentrations were assayed with a modified, solid-phase, 2-site fluoroimmunometric assay based on a direct sandwich technique (DELFIA method; Perkin Elmer, Turku, Finland). To measure the glycated hemoglobin content, a 3-mL blood sample was collected in EDTA-containing tubes and was analyzed by HPLC (Bio-Rad Diamat, Munich, Germany). After derivatization of the plasma samples, plasma [6,6-2H2]glucose enrichment was measured by electron ionization gas chromatography–mass spectrometry (Finnigan INCOS-XL; Finnigan Mat, Ilemel, Hemstead, United Kingdom).

Statistics
Data are expressed as means ± SEMs. The plasma responses were calculated as the area under the curve minus baseline values. To compare plasma metabolite concentrations and tracer kinetics over time between trials, a two-way repeated-measures analysis of variance (ANOVA) was performed. Subgroups were analyzed further whenever significant time-by-treatment interactions were observed. Changes in time within each group were checked for statistical significance with the use of a one-way repeated-measures ANOVA. When an F ratio was significant, a Scheffe's post hoc test was performed to locate specific differences. For non-time-dependent variables, a multiway ANOVA alone or with a Student's t test for unpaired observations was used. Significance was set at P < 0.05. All calculations were performed with STATVIEW 5.0 (SAS Institute Inc, Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Insulin
Plasma insulin concentrations in subjects that had fasted overnight were similar in both groups and in both trials. Insulin concentrations increased significantly in both groups after the ingestion of carbohydrate alone and carbohydrate with the protein and amino acid mixture (P < 0.001; Figure 1Go A). A repeated-measures ANOVA showed a significant time-by-treatment interaction for plasma insulin concentrations (P < 0.01). After t = 60 min, plasma insulin concentrations in the diabetes group were higher in the CHO+PRO trial than in the CHO trial (P < 0.05). No significant differences were found between trials in the control group. After the insulin response was expressed as the area under the curve (minus baseline values), significantly greater plasma insulin responses were observed in the CHO+PRO trial than in the CHO trial in both groups (P < 0.01, Figure 1BGo). Plasma insulin responses were 299 ± 64% and 132 ± 63% greater in the CHO+PRO trial than in the CHO trial in the diabetes and control groups, respectively (P < 0.01).



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  		FIGURE 1.. Mean (±SEM) plasma insulin concentrations (A) and responses (B) over a 3-h period after the ingestion of carbohydrate (CHO; open symbols) or carbohydrate and a protein hydrolysate and amino acid mixture (CHO+PRO; filled symbols) in patients with type 2 diabetes (n = 10) and in healthy, matched control subjects (n = 9). (A) Insulin concentrations increased significantly over time in both groups and trials, P < 0.001. No significant differences were found between trials in the control group. There was a significant time x treatment interaction for insulin (P < 0.01). *Significantly different from the CHO trial in the diabetes group, P < 0.05 (Scheffe's adjustment). (B)*Significantly different from the CHO trial, P < 0.05 (ANOVA).

 
C-peptide and proinsulin
Plasma C-peptide concentrations in fasting subjects were similar in both groups. A repeated-measures ANOVA showed a significant time-by-treatment interaction for plasma C-peptide concentrations (P < 0.01). In both trials, C-peptide concentrations increased significantly over time (P < 0.05; Figure 2Go). After t = 60 min, plasma C-peptide concentrations in the diabetes group were significantly higher in the CHO+PRO trial than in the CHO trial (P < 0.05). When expressed as the area under the curve, significantly greater C-peptide responses were observed in the CHO+PRO trial than in the CHO trial in both groups (P < 0.01). Plasma C-peptide responses were 98 ± 18% and 56 ± 26% greater in the CHO+PRO trial than in the CHO trial in the diabetes and control groups, respectively (P < 0.01). Plasma C-peptide concentrations correlated well with plasma insulin concentrations (r = 0.89, P < 0.001).



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  		FIGURE 2.. Mean (±SEM) plasma C-peptide and proinsulin concentrations over a 3-h period after the ingestion of carbohydrate (CHO) or carbohydrate and a protein hydrolysate and amino acid mixture (CHO+PRO) in patients with type 2 diabetes (n = 10) and in healthy, matched control subjects (n = 9). We found a significant time x treatment interaction for C-peptide and proinsulin (P < 0.01 for all). C-peptide concentrations increased significantly over time in both trials and groups, P < 0.05. After t = 60 min, C-peptide concentrations were significantly different between trials in the diabetes group (Scheffe's adjustment); no significant differences were found between trials in the control group. Proinsulin concentrations increased significantly over time in both trials and groups, P < 0.01. After t = 120 min, proinsulin concentrations were significantly different between trials in the diabetes group (Scheffe's adjustment); no significant differences were found between trials in the control group. *Significantly different from the CHO trial, P < 0.05.

 
Plasma proinsulin concentrations in fasting subjects were higher in the type 2 diabetes group than in the normoglycemic control subjects (28.3 ± 2.9 mmol/L compared with 7.5 ± 0.5 mmol/L, respectively P < 0.01). In both trials, proinsulin concentrations increased significantly over time (P < 0.01; Figure 2Go) and showed a significant time-by-treatment interaction (P < 0.01). After t = 120 min, plasma proinsulin concentrations in the diabetes group were higher in the CHO+PRO trial than in the CHO trial (P < 0.05). No significant differences were observed between trials in the control group. When expressed as the area under the curve, significantly greater proinsulin responses were observed in the CHO+PRO trial than in the CHO trial in both groups (P < 0.05). The plasma proinsulin responses were 151 ± 28% and 84 ± 37% greater in the CHO+PRO trial than in the CHO trial in the diabetes and the control groups, respectively (P < 0.05). Plasma proinsulin concentrations correlated with both plasma insulin and plasma C-peptide concentrations (r = 0.79 and r = 0.85, respectively; P < 0.001)

Glucose
Plasma glucose concentrations in fasting subjects were higher in the type 2 diabetic patients than in the normoglycemic control subjects (9.7 ± 0.3 mmol/L compared with 5.7 ± 0.1 mmol/L, respectively; P < 0.01). A repeated-measures ANOVA showed a significant time-by-treatment interaction for plasma glucose concentrations (P < 0.01). In the type 2 diabetic patients, plasma glucose concentrations in the CHO trial increased after carbohydrate ingestion until t = 150 min, after which values reached a plateau. In the CHO+PRO trial, glucose concentrations increased significantly during the first 90 min (P < 0.01), after which they either reached a plateau or tended to decline (Figure 3Go A). At t = 180 min, the plasma glucose concentration was significantly lower in the CHO+PRO trial than in the CHO trial (P < 0.05) for the type 2 diabetes group. In the control group, plasma glucose concentrations slightly increased during the first 60 min in both trials and then returned to baseline levels over the next 2 h (Figure 3AGo). Plasma glucose concentrations were significantly higher in the type 2 diabetic patients than in the matched control subjects (P < 0.05). After expressing the plasma glucose response as the area under the curve, we observed a significantly higher plasma glucose response in the type 2 diabetic patients than in the matched normoglycemic control subjects (P < 0.001; Figure 3BGo). In both groups, significantly lower plasma glucose responses were observed in the CHO+PRO trial than in the CHO trial (P < 0.001; Figure 3BGo). The plasma glucose response was 28 ± 6% and 33 ± 3% lower in the CHO+PRO trial than in the CHO trial in the diabetes and matched control groups, respectively (P < 0.001).



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  		FIGURE 3.. Mean (±SEM) plasma glucose concentrations (A) and responses (B) over a 3-h period after the ingestion of carbohydrate (CHO; open symbols) or carbohydrate and a protein hydrolysate and amino acid mixture (CHO+PRO; filled symbols) in patients with type 2 diabetes (n = 10) and in healthy, matched control subjects (n = 9). (A) Glucose concentrations increased significantly over time in both trials for the diabetic patients, P < 0.01. There was a significant time x treatment interaction for glucose, P < 0.01. *Significantly different from the CHO trial for both groups, P < 0.05 (Scheffe's adjustment). (B)*,#Significantly different from the CHO trial (ANOVA): *P < 0.05, #P < 0.001.

 
Glucose tracer kinetics
In the type 2 diabetes group, the plasma glucose Ra was stable over the entire testing period and averaged 42.4 ± 0.8 and 41.2 ± 1.1 µmol · kg–1 · min–1 in the CHO and CHO+PRO trials, respectively. In the control group, the plasma glucose Ra was also stable and averaged 39.8 ± 0.7 and 37.9 ± 0.8 µmol · kg–1 · min–1 in the CHO and CHO+PRO trials, respectively (Table 2Go and Figure 4Go A and B). No significant differences in the plasma glucose Ra were observed between trials or groups.


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  		TABLE 2. Plasma glucose kinetics1

 


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  		FIGURE 4.. Mean (±SEM) plasma glucose rate of appearance (Ra: A and B) and disappearance (Rd: C and D) over time in patients with type 2 diabetes (A and C) and in healthy, matched control subjects (B and D) after the ingestion of carbohydrate (CHO) or carbohydrate and a protein hydrolysate and amino acid mixture (CHO+PRO) in patients with type 2 diabetes (n = 10) and in healthy, matched control subjects (n = 9). No significant differences in Ra were observed between trials or groups. Glucose Rd increased over time in both trials in both groups, P < 0.05. The increase in Rd over time was significantly different between groups (P < 0.05) but not between trials. *Significantly different from t = 30 min, P < 0.05.

 
The glucose Rd increased over time in both trials and in both groups (P < 0.05; Figure 4C and DGo). In the diabetes group, the Rd averaged 19.7 ± 2.4 and 20.4 ± 2.8 µmol · kg–1 · min–1 at t = 30 min and increased over time to reach 45.1 ± 1.8 and 45.4 ± 3.6 µmol · kg–1 · min–1 in the CHO and CHO+PRO trials, respectively (Figure 4CGo). In the control group, the Rd averaged 14.7 ± 1.4 and 19.4 ± 1.7 µmol · kg–1 · min–1 at t = 30 min and increased to 45.4 ± 2.4 and 44.8 ± 2.2 µmol · kg–1 · min–1 in the CHO and CHO+PRO trials, respectively (Figure 4DGo). The increase in the Rd over time was significantly different between groups (P < 0.05).

Plasma glucose disposal, expressed as the percentage of the appearing glucose that disappears from the circulation, was significantly lower in the diabetic patients than in the matched control subjects (P < 0.001; Table 2Go). In the diabetes group, plasma glucose disposal was 12.5 ± 3.1% higher in the CHO+PRO trial than in the CHO trial (P < 0.01). In the control group, plasma glucose disposal was not significantly improved in the CHO+PRO trial (3.4 ± 2.2%; P = 0.2; Table 2Go).

In the diabetes group, the glucose disposal rate was significantly improved by 15.8 g ({approx}88 mmol) over the 150-min period in the CHO+PRO trial compared with the CHO trial (P < 0.01). In the control group, an additional 11.7 g ({approx}65 mmol) glucose was disposed of during the 150-min period in the CHO+PRO trial compared with the CHO trial (P = 0.2).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study showed that co-ingestion of carbohydrate with a mixture containing casein hydrolysate, leucine, and phenylalanine substantially increased insulin secretion when compared with the ingestion of carbohydrate alone. The substantial 3–4-fold greater insulin response significantly improved postprandial glucose disposal and resulted in lower plasma glucose concentrations in type 2 diabetic patients. This study indicates that nutritional interventions that improve endogenous insulin secretion can be practical and effective tools in the treatment of type 2 diabetes.

The synergistically stimulating effect of the combined ingestion of carbohydrate and intact protein on plasma insulin release was first reported in the late 1960s (1, 2) and was later confirmed in both healthy subjects (3) and type 2 diabetic patients (4-6). Floyd et al (7-9, 32) investigated the effects of intravenous infusions of various amino acids on plasma insulin secretion and reported that arginine, leucine, and phenylalanine were the most insulinotropic amino acids. We have confirmed many of these findings after testing the oral administration of these amino acids in combination with carbohydrate (18, 19). Consequently, we defined a practical and optimal insulinotropic amino acid and protein mixture composed of a protein hydrolysate, free leucine, and phenylalanine (18, 19). Recently, we investigated the insulinotropic properties of this mixture in patients with a long-term diagnosis of type 2 diabetes and reported a 189% greater plasma insulin response in these patients when the mixture was co-ingested with carbohydrate than when carbohydrate was ingested alone (27). Although that study clearly showed that endogenous insulin secretion can be substantially increased in patients with a long-term diagnosis of type 2 diabetes, the clinical relevance of these findings had not yet been established. Therefore, in the present study, we investigated plasma glucose disposal after the ingestion of carbohydrate with or without the addition of such an insulinotropic protein hydrolysate and amino acid mixture in healthy subjects and in type 2 diabetic patients.

The patients with type 2 diabetes who were selected for this study had been diagnosed with type 2 diabetes for ≥10 y. Basal fasting glucose concentrations, oral-glucose-tolerance test values, glycated hemoglobin content, and the homeostasis model assessment insulin resistance index values confirmed their type 2 diabetic state (Table 1Go). Hyperinsulinemia, a compensatory response to the prevailing hyperglycemia, was no longer present in these patients (Table 1Go and Figure 1Go). After ingestion of only carbohydrate in the CHO trial, insulin responses were substantially lower in the diabetic patients than in the control subjects (Figure 1BGo). This finding clearly illustrates the reduced sensitivity of the ß cell to glucose in the type 2 diabetic state (26). Interestingly, co-ingestion of carbohydrate with the protein hydrolysate and amino acid mixture in the CHO+PRO trial significantly increased the plasma insulin response by 299 ± 64% and 132 ± 63% in the diabetic patients and the normoglycemic control subjects, respectively (P < 0.01; Figure 1BGo). The insulin response in the CHO+PRO trial in the type 2 diabetic patients was similar to the insulin response reported in the CHO trial in the healthy subjects (Figure 1BGo). In other words, although the sensitivity of the pancreas to carbohydrate intake is significantly reduced in patients with a long-term diagnosis of type 2 diabetes, the capacity to secrete insulin in response to other stimuli (such as amino acids) remains intact. Therefore, the defects in the insulin response after the ingestion of a meal in these patients are mainly attributed to the reduced sensitivity of the ß cell to glucose and not to an overall defect in the capacity to produce or to secrete insulin.

To confirm that the elevated plasma insulin concentrations in the CHO+PRO trial are indeed secondary to increased insulin production, we measured plasma C-peptide and proinsulin concentrations according to the method of Hovorka and Jones (33). In the process of insulin production, the precursor proinsulin is cleaved into insulin and the 31-kD residue connecting peptide (C-peptide). Insulin, C-peptide, and a small amount of residual proinsulin are stored in the secretory granules of the ß cell until secretion (34). In the present study, we observed a significant increase in plasma C-peptide and proinsulin concentrations over time in all trials (Figure 2Go). Significantly greater plasma C-peptide responses were observed in the CHO+PRO than in the CHO trial (98 ± 18% and 56 ± 26% in the diabetic patients and healthy control subjects, respectively; P < 0.01). Similarly, plasma proinsulin responses were also 151 ± 28% and 84 ± 37% greater in the CHO+PRO trial than in the CHO trial in the diabetes and control groups, respectively (P < 0.05). Both C-peptide and proinsulin concentrations correlated well with plasma insulin concentrations (r = 0.89 and r = 0.79, respectively: P < 0.001). Thus, these data further support the observation that co-ingestion of carbohydrate with the protein and amino acid mixture in the CHO+PRO trial effectively stimulates de novo insulin production.

In response to the increased insulin production and secretion rate in the CHO+PRO trial, plasma glucose concentrations were significantly decreased when compared with values observed in the CHO trial (Figure 3AGo). In the CHO+PRO trial, plasma glucose responses were decreased by as much as 28 ± 6% and 33 ± 3% in the diabetic patients and normoglycemic control subjects, respectively, compared with responses in the CHO trial (P < 0.001). This decrease in the plasma glucose response is much more prominent than in our earlier observations (27), which can be explained by the longer trial duration in the present study. Interventions that effectively reduce the postprandial rise in plasma glucose concentrations after carbohydrate intake are of clinical significance and have been associated with a reduced risk of developing diabetic and cardiovascular complications (20, 21). Many food components or pharmacologic agents that effectively lower postprandial glucose concentration after meal ingestion inhibit gastric emptying or intestinal uptake of glucose or both (35-37). In the present study, we applied a continuous infusion of a [6,6-2H2]glucose tracer to measure the Ra of glucose in the circulation. Plasma glucose Ras were similar in both groups and trials and remained constant throughout the trials (Table 2Go, Figure 4A and BGo). This finding indicates that inhibition of gastrointestinal uptake of glucose is not responsible for the observed decline in the postprandial blood glucose response after co-ingestion of carbohydrate with the protein and amino acid mixture.

Whereas the plasma glucose Ra remained stable throughout the trials, the plasma glucose Rd from the circulation significantly increased over time in both trials (Figure 4Go; P < 0.01). In contrast with the Ra values, the plasma glucose Rd was strikingly different between the diabetic patients and the healthy, matched control subjects (Figure 4C and DGo). Whereas Rd values increased exponentially in the control subjects, a more gradual rise in the glucose Rd was observed in the diabetic patients (P < 0.01). It took about twice as long in the diabetic patients as in the healthy subjects for the plasma glucose Ra to be matched by its Rd (P < 0.01). Consequently, plasma glucose disposal (calculated as Rd expressed as a percentage of Ra) was significantly lower in the diabetic patients than in the normoglycemic control subjects (Table 2Go; P < 0.01). In both groups, the time for the Rd to match the Ra was significantly reduced in the CHO+PRO trial (Table 2Go; P < 0.01). Accordingly, plasma glucose disposal after co-ingestion of carbohydrate with the protein and amino acid mixture improved plasma glucose disposal by 13 ± 3% (P < 0.01) and 3 ± 2% (P = 0.2) in diabetic patients and healthy control subjects, respectively.

In conclusion, ingestion of a protein hydrolysate, leucine, and phenylalanine mixture can substantially augment insulin responses after carbohydrate intake. In patients with a long-term diagnosis of type 2 diabetes, co-ingestion of carbohydrate with such a mixture can induce a 3–4-fold greater plasma insulin response than ingestion of carbohydrate alone. This response effectively improves plasma glucose disposal and thereby reduces the postprandial plasma glucose concentration. The combined ingestion of an amino acid and protein mixture with carbohydrate represents an effective interventional strategy in the treatment of type 2 diabetes.


   ACKNOWLEDGMENTS
 
We thank Jos Stegen, Joan Senden, and Annemie Gijsen for their expert analytic assistance and thank the subjects who volunteered to participate in these trials for their enthusiastic support.

RJFM, AJMW, and LJCvL designed the study. RJFM organized and carried out the clinical trials with the assistance of RK and AHGZ. RJFM performed the statistical analysis and wrote the manuscript together with LJCvL. PPCAM performed the plasma proinsulin, insulin, and C-peptide analyses. NCS and WHMS provided medical assistance. None of the authors had a personal or financial conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pallotta JA, Kennedy PJ. Response of plasma insulin and growth hormone to carbohydrate and protein feeding. Metabolism 1968;17:901–8.[Medline]
  2. Rabinowitz D, Merimee TJ, Maffezzoli R, Burgess JA. Patterns of hormonal release after glucose, protein, and glucose plus protein. Lancet 1966;2:454–6.[Medline]
  3. Nuttall FQ, Gannon MC, Wald JL, Ahmed M. Plasma glucose and insulin profiles in normal subjects ingesting diets of varying carbohydrate, fat, and protein content. J Am Coll Nutr 1985;4:437–50.[Abstract]
  4. Nuttall FQ, Mooradian AD, Gannon MC, Billington C, Krezowski P. Effect of protein ingestion on the glucose and insulin response to a standardized oral glucose load. Diabetes Care 1984;7:465–70.[Abstract]
  5. Gannon MC, Nuttall FQ, Lane JT, Burmeister LA. Metabolic response to cottage cheese or egg white protein, with or without glucose, in type II diabetic subjects. Metabolism 1992;41:1137–45.[Medline]
  6. Gannon MC, Nuttall FQ, Grant CT, Ercan-Fang S, Ercan-Fang N. Stimulation of insulin secretion by fructose ingested with protein in people with untreated type 2 diabetes. Diabetes Care 1998;21:16–22.[Abstract]
  7. Floyd JC Jr, Fajans SS, Conn JW, Knopf RF, Rull J. Stimulation of insulin secretion by amino acids. J Clin Invest 1966;45:1487–502.
  8. Floyd JC Jr, Fajans SS, Pek S, Thiffault CA, Knopf RF, Conn JW. Synergistic effect of essential amino acids and glucose upon insulin secretion in man. Diabetes 1970;19:109–15.[Medline]
  9. Floyd JC Jr, Fajans SS, Pek S, Thiffault CA, Knopf RF, Conn JW. Synergistic effect of certain amino acid pairs upon insulin secretion in man. Diabetes 1970;19:102–8.[Medline]
  10. Blachier F, Leclerc Qmeyer V, Marchand J, et al. Stimulus-secretion coupling of arginine-induced insulin release. Functional response of islets to L-arginine and L-ornithine. Biochim Biophys Acta 1989;1013:144–51.[Medline]
  11. Sener A, Hutton JC, Malaisse WJ. The stimulus-secretion coupling of amino acid-induced insulin release. Synergistic effects of L-glutamine and 2-keto acids upon insulin secretion. Biochim Biophys Acta 1981;677:32–8.[Medline]
  12. Malaisse WJ, Plasman PO, Blachier F, Herchuelz A, Sener A. Stimulus-secretion coupling of arginine-induced insulin release: significance of changes in extracellular and intracellular pH. Cell Biochem Funct 1991;9:1–7.[Medline]
  13. Lajoix AD, Reggio H, Chardes T, et al. A neuronal isoform of nitric oxide synthase expressed in pancreatic beta-cells controls insulin secretion. Diabetes 2001;50:1311–23.[Abstract/Free Full Text]
  14. McClenaghan NH, Barnett CR, O'Harte FP, Flatt PR. Mechanisms of amino acid-induced insulin secretion from the glucose-responsive BRIN-BD11 pancreatic B-cell line. J Endocrinol 1996;151:349–57.[Abstract/Free Full Text]
  15. Pipeleers DG, Schuit FC, in't Veld PA, et al. Interplay of nutrients and hormones in the regulation of insulin release. Endocrinology 1985;117:824–33.[Abstract/Free Full Text]
  16. Xu G, Kwon G, Cruz WS, Marshall CA, McDaniel ML. Metabolic regulation by leucine of translation initiation through the mTOR-signaling pathway by pancreatic beta-cells. Diabetes 2001;50:353–60.[Abstract/Free Full Text]
  17. Schwanstecher C, Meyer M, Schwanstecher M, Panten U. Interaction of N-benzoyl-D-phenylalanine and related compounds with the sulphonylurea receptor of beta-cells. Br J Pharmacol 1998;123:1023–30.[Medline]
  18. van Loon LJC, Saris WHM, Verhagen H, Wagenmakers AJM. Plasma insulin responses following the ingestion of different amino acid or protein carbohydrate mixtures. Am J Clin Nutr 2000;72:96–105.[Abstract/Free Full Text]
  19. van Loon LJC, Kruijshoop M, Verhagen H, Saris WHM, Wagenmakers AJM. Ingestion of protein hydrolysate and amino acid–carbohydrate mixtures increases postexercise plasma insulin response in men. J Nutr 2000;130:2508–13.[Abstract/Free Full Text]
  20. Ceriello A. The possible role of postprandial hyperglycemia in the pathogenesis of diabetic complications. Diabetologia 2003;46(suppl):M9–16.
  21. Ceriello A. Impaired glucose tolerance and cardiovascular disease: the possible role of post-prandial hyperglycemia. Am Heart J 2004;147:803–7.[Medline]
  22. Biolo G, Williams BD, Fleming RY, Wolfe RR. Insulin action on muscle protein kinetics and amino acid transport during recovery after resistance exercise. Diabetes 1999;48:949–57.[Abstract]
  23. Volpi E, Ferrando AA, Yeckel CW, Tipton KD, Wolfe RR. Exogenous amino acids stimulate net muscle protein synthesis in the elderly. J Clin Invest 1998;101:2000–7.[Medline]
  24. Charlton M, Nair KS. Protein metabolism in insulin-dependent diabetes mellitus. J Nutr 1998;128(suppl):S323–7.
  25. Porte D Jr, Kahn SE. Beta-cell dysfunction and failure in type 2 diabetes: potential mechanisms. Diabetes 2001;50(suppl):S160–3.[Free Full Text]
  26. Polonsky KS, Sturis J, Bell GI. Seminars in medicine of the Beth Israel Hospital, Boston. Non-insulin-dependent diabetes mellitus—a genetically programmed failure of the beta cell to compensate for insulin resistance N Engl J Med 1996;334:777–83.[Free Full Text]
  27. van Loon LJC, Kruijshoop M, Menheere PPCA, Wagenmakers AJM, Saris WHM, Keizer HA. Amino acid ingestion strongly enhances insulin secretion in patients with long-term type 2 diabetes. Diabetes Care 2003;26:625–30.[Abstract/Free Full Text]
  28. Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabet Med 1998;15:539–53.[Medline]
  29. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–9.[Medline]
  30. Steele R. Influences of glucose loading and of injected insulin on hepatic glucose output. Ann N Y Acad Sci 1959;82:420–30.
  31. Wolfe RR, Jahoor F. Recovery of labeled CO2 during the infusion of C-1- vs. C-2-labeled acetate: implications for tracer studies of substrate oxidation. Am J Clin Nutr 1990;51:248–52.[Abstract/Free Full Text]
  32. Floyd JC Jr, Fajans SS, Conn JW, Thiffault C, Knopf RF, Guntsche E. Secretion of insulin induced by amino acids and glucose in diabetes mellitus. J Clin Endocrinol Metab 1968;28:266–76.[Abstract/Free Full Text]
  33. Hovorka R, Jones RH. How to measure insulin secretion. Diabetes Metab Rev 1994;10:91–117.[Medline]
  34. Steiner D, Kemmler W, Clark J, et al. The biosynthesis of insulin. In: Steiner D, Freinkel N, eds. Handbook of physiology-endocrinology. Baltimore: Wiliams & Wilkins, 1972:175.
  35. Pelletier X, Thouvenot P, Belbraouet S, et al. Effect of egg consumption in healthy volunteers: influence of yolk, white or whole-egg on gastric emptying and on glycemic and hormonal responses. Ann Nutr Metab 1996;40:109–15.[Medline]
  36. Ranganath L, Norris F, Morgan L, Wright J, Marks V. Delayed gastric emptying occurs following acarbose administration and is a further mechanism for its anti-hyperglycaemic effect. Diabet Med 1998;15:120–4.[Medline]
  37. Liljeberg H, Bjorck I. Delayed gastric emptying rate may explain improved glycaemia in healthy subjects to a starchy meal with added vinegar. Eur J Clin Nutr 1998;52:368–71.[Medline]
Received for publication December 15, 2004. Accepted for publication March 21, 2005.




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