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Nutrition and Disease

Co-Ingestion of a Protein Hydrolysate with or without Additional Leucine Effectively Reduces Postprandial Blood Glucose Excursions in Type 2 Diabetic Men¹

Ralph J. Manders^*,2, René Koopman^*, Wendy E. Sluijsmans^*, Robin van den Berg^**, Kees Verbeek^**, Wim H. Saris^*, Anton J. Wagenmakers and Luc J. van Loon^*,

^* Department of Human Biology and Department of Movement Sciences, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University, 6200 MD Maastricht, the Netherlands; ^** TNO Nutrition and Food Research, Department Analytical Sciences, 3704 HE Zeist, the Netherlands; and School of Sport and Exercise Sciences, University of Birmingham, Birmingham, B15 2TT, UK

² To whom correspondence should be addressed. E-mail: R.Manders{at}HB.unimaas.nl.

	ABSTRACT

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
This study examined postprandial plasma insulin and glucose responses after co-ingestion of an insulinotropic protein (Pro) hydrolysate with and without additional free leucine with a single bolus of carbohydrate (Cho). Male patients with long-standing Type 2 diabetes (n = 10) and healthy controls (n = 10) participated in 3 trials in which plasma glucose, insulin, and amino acid responses were determined after the ingestion of beverages of different composition (Cho: 0.7 g/kg carbohydrate, Cho+Pro: 0.7 g/kg carbohydrate with 0.3 g/kg protein hydrolysate, or Cho+Pro+Leu: 0.7 g/kg carbohydrate, 0.3 g/kg protein hydrolysate and 0.1 g/kg free leucine). Plasma insulin responses [expressed as area under the curve (AUC)] were 141 and 204% greater in patients with Type 2 diabetes and 66 and 221% greater in the controls in the Cho+Pro and Cho+Pro+Leu trials, respectively, compared with those in the Cho trial (P < 0.05). The concomitant plasma glucose responses were 15 and 12% lower in the patients with Type 2 diabetes and 92 and 97% lower in the control group in the Cho+Pro and Cho+Pro+Leu trials, respectively, compared with those in the Cho trial (P < 0.05). Plasma leucine concentrations correlated with the insulin response in all subjects (r = 0.43, P < 0.001). We conclude that co-ingestion of a protein hydrolysate with or without additional free leucine strongly augments the insulin response after ingestion of a single bolus of carbohydrate, thereby significantly reducing postprandial blood glucose excursions in patients with long-standing Type 2 diabetes.

KEY WORDS: • Type 2 diabetes • amino acids • insulin • postprandial glucose

Many studies have reported the stimulating effect of the combined ingestion of carbohydrate and protein on insulin release in vivo in humans (1,2). In addition, strong insulinotropic responses were reported after i.v. administration of various free amino acids (3–6). Leucine was identified as a particularly interesting insulin secretagogue because it both induces and enhances pancreatic ß-cell insulin secretion through its oxidative decarboxylation, and by its ability to allosterically activate glutamate dehydrogenase (7–10). In accordance, we showed that a mixture containing a protein hydrolysate with additional free leucine and/or phenylalanine has strong insulinotropic properties in humans. In healthy men, co-ingestion of this mixture with carbohydrate augments the insulin response 2- to 3-fold compared with the ingestion of only carbohydrate (11,12).

In patients with long-standing Type 2 diabetes, hyperglycemia is no longer accompanied by compensatory hyperinsulinemia. Therefore, it is generally thought that the absolute insulin secreting capacity of the pancreatic ß-cell is substantially impaired in these individuals (13,14). As a consequence, it was questioned whether amino acid–induced insulin secretion could represent an effective strategy with which to improve blood glucose homeostasis in Type 2 diabetes (11,12). We showed recently that the insulin response after continuous ingestion of large amounts of carbohydrate can be increased 2- to 4-fold in patients with long-standing Type 2 diabetes, simply by co-ingesting a protein hydrolysate, leucine, and phenylalanine mixture (15,16). Furthermore, we showed that the greater endogenous insulin response after protein/amino acid co-ingestion is accompanied by an increase in blood glucose disposal rate, resulting in a 30% lower blood glucose response compared with the ingestion of only carbohydrate (16). Even though these proof-of-principle studies suggest that protein and/or amino acid co-ingestion represents a promising strategy to improve blood glucose homeostasis in Type 2 diabetes, it should be noted that these and other findings (11,12,15–18) were all obtained in a setting in which excessive amounts of carbohydrate were continuously administered. It remains to be established whether co-ingestion of a protein and/or leucine mixture can lower postprandial blood glucose responses after the ingestion of a single, meal-like amount of carbohydrate.

In the present study, we determined the postprandial insulin, glucose, and amino acid responses after the ingestion of a single bolus of carbohydrate with or without the addition of a protein hydrolysate or a protein hydrolysate/leucine mixture in both patients with long-standing Type 2 diabetes and healthy, normoglycemic controls.

	SUBJECTS AND METHODS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Subjects. Ten male patients with long-standing Type 2 diabetes (n = 10) and 10 healthy matched control subjects participated in this study (Table 1). Exclusion criteria were impaired renal or liver function, obesity (BMI > 35 kg/m²), cardiac disease, hypertension, diabetes complications, and exogenous insulin therapy. All patients with Type 2 diabetes were using metformin only (n = 3), or metformin in combination with sulfonylurea derivatives (n = 7). Blood glucose–lowering medication was withheld for 2 d before the screening, and sulfonylureas were withheld for 2 d before each trial. 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 Medical Ethical Committee of the Academic Hospital of Maastricht.

    Design. Each subject participated in 3 trials, separated by at least 7 d, in which plasma glucose, insulin, and amino acid responses were determined after the ingestion of 3 different beverages (Cho: carbohydrate, Cho+Pro: carbohydrate with a casein protein hydrolysate, or Cho+Pro+Leu: carbohydrate, a casein protein hydrolysate, and leucine). Subjects rested in a supine position in a reclining chair for 4 h. The test beverages were provided in a randomized order and double-blind fashion.

    Protocol. After an overnight fast, subjects reported to the laboratory at 0800 by car or public transportation. A Teflon catheter (Baxter BV, Utrecht, the Netherlands) was inserted into an antecubital vein for venous blood sampling and a resting blood sample was collected. At 0 min subjects drank a single bolus (4 mL/kg) of the experimental beverage. Blood samples were drawn every 15 min for the first hour after which blood was sampled at 30-min intervals up to 240 min for measurement of plasma glucose and insulin concentrations. Plasma amino acid concentrations were determined at 1-h intervals.

    Diet and activity before testing. All subjects maintained their normal dietary and physical activity patterns throughout the entire experimental period. In addition, subjects refrained from heavy physical labor and/or exercise training for at least 3 d before each trial. Subjects filled out a food intake diary for 2 d before the first trial and were instructed to consume the same foods on the 2 d before the other 2 trials. Furthermore, the evening before each trial, all subjects consumed the same standardized meal (43.8 kJ/kg body weight; 60 Energy % (En%) carbohydrate, 28 En% fat, and 12 En% protein).

    Beverages. The subjects were administered a single bolus (4 mL/kg) containing 0.7 g/kg body weight (BW) carbohydrate (50% glucose and 50% maltodextrin, Cho) with 0.3 g/kg BW of a casein protein hydrolysate (Cho+Pro) or 0.3 g/kg BW of a casein protein hydrolysate and 0.1 g/kg BW of leucine (Cho+Pro+Leu). To investigate the modulating effect of additional protein hydrolysate ingestion, carbohydrate ingestion was the same in all trials. In accordance, free leucine was added in the Cho+Pro+Leu trial without reducing the amount of protein ingested, to evaluate whether the insulinotropic response to protein co-ingestion could be further enhanced. As such, the beverages provided were neither isocaloric nor isonitrogenous. Glucose and maltodextrin were obtained from AVEBE, crystalline leucine from BUFA, and the casein protein hydrolysate was prepared by DSM Food Specialties. The casein hydrolysate (Insuvital^TM) was obtained by enzymatic hydrolysis of sodium caseinate using a proprietary mix of proteases. Drinks were uniformly flavored by adding 0.2 g sodium saccharinate, 1.8 g citric acid, and 5 g cream vanilla flavor (Quest International) per liter of beverage.

    Blood sample analysis. Blood was collected in EDTA-containing tubes and centrifuged at 1000 x g at 4°C for 10 min. Aliquots of plasma were immediately frozen in liquid nitrogen and stored at –80°C until analyses. Glucose concentrations (Uni Kit III) were analyzed with the COBAS FARA semiautomatic analyzer (Roche). Plasma insulin was determined by RIA (HI-14K, Linco research). Free amino acids were analyzed using ion-exchange chromatography (JEOL, AminoTac JLC-500/V) with postcolumn ninhydrin derivatization with norvaline as an internal standard. Before analysis, samples were deproteinated with 5-sulfosalicylic acid. To determine glycosylated hemoglobin (HbA1c) content, a 3-mL blood sample was collected in EDTA-containing tubes and analyzed by HPLC (Bio-Rad Diamat).

    Statistics. Data are expressed as means ± SEM. Primary outcome measures were plasma glucose, insulin, and amino acid concentrations and plasma responses, calculated as AUC above baseline values. To compare plasma metabolite concentrations over time and between trials, a 2-way repeated-measures ANOVA was applied. Changes over time within each group were tested using 1-way repeated-measures ANOVA. Scheffé's post hoc test was applied in the case of a significant F-ratio to locate specific differences. Paired student's t tests were used to compare fasting and 2 h OGTT values. Significance was set at the 0.05 level of confidence. All calculations were performed using StatView 5.0 (SAS Institute).

	RESULTS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Plasma insulin concentrations. Baseline plasma insulin concentrations did not differ between groups and trials with mean values of 102.8 ± 9.4 and 102.1 ± 19.6 pmol/L for the Type 2 diabetes and control group respectively (Fig. 1A). In the patients with Type 2 diabetes, plasma insulin concentrations increased significantly only in the Cho+Pro and the Cho+Pro+Leu trials (P < 0.05). In the control group, strong increases in plasma insulin concentrations were reported in all trials (P < 0.05). This increase was more pronounced in the Cho+Pro and the Cho+Pro+Leu trials than in the Cho trial. Insulin responses (expressed as AUC) in the diabetes group were 141 ± 40 and 204 ± 37% greater in the Cho+Pro and the Cho+Pro+Leu trials, respectively, compared with the Cho trial (P < 0.05, Fig 1B). In the control group, insulin responses were 66 ± 20 and 221 ± 82% greater in the Cho+Pro and Cho+Pro+Leu trials, respectively, than in the Cho trial (P < 0.05). Furthermore, in the control group, the insulin response in the Cho+Pro+Leu trial was greater than the Cho+Pro trial (P < 0.05). The insulin response did not differ between groups within the same trial.

View larger version (18K): [in this window] [in a new window]   FIGURE 1  Plasma insulin concentrations (panel A) and responses (AUC, panel B) over a 4-h period after the ingestion of Cho, Cho+Pro, and Cho+Pro+Leu in patients with Type 2 diabetes (T2D) and healthy control subjects (CON). Values are means ± SEM, n = 10/group. (A) *Different from the Cho trial P < 0.05; #different from the Cho+Pro trial, P < 0.05. (B) Within a group, means without a common letter differ, P < 0.05. Groups within the same trial did not differ.

View larger version (16K): [in this window] [in a new window]   FIGURE 2  Plasma glucose concentrations (panel A) and response (AUC, panel B) over a 4-h period after the ingestion of Cho, Cho+Pro, and Cho+Pro+Leu in patients with Type 2 diabetes (T2D) and healthy control subjects (CON). Values are means ± SEM, n = 10/group. (A) *Different from the Cho trial, P < 0.05. (B) Within a group, means without a common letter differ, P < 0.05. *Different from the Type 2 diabetes group within the same trial, P < 0.05.

View larger version (13K): [in this window] [in a new window]   FIGURE 3  Plasma EAA without leucine and NEAA responses, expressed as AUC, over a 4-h period after the ingestion of Cho, Cho+Pro, and Cho+Pro+Leu in patients with Type 2 diabetes (A) and healthy control subjects (B). Values are means ± SEM, n = 10/group. Within a group, means without a common letter differ, P < 0.05. Groups within the same trial did not differ .

	DISCUSSION

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Numerous in vitro studies using primary pancreatic islet cells or ß-cell lines reported strong insulinotropic effects for arginine, leucine, isoleucine, alanine, and phenylalanine, but the various mechanisms by which amino acids can stimulate insulin secretion have not yet been fully elucidated (10). Both in vivo and in vitro work identified leucine as a particularly interesting insulin secretagogue because leucine both induces and enhances pancreatic ß-cell insulin secretion through its oxidative decarboxylation and its ability to allosterically activate glutamate dehydrogenase (7–10). These findings generally agree with recent in vivo observations in healthy men, showing that co-ingestion of relatively small amounts of free leucine further augments the insulin response after the combined ingestion of carbohydrate and protein (17). Furthermore, Xu et al. (9) suggested that the signals that stimulate insulin release are also responsible for the leucine-induced activation of the mammalian target of rapamycin (mTOR) signaling pathway in the pancreatic ß-cell. The latter was proposed to enhance ß-cell function through the maintenance of ß-cell mass. As such, leucine administration was suggested to be an excellent candidate to optimize the insulinotropic effects of protein co-ingestion. Therefore, we determined the postprandial plasma insulin, glucose, and amino acid responses after co-ingestion of a casein protein hydrolysate with and without additional leucine, together with a single, meal-like bolus of carbohydrate in patients with long-standing Type 2 diabetes and healthy controls.

Ingestion of carbohydrate only (Cho) resulted in a blunted insulin response in patients with Type 2 diabetes compared with the normoglycemic controls, thereby clearly demonstrating the reduced sensitivity of the pancreas to glucose ingestion in the Type 2 diabetic state (14). Co-ingestion of the casein hydrolysate (Cho+Pro) resulted in 140 and 70% greater insulin responses compared with the Cho trial in patients with Type 2 diabetes and the normoglycemic controls, respectively. The additional administration of free leucine (Cho+Pro+Leu) further stimulated insulin release, resulting in a >200% greater insulin response in both the diabetic and control group compared with the Cho trial. The insulin responses in the Cho+Pro and Cho+Pro+Leu trials in patients with Type 2 diabetes were of similar magnitude to those reported in the Cho and Cho+Pro trials in the healthy controls. Thus, even though the sensitivity of the pancreas to carbohydrate was significantly impaired in patients with long-standing Type 2 diabetes, their capacity to secrete insulin in response to both glucose and amino acids is still highly functional. These data imply that the impaired insulin response after carbohydrate ingestion in patients with Type 2 diabetes is attributed to a reduced sensitivity of the ß-cell to glucose, and does not necessarily represent an overall defect in the capacity of the pancreas to produce and/or secrete insulin.

The greater insulin response after protein or protein/leucine co-ingestion reduced the glucose response. The differences in glucose responses between the Cho and Cho+Pro or Cho+Pro+Leu trials were of similar magnitude in both groups. However, expressed relatively, the reductions in the glucose response were 15 ± 5 and 12 ± 3% in the Type 2 diabetes group, and 92 ± 2 and 97 ± 3% in the control group, respectively, compared with the Cho trial. These data extend previous findings (15,16), and show that protein/leucine co-ingestion represents an effective strategy for reducing postprandial blood glucose excursions after the ingestion of a single bolus of carbohydrate, resembling the amount of carbohydrate in a low-fat meal. Consequently, our data imply that such nutritional interventions can be applied to improve postprandial blood glucose homeostasis in patients with Type 2 diabetes. We speculated that co-ingestion of protein/leucine with every main meal could improve blood glucose homeostasis over more prolonged periods. However, because daily food intake generally includes 3 main meals with various between-meal snacks, more studies are warranted to establish the potential of protein/amino acid co-ingestion as a strategy to improve blood glucose homeostasis under daily, free-living conditions.

Increasing postprandial insulin secretion could also have other benefits for patients with Type 2 diabetes. Skeletal muscle protein breakdown rates were reported to be elevated in uncontrolled Type 2 diabetes (21). The administration of protein and/or leucine with carbohydrate may also represent an effective strategy to prevent this loss of skeletal muscle mass by increasing postprandial insulin concentrations and by providing ample precursors for protein synthesis (22). In addition, leucine administration was suggested to stimulate muscle protein synthesis by insulin-independent activation of the mTOR signaling pathway (23,24). Although we did not aim to assess the muscle protein anabolic response to protein and/or leucine co-ingestion, some interesting results were observed when evaluating the plasma amino acid concentrations. Although amino acid responses were negative after ingesting only carbohydrate, co-ingestion of the protein hydrolysate in the Cho+Pro trial produced a positive plasma amino acid response. Interestingly, additional supplementation with leucine (Cho+Pro+Leu) resulted in an 60% lower plasma EAA-Leu response compared with the Cho+Pro trial, even though the same amount of protein was ingested. These findings generally agree with those of Nair et al. (25,26), showing that leucine administration reduces most plasma amino acid concentrations. The lower amino acid responses in the Cho+Pro+Leu trial could be attributed to the proposed capacity of leucine to inhibit proteolysis and/or to stimulate protein synthesis (17,23). In addition, the lower amino acid response could also be explained by the effects of elevated insulin levels on amino acid oxidation and/or on splanchnic sequestration of amino acids. Clearly, more research is warranted to determine the effects of leucine administration on muscle protein balance in vivo in humans.

We conclude that co-ingestion of a protein hydrolysate with or without additional leucine augments endogenous insulin secretion after the consumption of a single bolus of carbohydrate, thereby substantially reducing postprandial blood glucose excursions in patients with long-standing Type 2 diabetes. Co-ingestion of a protein hydrolysate and/or leucine mixture represents an effective strategy for improving postprandial blood glucose homeostasis in patients with Type 2 diabetes.

	FOOTNOTES

 
¹ Supported by a grant from DSM Food Specialties (Delft, The Netherlands).

³ Abbreviations used: AUC, area under the curve above baseline; BW, body weight; Cho, carbohydrate trial; Cho+Pro, carbohydrate+protein trial; Cho+Pro+Leu, carbohydrate+protein+leucine trial; EAA, essential amino acid; EAA-Leu, essential amino acid minus leucine; En%, energy percent; HbA1c, glycosylated hemoglobin; OGIS, oral glucose sensitivity; OGTT, oral glucose tolerance test.

Manuscript received 5 October 2005. Initial review completed 26 October 2005. Revision accepted 8 February 2006.

	LITERATURE CITED

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