Evaluate the efficacy of protein hydrolysate co-ingestion as a dietary strategy to improve blood glucose homeostasis under free-living conditions in long-standing type 2 diabetes patients.
A total of 13 type 2 diabetes patients were enrolled in a randomized, double-blind cross-over design and studied on two occasions for 40 h under strict dietary standardization but otherwise normal, free-living conditions. In one trial, subjects ingested a protein hydrolysate (0.4 g kg−1 bw casein hydrolysate, PRO) with every main meal. In the other trial, a placebo was ingested (PLA). Blood glucose concentrations were assessed by continuous glucose monitoring.
Average 24 h glucose concentrations were similar between the PLA and the PRO trials (8.9±0.8 vs 9.2±0.7 mmol l−1, respectively). Hyperglycemia (glucose concentrations >10 mmol l−1) was experienced 34±9% of the time (8±2 h per 24 h) in the PLA trial. Protein hydrolysate co-ingestion with each main meal (PRO) did not reduce the prevalence of hyperglycemia (39±10%, 9±2 h per 24 h; P=0.2).
Co-ingestion of a protein hydrolysate with each main meal does not improve glucose homeostasis over a 24 h period in long-standing type 2 diabetes patients.
Glycemic control is the main objective in type 2 diabetes management. Both the UK Prospective Diabetes Study (UKPDS, 1998a, 1998b; Stratton et al., 2000) and the Diabetes Control and Complications Trial (DCCT, 1993) have shown that improved glycemic control effectively reduces the risk of developing micro- and macrovascular complications and cardiovascular disease. Glycemic control is generally assessed by measuring either HbA1c content or fasting glucose concentrations. However, these parameters provide little information on the prevalence of hyperglycemia throughout the day. Recent data applying continuous glucose monitoring in well-controlled type 2 diabetes patients have shown that daily postprandial glucose excursions leading to hyperglycemia are more prevalent than expected (Praet et al., 2006). These postprandial glucose excursions represent a direct and independent risk factor for the development of cardiovascular complications in patients with type 2 diabetes (Ceriello, 2003; Heine et al., 2004; Ceriello. 2005). Therefore, therapeutic strategies in the treatment of type 2 diabetes should aim to reduce postprandial hyperglycemia.
In long-standing type 2 diabetes patients, hyperglycemia is no longer accompanied by compensatory hyperinsulinemia and, as such, it is generally assumed that insulin secretory capacity of the β-cell is severely impaired. Such a defect is indicative of a progressive insensitivity of the β-cell to glucose (Porte and Kahn, 2001). Even though insulin secretion in response to glucose ingestion is blunted in long-standing type 2 diabetes patients, we have shown that co-ingestion of a protein hydrolysate/leucine mixture can stimulate endogenous insulin secretion resulting in a 2–4-fold greater postprandial insulin response (van Loon et al., 2003; Manders et al., 2005, 2006a). This increased postprandial insulin response has been shown to accelerate glucose disposal, attenuating the postprandial rise in blood glucose concentrations in these type 2 diabetes patients (Manders et al., 2005). More recently, we reported that co-ingestion of a protein hydrolysate/leucine mixture after each main meal improves 24 h glucose homeostasis in long-standing type 2 diabetes patients by reducing the prevalence of hyperglycemia by as much as 26% (Manders et al., 2006b).
Ample evidence has been provided to support that co-ingestion of a mixture of a protein hydrolysate with free leucine represents an effective nutritional strategy to improve glucose homeostasis in type 2 diabetes patients. Due to the ongoing debate on the safety of free amino acids as dietary supplements (Anderson and Raiten, 1992; Matthews, 2005), the addition of free amino acids to improve protein quality and/or function is either restricted or even prohibited in most countries. Consequently, at present, free leucine co-ingestion does not seem to represent a practical interventional strategy. However, co-ingestion of a protein hydrolysate without additional free leucine has also been shown to substantially elevate the postprandial insulin response (Manders et al., 2006a). Therefore, in the present study, we assessed the efficacy of co-ingestion of a protein hydrolysate without additional free leucine to modulate 24 h glucose homeostasis in long-standing type 2 diabetes patients.
Subjects and methods
A total of 13 long-standing, male type 2 diabetes patients participated in this study (age: 62±2 years; bodyweight: 87±4 kg; body mass index (BMI): 28±1 kg m−2; HbA1c: 7.8±0.3%). Exclusion criteria were impaired renal or liver function, extreme obesity (BMI>35 kg m−2), cardiac disease, hypertension, diabetes complications and exogenous insulin therapy. Type 2 diabetes patients were using either metformin (n=2), a sulfonylurea derivative (n=2) or metformin in combination with sulfonylureas (n=9). All subjects were informed about the nature and the risks of the experimental procedures before their written informed consent was obtained. The Medical Ethical Committee of the Academic Hospital in Maastricht approved all clinical trials.
Medication, diet and activity before testing
Blood glucose lowering medication was withheld for 2 days prior to the screening but continued throughout the experimental trials. All subjects maintained normal dietary and physical activity patterns throughout the entire experimental period, and refrained from exhaustive physical labor and/or exercise training for at least 3 days prior to each trial. Food intake and physical activity questionnaires were collected for 2 days prior to the trials to keep dietary intake and physical activity as identical as possible. The evening before each trial, subjects received a standardized meal (see Diet & Physical activity).
Screening and study design
After an overnight fast, all subjects performed a standard 2 h, 75 g oral glucose tolerance test (OGTT). Type 2 diabetes was confirmed according to the 2003 ADA guidelines (ADA, 2003).
Each subject participated in a randomized, double-blind crossover design. Subjects were studied on two occasions for 40 h under strict dietary standardization but otherwise normal, free-living conditions with the use of a continuous glucose monitoring system (CGMS). In one trial, subjects received three beverages containing a protein hydrolysate (PRO), and in the other trial, a placebo beverage (PLA) was provided.
Prior to the start of the first 40 h assessment period, subjects reported to the laboratory in the afternoon and were given instructions regarding their standardized diet, the consumption of the experimental beverages and on the correct use of the food intake and physical activity questionnaires. All subjects received a short training in the use of the capillary blood sampling method (Glucocard Memory PC, A Menarini Diagnostics, Firenze, Italy). Next, a microdialysis fiber (Medica, Medolla, Italy) was inserted in the peri-umbilical region. The micro-fiber was subsequently connected to a portable continuous glucose-measuring device (CGMS; GlucoDay S, A Menarini Diagnostics, Firenze, Italy). Thereafter, subjects were provided with their diet and were allowed to return home and resumed their normal daily activities. The following day the subjects consumed their designated meals, drinks and snacks at the set time-points. Before consuming a meal, subjects obtained a capillary blood glucose sample and after finishing the meal the subjects drank a bolus beverage containing either PRO or PLA. The subsequent day, subjects reported back to the laboratory where the CGMS was removed. CGMS data of the second day (from 0700 to 0700 h) were used for data analyses.
Diet and physical activity
All meals, snacks and beverages were provided in preweighed packages and ingested at predetermined time-points to ensure fully standardized dietary modulation during the two 40 h test periods. On the evening before the 24 h analysis period, all subjects received a standardized meal (43.8 kJ kg−1 bw; consisting of 60 Energy % (En%) carbohydrate, 28 En% fat and 12 En% protein). The following day, subjects consumed their meals, drinks and snacks at set time points. The standardized diet (three meals and three snacks per day) provided 12.2±0.5 MJ d−1, and consisted of 64 En% carbohydrate, 25 En% fat and 11 En% protein. After consumption of each main meal, subjects ingested a prepackaged bottle containing either PRO or PLA. Ingestion of the protein hydrolysate beverages represented an additional daily energy intake of 1728 kJ per day, and modulated the macronutrient composition of the diet to 54 En% carbohydrate, 22 En% fat and 25 En% protein. During both test periods, subjects filled out food intake and exercise questionnaires. The rate of energy expenditure during the intervention day was determined using the Compendium of Physical Activities (Ainsworth et al., 2000) and averaged 11.7±0.8 and 11.5±0.6 MJ d−1 in the PLA and PRO trial, respectively (NS).
Beverages consisted of 4 ml kg−1 of water (PLA) or water containing 0.4 g kg−1 casein PRO and had to be ingested directly after each meal. The casein protein hydrolysate was prepared by DSM Food Specialties (Delft, the Netherlands). The casein hydrolysate (Insuvital) was obtained by enzymatic hydrolysis of sodium caseinate using a proprietary mix of proteases. Drinks were uniformly flavored by adding 0.2 g sucralose, 1.8 g citric acid and 5 g cream vanilla flavor (Quest International, Naarden, the Netherlands) per liter beverage to make the taste comparable in both trials.
Continuous glucose monitoring system
The GlucoDay S system is an ambulant continuous glucose monitoring system based on the microdialysis technique and allows continuous glucose monitoring for a period of 48 h (Maran et al., 2002). A detailed description of the device is provided elsewhere (Poscia et al., 2003; Varalli et al., 2003).
Blood sample analysis
Blood (10 ml) was collected in EDTA containing tubes and centrifuged at 1000 g and 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, Roche, Basel, Switzerland) were analyzed with the COBAS FARA semi-automatic analyzer (Roche). Plasma insulin was determined by radioimmunoassay (HI-14K, Linco Research Inc., St Charles, MO, USA). To determine HbA1c content a 3 ml blood sample was collected in EDTA containing tubes and analyzed by high-performance liquid chromatography (Bio-Rad Diamat, Munich, Germany).
Statistics and data analysis
The acquired data were downloaded from the CGMS to a personal computer with GlucoDay software (V3.0.5). Values reported by the CGMS were converted into glucose values using the SMBG values. The efficacy and the accuracy of the GlucoDay S has been validated for both diabetes patients (Maran et al., 2002; Wentholt et al., 2005) and healthy subjects (Maran et al., 2004). CGMS data of the second day (from 0700 to 0700 h) were used for data analyses and are expressed as means±s.e.m. All parameters were analyzed over the entire 24 h measuring period and postprandially (that is 4 h after breakfast and for 6 h after lunch and dinner). To quantify and compare the prevalence of hyperglycemia between the trials, the amount of time during which glucose concentrations were >10 mmol l−1 was calculated. An ANOVA or a Student's t-test for paired observations was applicable. Significance was set at the 0.05 level of confidence. All statistical calculations were performed using StatView 5.0 (SAS Institute Inc., Cary, NC, USA).
A total of 13 type 2 diabetes patients (age: 62±2 years; BMI: 28±1 kg m−2) were recruited to participate in this study. All patients had been diagnosed with type 2 diabetes for several years (7±1 years). HbA1c content averaged 7.8±0.3%. Fasting glucose concentrations averaged 10.8±0.8 mmol l−1 and were significantly increased at the end of the OGTT (18.9±1.5 mmol l−1; P<0.01). Fasting plasma insulin concentrations were within a normal range (19.6±3.6 mU l−1) Whole-body insulin sensitivity was calculated using the oral glucose insulin sensitivity -index (Mari et al., 2001) and averaged 238±16 ml min−1 m−2.
Average 24 h blood glucose concentrations are illustrated in Figure 1. Fasting glucose concentrations averaged 7.7±0.7 and 7.6±0.7 mmol l−1 in the PLA and PRO trial, respectively (P=0.9). Mean 24 h blood glucose concentration averaged 8.9±0.8 and 9.2±0.7 mmol l−1 in the PLA and PRO trial, respectively and did not differ between trials. No differences were observed in postprandial glucose concentrations between trials (Table 1). Although subjects continued their oral blood glucose-lowering medication and consumed a healthy diet during the intervention period, hyperglycemia (here defined as blood glucose concentrations >10 mmol l−1) was present 34±9% of the time (8±2 h) over the 24 h period in the PLA trial (Table 2). Ingestion of the PRO mixture did not modulate the prevalence of hyperglycemia (39±10%, 9±2 h; P=0.2).
The present study shows that type 2 diabetes patients who receive standard primary medical care experience hyperglycemia throughout the greater part of the day. Co-ingestion of an insulinotropic protein hydrolysate with each main meal does not reduce the prevalence of hyperglycemia in these long-standing type 2 diabetes patients.
With the use of a validated continuous glucose-monitoring system (Maran et al., 2002; Poscia et al., 2003; Varalli et al., 2003; Maran et al., 2004; Wentholt et al., 2005), we assessed 24 h blood glucose concentrations in long-standing type 2 diabetes patients under strict dietary standardization but otherwise free-living conditions. Despite a healthy, balanced diet and the continued use of oral blood glucose-lowering medication (ADA 2006), these type 2 diabetes patients showed substantial hyperglycemia throughout the greater part of the day. The prevalence of such elevated postprandial blood glucose excursions in type 2 diabetes patients imposes a direct and independent risk for the development of cardiovascular complications (Ceriello, 2003, 2005; Heine et al., 2004). In accordance, both the Diabetes Control and Complications Trial (DCCT, 1993) and the UK Prospective Diabetes Study (UKPDS, 1998a, 1998b; Stratton et al., 2000) reported that improving glycemic control effectively reduces the risk of developing micro- and macrovascular complications and cardiovascular disease. Therefore, therapeutic strategies for the treatment of type 2 diabetes should target to attenuate the postprandial hyperglycemic blood glucose excursions.
The blunted insulin secretory response following carbohydrate ingestion represents an important factor contributing to the elevated postprandial glucose excursions in these long-standing type 2 diabetes patients (van Loon et al., 2003). This reduced insulin response is attributed to the progressive insensitivity of the β-cell to glucose rather than a reduced insulin secretory capacity (Polonsky et al., 1996; van Loon et al., 2003). Therefore, other insulin secretagogues, like amino acids, can be applied to augment endogenous insulin release in these patients. Several groups have proposed that protein (hydrolysate) and/or amino-acid ingestion forms an effective strategy to augment postprandial endogenous insulin release thereby effectively reducing postprandial glucose excursions (Nuttall and Gannon, 2004; Frid et al., 2005; Van Loon, 2007). Both in vivo and in vitro work has identified leucine as a particularly interesting insulin secretagogue, as leucine both induces and enhances pancreatic β-cell insulin secretion and could also help to maintain β-cell mass (Xu et al., 2001; Nair and Short, 2005; Newsholme et al., 2005). As such, leucine co-ingestion has been suggested as an effective strategy to further augment the insulinotropic effects of protein co-ingestion. In accordance, we established that co-ingestion of a protein hydrolysate/leucine mixture with carbohydrate can be used to augment endogenous insulin secretion, accelerate blood glucose disposal and to attenuate the postprandial rise in glucose concentrations in type 2 diabetes patients (van Loon et al., 2003; Manders et al., 2005, 2006a). More recently, we showed that co-ingestion of such a protein/amino-acid mixture with every main meal can be used as an effective nutritional intervention strategy to reduce the prevalence of daily postprandial hyperglycemia by ∼26% in these patients (Manders et al., 2006b).
There has been an ongoing debate on whether the enrichment of foods with free amino acids should be allowed (Anderson and Raiten, 1992; HCN, 1999; Matthews, 2005). In 1992, it was concluded that there is insufficient data to evaluate the safety of amino-acid supplementation (Anderson and Raiten, 1992). For this reason, the addition of free amino acids to improve protein quality, or function as metabolically active components has become either restricted or even prohibited in most countries. In 1999, the Dutch Health Council endorsed the conclusions from the FASEB report prohibiting the addition of free amino acids to food as a dietary supplement, with the exception of a small number of highly specific applications (HCN, 1999). Therefore, at present, the proposed co-ingestion of free leucine following each main meal does not seem to represent a feasible dietary interventional strategy.
As co-ingestion of a protein hydrolysate without additional free leucine has also been shown to substantially elevate the postprandial insulin response (Manders et al., 2006a) we, therefore, aimed to assess the practical relevance of the co-ingestion of an insulinotropic protein hydrolysate as a nutritional intervention strategy to improve daily glycemic control in long-standing type 2 diabetes patients. We confirm our previous findings (Praet et al., 2006) showing that long-standing, well-controlled, type 2 diabetes patients are in a state of hyperglycemia throughout a considerable part of the day (8±2 h per 24 h), despite the continued use of oral blood glucose-lowering medication. These findings support the current belief that pharmacological treatment with oral blood glucose lowering medication does not provide adequate protection against hyperglycemia (Del Prato, 2002; Praet et al., 2006), and implies that additional strategies need to be developed to improve glycemic control in type 2 diabetes patients.
In the present study, we failed to observe any modulating effect of protein hydrolysate co-ingestion on daily glycemic control. No differences in the prevalence of hyperglycemia (8±2 versus 9±2 h per 24 h; P=0.2) were observed between the placebo and protein trials, respectively (Table 2). These data tend to be not in line with our previous findings (Manders et al., 2006b), in which we reported a 26±9% decline in hyperglycemia in a similar group type 2 diabetes patients. Except for the absence of additional free leucine co-ingestion, the applied intervention and research design were identical between studies. Although the use of continuous glucose-monitoring devices in an applied setting do not allow for concomitant insulin measurements, other studies (van Loon et al., 2003; Frid et al., 2005; Manders et al., 2005, 2006a; Gannon and Nuttall, 2006) have repeatedly shown that protein/amino-acid co-ingestion stimulates insulin secretion resulting in enhanced glucose disposal and a reduced postprandial glucose response. We speculate that co-ingestion of free leucine is instrumental to maximize the insulinotropic response (Koopman et al., 2005; Manders et al., 2006a) and, as such, to maximize the impact on glycemic control. However, it should be noted that in the present study protein hydrolysate was administered on top of the same standardized diet that was provided in the PLA trial. So we need to stress that similar 24 blood glucose kinetics were reported in the PRO trial, despite the fact that total energy intake was ∼14% greater in the PRO versus PLA trial. If the greater protein intake in the PRO trial would have been adjusted by reducing total carbohydrate intake in the diet, this would have resulted in a major impact on the overall glycemic response (Gannon and Nuttall, 2004).
In the present study, we investigated the acute response of protein hydrolysate co-ingestion on glycemic control on a daily basis. Besides the acute effects on endogenous insulin release, protein (hydrolysate) co-ingestion might also have a significant impact when implemented in the diet for a more prolonged intervention period. Increasing the amount of dietary protein has been suggested to increase satiety and reduce total energy intake, thereby stimulating weight loss (Halton and Hu, 2004). In a prolonged intervention study, Gannon and Nuttall (2004) reported improved glycemic control over a 5-week intervention after increasing the protein content in the diet. Though this substantially improved glycohemoglobin levels, it should be noted that dietary protein intake was increased at the expense of carbohydrate intake, thereby lowering postprandial blood glucose excursions. More research is warranted regarding the use of protein (hydrolysates) and/or specific amino acids as pharmaconutrients in the prevention and/or treatment of type 2 diabetes.
In conclusion, long-standing type 2 diabetes patients experience hyperglycemia throughout the greater part of the day, despite the use of oral blood glucose-lowering medication. co-ingestion of a protein hydrolysate after each main meal does not have a substantial effect on glycemic control in type 2 diabetes patients.
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We thank A Menarini Diagnostics BENELUX and Hanneke van Milligen for technical assistance and the subjects who volunteered to participate in these trials for their enthusiastic support. This study was supported by a grant from DSM Food Specialties (Delft, The Netherlands)
Contributors: RJFM, SFEP, WHMS and LJCvL designed the study. RJFM and MHV organized and carried out the clinical trials. RJFM performed all calculations and the statistical analyses. All authors contributed to the final version of the manuscript.
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Manders, R., Praet, S., Vikström, M. et al. Protein hydrolysate co-ingestion does not modulate 24 h glycemic control in long-standing type 2 diabetes patients. Eur J Clin Nutr 63, 121–126 (2009). https://doi.org/10.1038/sj.ejcn.1602891
- postprandial glycemia
- continuous glucose monitoring
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