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Nutrition and Disease

The Muscle Protein Synthetic Response to Carbohydrate and Protein Ingestion Is Not Impaired in Men with Longstanding Type 2 Diabetes^1–3,

Departments of ⁴ Human Biology and ⁵ Human Movement Sciences, Nutrition and Toxicology Research Institute Maastricht, Maastricht University, 6200 MD Maastricht, The Netherlands; and ⁶ Department of Clinical Chemistry, Academic Hospital Maastricht, 6229 HX Maastricht, The Netherlands

^* To whom correspondence should be addressed. E-mail: r.manders{at}hb.unimaas.nl.

	ABSTRACT

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
Protein ingestion stimulates muscle protein synthesis and improves net muscle protein balance. Insulin resistance has been suggested to result in a reduced muscle protein synthetic response to food intake. As such, we hypothesized that type 2 diabetes patients have a impaired muscle protein synthetic response to food ingestion. To test this hypothesis, 10 male type 2 diabetes patients using their normal oral glucose-lowering medication (68 ± 2 y) and 10 matched, normoglycemic men (65 ± 2 y) were randomly assigned to 2 crossover treatments in which whole body and muscle protein synthesis were measured following the consumption of either carbohydrate (CHO) or carbohydrate with a protein hydrolysate (CHO+PRO). Primed, continuous infusions with L-[ring-¹³C₆]phenylalanine and L-[ring-²H₂]tyrosine were applied and blood and muscle samples were collected to assess whole-body protein balance and mixed muscle protein fractional synthetic rate over a 6-h period. Whole-body phenylalanine and tyrosine flux were higher after the CHO+PRO treatment compared with the CHO treatment in the diabetes and control group (P < 0.01). Protein balance was negative following CHO but positive following CHO+PRO treatment in both groups. Muscle protein synthesis rates were higher in both groups following the CHO+PRO (0.086 ± 0.014%/h) treatment than in the CHO treatment (0.040 ± 0.003%/h; P < 0.01) with no difference between the diabetes patients and normoglycemic controls. We conclude that the muscle protein synthetic response to CHO or CHO+PRO ingestion is not substantially impaired in longstanding, type 2 diabetes patients treated with oral blood glucose-lowering medication.

	Introduction

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

Protein ingestion has been shown to promote muscle protein accretion in healthy, young individuals (10). Recent studies suggest that the muscle protein anabolic response to food intake is blunted in the elderly (11–13). This blunted anabolic response has been attributed to the fact that skeletal muscle protein synthesis is more resistant to the stimulating effects of elevated plasma amino acid and/or circulating insulin levels (14) and might be attributed to both impairments in insulin-stimulated muscle perfusion (14) and a reduced responsiveness of the mRNA translation initiation machinery (11,15). However, the relative contribution of each of these, along with other unidentified aspects, is still undefined. As the muscle protein synthetic response to combined hyperaminoacidemia and glucose-induced hyperinsulinemia is impaired in the elderly (12), we hypothesize that the anabolic response to protein ingestion is even more impaired in type 2 diabetes patients at a more advanced age.

Many studies have reported the stimulating effect of the combined ingestion of carbohydrate and protein on postprandial insulin release in vivo in humans (16,17). In our laboratory, we have shown that coingestion of a protein hydrolysate with carbohydrate can be applied to improve glucose homeostasis in longstanding type 2 diabetes patients by strongly stimulating endogenous insulin release (18,19). Insulin has also been reported to stimulate protein synthesis under conditions of hyperaminoacidemia (20,21) and can effectively reduce muscle proteolysis (22,23). Therefore, coingestion of an insulinotropic protein hydrolysate with carbohydrate could represent an effective strategy to stimulate the muscle protein anabolic response to food intake in longstanding, type 2 diabetes patients (24). In the present study, we assessed the muscle protein synthetic response to the ingestion of carbohydrate and carbohydrate plus protein in longstanding, type 2 diabetes patients under normal, practical conditions, in which blood glucose-lowering medication is maintained.

	Subjects and Methods

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
    Subjects. Ten longstanding (diagnosed with type 2 diabetes for over 5 y) male type 2 diabetes patients and 10 age- and BMI-matched, normoglycemic controls were selected to participate in this study (Table 1). Exclusion criteria were impaired renal or liver function, extreme obesity (BMI > 35 kg/m²), cardiac disease, hypertension, diabetes complications, and exogenous insulin therapy. The type 2 diabetes patients were using either metformin (n = 2), a sulfonylurea derivative (n = 1), metformin in combination with sulfonylureas (n = 5), or dietary modulation only (n = 2). All subjects were informed about the nature and 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 experiments.

    Medication, diet, and activity prior to testing. Blood glucose-lowering medication was withheld for 2 d prior to the screening but continued throughout the experiments. All subjects maintained normal dietary and physical activity patterns throughout the entire experimental period. Subjects refrained from heavy physical labor and/or exercise training for at least 3 d prior to each experiment and filled out a food intake questionnaire for 2 d prior to the first experiment to keep their dietary intake as identical as possible prior to the other experiment. The evening before each experiment, subjects received a standardized meal containing 44 kJ/kg body weight consisting of 60 energy % (En%) carbohydrate, 28 En% fat, and 12 En% protein.

    Experiments. Each subject participated in a randomized, double-blind, crossover design. All subjects were studied on 2 occasions, separated by 7 d, in which drinks containing carbohydrate (CHO) or carbohydrate plus protein hydrolysate (CHO+PRO) were administered. Each experiment lasted 8 h. Repeated boluses of a given test drink were administered to ensure a continuous supply of glucose and amino acids from the gut throughout the experiment. We collected plasma and muscle samples during a 6-h measurement period. We designed these experiments to simultaneously assess whole-body amino acid kinetics and mixed muscle protein fractional synthetic rate (FSR) in the m. vastus lateralis.

    Protocol. At 0800, after an overnight fast, subjects arrived at the laboratory by car or public transportation. Teflon catheters were inserted into an antecubital vein for stable isotope infusion and in a dorsal hand vein of the contra-lateral arm, which was placed in a hot-box (60°C) for arterialized blood sampling. After baseline blood sample collection, a single intravenous dose of L-[ring-¹³C₆] phenylalanine (2 µmol/kg) and L-[ring-²H₂] tyrosine (0.775 µmol/kg) was administered. Thereafter, continuous tracer infusion (0.049 ± 0.0004 µmol/(kg·min) for L-[ring-¹³C₆] phenylalanine and 0.018 ± 0.0002 µmol/(kg·min) for L-[ring-²H₂] tyrosine) was started. We chose L-[ring-¹³C₆] phenylalanine as opposed to L-[1-¹³C]leucine as amino tracer to study protein metabolism, because previous work (28) has shown that total amino acid oxidation rates are likely to be overestimated when using leucine as tracer. After a 2-h preinfusion period (0 min), an arterialized blood sample and a muscle biopsy from the m. vastus lateralis were collected. Subjects then received an initial bolus (1.5 mL/kg) of a test drink. Repeated boluses (1.5 mL/kg) were ingested every 30 min until 330 min. Arterialized blood samples were collected at 15, 30, 45, 60, 75, 90, 120, 150, 180, 210, 240, 270, 300, 330, and 360 min with a 2nd muscle biopsy taken at 360 min from the contra-lateral leg. Subjects remained in a resting, supine position throughout the entire experiment.

    Beverages. Subjects received 12 beverages with a volume of 1.5 mL/kg every 30 min over a 6-h period to ensure a given dose of 0.6 g carbohydrate/kg (50% glucose and 50% maltodextrin) with or without the addition of 0.3 g/kg of a casein protein hydrolysate per hour. In total, all subjects were provided with 1.3 L of water, 268 g carbohydrate, and 134 g protein hydrolysate. Repeated boluses were administered to ensure a continuous supply of amino acids in the circulation, preventing perturbations in L-[ring-¹³C₆] phenylalanine and L-[ring-²H₂] tyrosine enrichments. Glucose and maltodextrin were obtained from AVEBE. The casein protein hydrolysate was prepared by DSM Food Specialties 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 beverage.

    Analysis. Blood samples were collected in EDTA-containing tubes and centrifuged at 1000 x g; 10 min at 4°C. Aliquots of plasma were frozen in liquid nitrogen and stored at –80°C. Plasma glucose concentrations (Uni Kit III, 07367204, Roche) were analyzed with the COBAS-FARA semiautomatic analyzer (Roche). To measure the HbA1c concentration, a 3-mL blood sample was collected in EDTA-containing tubes and analyzed by HPLC (Bio-Rad Variant II). Insulin was measured by radio immunoassay (HI-14K, Linco Research). Plasma (500 µL) was deproteinized with 5-sulphosalicylic acid for determination of amino acid concentrations as described previously (29). The phenylalanine and tyrosine concentrations in the infusates were 4.66 ± 0.04 and 1.73 ± 0.02 mmol/L, respectively. Plasma phenylalanine and tyrosine were derivatized to their t-butyldimethylsilyl derivatives and their ¹³C and/or ²H enrichments were determined by electron ionization GC-MS (Agilent 6890N GC/5973N MSD) as described elsewhere (21).

For measurement of L-[ring-¹³C₆] phenylalanine enrichment in both the free amino acid and mixed muscle protein pool, 55 mg of wet muscle was freeze-dried and processed as described previously (21). The free amino acid concentrations in the muscle supernatant were measured by HPLC, after precolumn derivatization with o-phthaldialdehyde (30), whereas intracellular free L-[ring-¹³C₆] phenylalanine, L-[ring-²H₂] tyrosine, and L-[ring-¹³C₆] tyrosine enrichments were measured using their t-butyldimethylsilyl derivatives on a GC-MS (21). Muscle-bound phenylalanine enrichment was determined using its N(O,S)-ethoxycarbonyl ethyl ester for the determination of ¹³C:¹²C ratios on a gas chromatography isotope ratio mass spectrometer (31).

    Calculations. Infusion of L-[ring-¹³C₆] phenylalanine and L-[ring-²H₂] tyrosine with muscle and arterialized blood sampling was used to simultaneously assess whole-body amino acid kinetics and FSR of mixed muscle protein. Whole-body kinetics for phenylalanine and tyrosine were calculated based on the equations described by Short et al. (32). FSR of mixed muscle protein synthesis was calculated by dividing the increment in enrichment in the product, i.e. protein-bound L-[ring-¹³C₆] phenylalanine, by the enrichment of the precursor (plasma L-[ring-¹³C₆] phenylalanine enrichment) as described previously (21).

    Statistics. All data are expressed as means ± SEM. Plasma essential amino acid, insulin, and glucose responses were calculated as area under the curve above baseline values. A 3-factor repeated-measure ANOVA with group, time, and treatment as factors was used to compare differences between treatments over time between groups. For nontime dependent variables, a 2-factor ANOVA with group and treatment as factors was used to compare differences in treatment effects between groups. In case of significant difference between treatments, we applied the Scheffé post hoc test to locate these differences. Paired student's t tests were used to compare fasting and 2-h OGTT values. Statistical significance was set at P < 0.05. All calculations were performed using StatView 5.0 (SAS Institute).

	Results

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
    Plasma analyses. Fasting plasma insulin concentrations during the experiments did not differ between the type 2 diabetes patients (79 ± 7 pmol/L) and normoglycemic controls (98 ± 12 pmol/L) (P = 0.19). Overall, plasma insulin responses, expressed as area under the curve above baseline values, were higher after the CHO+PRO treatment than after the CHO treatment (Table 2; estimated marginal means: 176 ± 24 vs. 125 ± 14 nmol/L·6 h), respectively; P < 0.05). Fasting plasma glucose concentrations were higher in the diabetes patients (9.3 ± 1.0 mmol/L) than in the controls (5.7 ± 0.1 mmol/L) (P < 0.01). In the diabetes and control groups, plasma glucose responses were 32 ± 8 and 32 ± 9% lower after the CHO+PRO trial than after the CHO trial, respectively (P < 0.05; Table 2). Basal plasma phenylalanine, tyrosine, or branched-chain amino acid (leucine, isoleucine, and valine) concentrations did not differ between groups. A complete overview of the subsequent plasma amino acid responses is provided in Table 3.

Mean plasma amino acid enrichments during the last 4 h of the experiment, muscle free amino acid enrichments in the 6-h muscle biopsy, and the increments in muscle protein enrichment are presented in Table 4. In the muscle biopsies collected at 6 h, free L-[ring-¹³C₆] phenylalanine, L-[ring-²H₂] tyrosine, and L-[ring-¹³C₆] tyrosine enrichments did not differ between the type 2 diabetes patients and normoglycemic controls. Overall, free L-[ring-¹³C₆] phenylalanine, L-[ring-²H₂] tyrosine, and L-[ring-¹³C₆] tyrosine enrichments in the 6-h muscle biopsy were higher after the CHO treatment than after the CHO+PRO treatment (P < 0.05). The increase in muscle protein-bound L-[ring-¹³C₆] phenylalanine enrichment after the CHO and CHO+PRO treatments did not differ between the type 2 diabetes patients and control group.

View larger version (24K): [in this window] [in a new window]   FIGURE 1  Rate of whole-body protein breakdown, synthesis, oxidation, and net protein balance (A) and fractional rate of mixed muscle protein synthesis (B) in type 2 diabetes patients and normoglycemic controls after CHO and CHO+PRO treatments. Values are means ± SEM, n = 10. Results of 2-way ANOVA (P-values), A: Breakdown; group 0.38, treatment < 0.01, interaction 0.41; Synthesis; group 0.66, treatment < 0.001, interaction, 0.95; Oxidation; group 0.47, treatment < 0.01, interaction 0.29; Net balance; group 0.47, treatment < 0.01, interaction 0.29. B: group 0.45, treatment < 0.05, interaction 0.21.

	Discussion

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
This study shows that coingestion of protein with carbohydrate improves whole-body protein balance and augments mixed muscle protein synthesis rates in longstanding type 2 diabetes patients and matched, normoglycemic controls.

Insulin resistance and type 2 diabetes are characterized by impairments in glucose and fat metabolism (25). In addition, impairments in insulin sensitivity may also extend to protein metabolism in the insulin-resistant and/or type 2 diabetes state (7,33). However, in the fasted state, whole-body leucine (4,34–36), phenylalanine (4,37), and tyrosine fluxes (4), as well as leucine oxidation (4,34,35) and phenylalanine hydroxylation rates (4), do not differ in type 2 diabetes patients compared with matched, normoglycemic controls. Moreover, fasting mixed muscle protein synthesis rates (4) and net muscle protein balance (33) also do not seem to be altered in type 2 diabetes patients. Therefore, basal fasting protein metabolism does not appear substantially impaired in the type 2 diabetic state.

The development of glucose intolerance, insulin resistance, and/or type 2 diabetes at an advanced age is generally associated with a substantial loss of skeletal muscle mass. As skeletal muscle tissue is responsible for up to 80% of whole-body glucose uptake, it is evident that the gradual decline in muscle mass lowers blood glucose disposal capacity. As the basal muscle protein turnover rates do not seem to be affected by either age (38) or the presence of the insulin-resistant state, there has been increasing interest in the effect of aging and insulin resistance on the muscle protein synthetic response to major anabolic stimuli (i.e. food intake and physical activity). The gradual loss of muscle mass with aging is believed to be attributed to a more blunted muscle protein synthetic response to food intake (11–13). The latter has been suggested to be due to a reduced sensitivity of muscle protein synthesis to the stimulating effects of elevated plasma amino acid and/or insulin concentrations (14). As the muscle protein synthetic response to combined hyperaminoacidemia and glucose-induced hyperinsulinemia is impaired in the elderly (12), we hypothesized that the muscle protein synthetic response to food intake is even further reduced in longstanding, type 2 diabetes patients at a more advanced age.

In this study, we assessed the impact of ingesting either carbohydrate or carbohydrate plus protein (hydrolysate) on whole-body protein turnover and muscle protein synthesis rates in longstanding, type 2 diabetes patients and healthy, matched controls at a more advanced age. The anabolic response to carbohydrate and/or protein ingestion did not differ between groups (Table 4; Fig. 1B). Consistent with previous findings (4,35), we did not detect differences in basal plasma and/or muscle free amino acid concentrations between type 2 diabetes patients and healthy, matched controls. In addition, the rate of phenylalanine hydroxylation did not differ between groups following the ingestion of carbohydrate only (Fig. 1A). Whole-body net protein balance remained negative when only carbohydrate was ingested (Fig. 1A). The latter is in accordance with previous observations in healthy subjects showing net protein balance remained negative (21,24,39) in the absence of protein and/or amino acid coingestion (21,24,40,41).

Administration of protein (hydrolysate) and/or amino acids with carbohydrate rapidly increases muscle protein synthesis in both the young and elderly (21,42). The stimulating effect of protein/amino acid administration can be attributed to the function of amino acids as building blocks for de novo protein synthesis (10), the potential of amino acids to stimulate insulin secretion (19,43), and the property of amino acids to directly stimulate protein synthesis by activating the mRNA translational machinery (44). As such, ingestion of a mixture containing both carbohydrate and protein represents an effective nutritional intervention to stimulate the muscle protein synthetic response to food ingestion in type 2 diabetes patients. Coingestion of protein significantly suppressed whole-body protein breakdown and increased protein synthesis rates in both the type 2 diabetes patients and normoglycemic controls, with no apparent differences between groups (Fig. 1A). Coingestion of the protein hydrolysate increased whole-body protein synthesis rates by 85% compared with the ingestion of carbohydrate only. As a result, whole-body protein net balance became positive after the CHO+PRO treatment (Fig. 1A), with no apparent differences between groups. The latter underlines the fact that protein coingestion is essential for net muscle protein accretion to occur. Consistent with the whole-body estimates of muscle protein synthesis, skeletal muscle protein synthesis rates in the vastus lateralis muscle were higher following protein coingestion in both the type 2 diabetes patients and normoglycemic controls (Fig. 1B). Because no apparent differences in the muscle protein synthetic response to carbohydrate and/or protein ingestion were observed between groups, we conclude that the muscle protein synthetic response to carbohydrate and carbohydrate plus protein administration is preserved in longstanding type 2 diabetes patients receiving standard medical care, i.e. while using oral blood glucose-lowering medication. The latter condition was specifically selected to allow a comparison between healthy, normoglycemic men and type 2 diabetes patients under normal, practical conditions in which these patients generally consume their diet. Despite their medication, the type 2 diabetes patients who participated in this study had substantially higher basal plasma glucose concentrations, HbA1c concentrations, and HOMA-IR values compared with the normoglycemic controls (Table 1). In addition, the insulin response following the intake of carbohydrate with or without protein seemed to be blunted in the type 2 diabetes patients. The latter is not surprising, because longstanding type 2 diabetes patients were selected, in whom compensatory hyperglycemia was no longer apparent. Accordingly, the accompanying plasma glucose responses were substantially greater in the diabetes patients compared with the controls (Table 2).

Although glucose and fat metabolism are impaired in the insulin-resistant state, our data imply that the muscle protein synthetic response to food intake is largely unaffected in these type 2 diabetes patients. The latter seems to be in contrast with previous reports indicating that insulin resistance and impaired myocellular signaling are key factors in the etiology of muscle loss in the elderly (12,14,15). However, our data are consistent with Bell et al. (33), showing that the muscle protein synthetic response during a high energy-hyperinsulinemic clamp is preserved in poorly controlled, type 2 diabetes patients. The apparent discrepancy in the literature is likely attributed to the differences in the amount of nutrients that were administered (45,46) and the medication that was prescribed in the selected patients. In the present study, we aimed to assess the maximally stimulated muscle protein synthetic response to food intake. Therefore, we provided subjects with relatively large amounts of carbohydrate [0.6 g/(kg·h)] and protein [0.3 g/(kg·h)] during the 6-h test period. Future studies investigating the muscle protein synthetic response to the ingestion of smaller, meal-like amounts of carbohydrate, fat, and/or protein are warranted in various diabetes subpopulations under different pharmaceutical treatments.

In conclusion, coingestion of a protein hydrolysate with carbohydrate improves whole-body protein balance and augments muscle protein synthesis rate to a similar extent in longstanding, type 2 diabetes patients and normoglycemic controls. The skeletal muscle protein synthetic response to carbohydrate and/or protein ingestion is not substantially impaired in longstanding, type 2 diabetes patients treated with oral blood glucose-lowering medication.

	FOOTNOTES

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

² Author disclosures: R. J. Manders, R. Koopman, M. Beelen, A. P. Gijsen, W. K. Wodzig, W. H. Saris, and L. J. van Loon, no conflicts of interest.

³ Supplemental Calculations and References are available with the online posting of this paper at jn.nutrition.org.

⁷ Abbreviations used: CHO, carbohydrate treatment; CHO+PRO, carbohydrate and protein treatment; En%, energy percent; FSR, fractional synthetic rate; HbA1c, glycosylated hemoglobin; HOMA-IR, homeostasis model assessment insulin resistance; OGTT, oral glucose tolerance test; TTR, tracer trace ratio.

Manuscript received 8 November 2007. Initial review completed 30 November 2007. Revision accepted 27 March 2008.

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