sarcopenia, elderly men, skeletal muscle protein synthesis, casein, milk serum, dietary protein

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1 The Journal of Nutrition Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions Ingestion of Casein in a Milk Matrix Modulates Dietary Protein Digestion and Absorption Kinetics but Does Not Modulate Postprandial Muscle Protein Synthesis in Older Men 1 3 Tyler A Churchward-Venne, Tim Snijders, Armand MA Linkens, Henrike M Hamer, Janneau van Kranenburg, and Luc JC van Loon* Department of Human Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre+ (MUMC+), Maastricht, The Netherlands Abstract Background: The slow digestion and amino acid absorption kinetics of isolated micellar casein have been held responsible for its relatively lower postprandial muscle protein synthetic response compared with rapidly digested proteins such as isolated whey. However, casein is normally consumed within a milk matrix. We hypothesized that protein digestion and absorption kinetics and the subsequent muscle protein synthetic response after micellar casein ingestion are modulated by the milk matrix. Objective: The aim of this study was to determine the impact of a milk matrix on casein protein digestion and absorption kinetics and postprandial muscle protein synthesis in older men. Methods: In a parallel-group design, 32 healthy older men (aged y) received a primed continuous infusion of L-[ring- 2 H 5 ]-phenylalanine, L-[ring-3,5-2 H 2 ]-tyrosine, and L-[1-13 C]-leucine, and ingested 25 g intrinsically L-[1-13 C]- phenylalanine and L-[1-13 C]-leucine labeled casein dissolved in bovine milk serum (Cas+Serum) or water (Cas). Plasma samples and muscle biopsies were collected in the postabsorptive state and for 300 min in the postprandial period to examine whole-body and skeletal muscle protein metabolism. Results: Casein ingestion increased plasma leucine and phenylalanine concentrations and L-[1-13 C]-phenylalanine enrichments, with a more rapid rise after Cas vs. Cas+Serum. Nonetheless, dietary protein derived phenylalanine availability did not differ between Cas+Serum (47 6 2%, mean 6 SEM) and Cas (46 6 3%) when assessed over the 300-min postprandial period (P = 0.80). The milk matrix did not modulate postprandial myofibrillar protein synthesis rates from 0 to 120 min ( vs %/h) or from 120 to 300 min ( vs %/h) after Cas+Serum vs. Cas. Similarly, no treatment differences in muscle protein bound L-[1-13 C]-phenylalanine enrichments were observed at 120 min ( vs ) or 300 min ( vs mole percent excess) after Cas+Serum vs. Cas. Conclusions: Casein ingestion in a milk matrix delays protein digestion and absorption but does not modulate postprandial muscle protein synthesis when compared to the ingestion of micellar casein only in healthy older men. This trial was registered at Nederlands Trial Register as NTR4429. J Nutr 2015;145: Keywords: Introduction sarcopenia, elderly men, skeletal muscle protein synthesis, casein, milk serum, dietary protein Aging is associated with a progressive decline in skeletal muscle mass, strength, and physical performance, a geriatric condition termed sarcopenia (1). Although the etiology of sarcopenia is 1 Supported by the National Dairy Council, Rosemont, IL. 2 Author disclosures: TA Churchward-Venne, T Snijders, AMA Linkens, HM Hamer, J van Kranenburg, and LJC van Loon, no conflicts of interest. 3 Supplemental Methods and Supplemental Figures 1 3 are available from the Online Supporting Material link in the online posting of the article and from the same link in the online table of contents at * To whom correspondence should be addressed. L.vanLoon@ maastrichtuniversity.nl. multifactorial in nature (2), it is partly attributed to a blunted muscle protein synthetic response after protein ingestion (3). As a consequence, there has been an increasing interest in identifying the characteristics that determine the postprandial rise in muscle protein synthesis rates. This work has addressed the impact of the type (4 7) and dose (5, 8 14) of protein, as well as the timing (15) and the matrix (16, 17) in which the protein is ingested. More insight into the characteristics that modulate postprandial muscle protein synthesis will allow us to define more anabolic food products and may support the development of more effective dietary strategies to prevent and/or treat muscle loss with aging. ã 2015 American Society for Nutrition Manuscript received March 16, Initial review completed April 12, Revision accepted May 4, First published online May 27, 2015; doi: /jn

2 Dairy proteins provide an important source of dietary protein, and the capacity of bovine milk to stimulate postprandial muscle protein synthesis has been well studied (18, 19). Casein is the most abundant protein fraction found in bovine milk (;80% casein and 20% whey) and contains a complete profile of essential amino acids (20). Casein has been characterized as slow protein (21) based on its protein digestion and amino acid absorption kinetics. For example, ingestion of intact casein is followed by an attenuated but more sustained postprandial hyperaminoacidemia, an inhibition of whole-body protein breakdown, and a modest increase in whole-body protein synthesis rates (21 23). The slower digestion rate of intact micellar casein has been attributed to its unique property to coagulate when it interacts with gastric acid (24). Caseins are phosphate-containing proteins that occur as micelles in their native form. When casein enters the stomach and is brought to a ph of 4.6 or lower, casein precipitates, whereas whey protein will remain in solution (24). Several studies have demonstrated that the ingestion of isolated casein has a reduced capacity to stimulate postprandial muscle protein synthesis when compared to the ingestion of whey (4, 6, 7) or plant-based soy protein (7). Furthermore, more prolonged casein supplementation during resistance-type exercise training has been shown to result in a lower magnitude of lean body mass accretion when compared to whey protein supplementation (25). The reduced anabolic properties of isolated casein are likely attributed to its slower digestion and absorption kinetics (24, 26) as well as differences in amino acid composition (4). Protein digestion rate has been established as an independent variable influencing postprandial protein deposition (27) and the partitioning of amino acids between splanchnic and nonsplanchnic tissues (21, 22, 28). To date, postprandial aminoacidemia and the subsequent muscle protein synthetic response has only been assessed after ingestion of isolated casein in the absence of the milk matrix in which it naturally occurs. It is currently unknown how casein behaves when it is ingested within a milk matrix. There are indications that casein may behave differently when it is ingested within a milk matrix (24, 29). The milk matrix may modulate gastric ph and/or emptying and thus the bioavailability of casein-derived amino acids and the subsequent postprandial muscle protein synthetic response. We hypothesized that casein ingested in a milk matrix would result in a more rapid casein digestion and amino acid absorption, greater amino acid availability, greater whole-body protein accretion, and greater postprandial muscle protein synthesis when compared to the ingestion of isolated casein dissolved in water. Therefore, we used intrinsically L-[1-13 C]-phenylalanine and L-[1-13 C]-leucine labeled casein (30) to allow for the assessment of protein digestion and absorption kinetics, wholebody and skeletal muscle protein synthesis rates, and the use of dietary protein derived amino acids for de novo muscle protein synthesis in vivo. The objective of the present study was to compare the digestibility, bioavailability, and subsequent wholebody and myofibrillar protein synthetic response to the ingestion of micellar casein provided as an isolate in water or within a milk matrix in healthy older men. Methods In vitro experiment. In an in vitro pilot experiment, we assessed the buffer capacity of skim milk vs. an isonitrogenous solution of isolated casein, isolated whey protein, and a combination of both isolated casein and whey protein in the ratio in which these proteins are present in milk (80:20). These data demonstrated that milk has a greater buffer capacity compared to a solution of isolated micellar casein or whey or a combination of both proteins in water (Supplemental Figure 1). Participants. Thirty-two healthy older men (aged y) participated in this parallel-group, randomized controlled trial. The trial was conducted between April and June 2014, at Maastricht University in Maastricht, The Netherlands. The characteristics of the study participants are presented in Table 1. None of the participants had a history of participating in a regular exercise program in the last 5 y. All participants were informed about the purpose of the study, the experimental procedures, and all known risks before providing informed written consent to participate. The study was approved by the Medical Ethics Committee from the Academic Hospital Maastricht. All procedures were carried out in accordance with the ethical standards outlined in the most recent version of the Helsinki Declaration. Pretesting. Before the experimental infusion trial, participants underwent a medical screening to assess glycated hemoglobin, glucose tolerance (as assessed via a 2-h oral glucose tolerance test), systolic and diastolic blood pressure, body weight, height, and body composition (by DXA, Discovery A; Hologic). All participants were deemed healthy based on their responses to a routine medical screening questionnaire. Following the pretesting visit, participants were randomly assigned to 1 of 2 possible treatment groups (see below). Treatment randomization was computer generated ( and both participants and researchers were blinded to the treatment allocation. Diet and physical activity control. All participants avoided strenuous physical activity and maintained their usual diet for 2 d immediately before the experimental infusion trial. On the evening before the infusion trial, all participants consumed a prepackaged standardized meal containing 55% energy as carbohydrate, 30% energy as fat, and 15% energy as protein. Infusion protocol. Participants arrived at the laboratory by car or public transport at ;0800 after an overnight fast. A Teflon catheter was inserted into an antecubital vein for the stable isotope infusion. Subsequently, a second Teflon catheter was inserted into a dorsal hand vein on the contralateral arm and placed in a hot-box (60 C) for repeated arterialized blood sampling during the course of the infusion trial. After taking a baseline blood sample, participants received a priming dose of L-[ring- 2 H 5 ]- phenylalanine (2 mmol kg 21 ), L-[ring-3,5-2 H 2 ]-tyrosine (0.615 mmol kg 21 ), and L-[1-13 C]-leucine (4 mmol kg 21 ) followed by the initiation of a continuous infusion (t = 2240 min) of L-[ring- 2 H 5 ]-phenylalanine ( mmol kg 21 min 21 ), L-[ring-3,5-2 H 2 ]-tyrosine ( mmol kg 21 min 21 ), and L-[1-13 C]-leucine (0.10 mmol kg 21 min 21 ), respectively. After resting in a supine position for 120 min, a second arterialized blood sample was drawn and a biopsy was collected from the vastus lateralis (t = 2120 min) of a randomly selected leg (www. randomization.com). To determine basal muscle protein synthesis rates, a TABLE 1 Subject characteristics of healthy older men who ingested 25-g intrinsically labeled casein with or without milk serum 1 Cas Cas+Serum P Age, y Height, m Weight, kg BMI, kg m Systolic blood pressure, mm Hg Diastolic blood pressure, mm Hg Fat, % Lean body mass, kg Leg lean mass, kg Hb A 1c, % Basal glucose, mmol/l HOMA-IR Values are means 6 SEMs, n = 16. No significant differences were observed between groups. Cas, casein dissolved in water; Cas+Serum, casein dissolved in bovine milk serum; Hb A 1c, glycated hemoglobin. Casein and muscle protein synthesis in older men 1439

3 second muscle biopsy from the same leg was collected 120 min after the first biopsy (t = 0 min). Subsequently, participants received a beverage containing 25-g intrinsically L-[1-13 C]-phenylalanine and L-[1-13 C]- leucine labeled casein dissolved in bovine milk serum (Cas+Serum) 4 or water (Cas) (t = 0 min). The total beverage volume was 600 ml, which subjects were instructed to consume within 5 min. The milk serum concentration was adjusted to match the lactose concentration of normal bovine skimmed milk (;5%). Arterialized blood samples were drawn at t = 2240, 2120, 260, 0, 15, 30, 45, 60, 90, 120, 150, 180, 240, and 300 min. A third and fourth muscle biopsy was collected from the contralateral leg at t = 120 and t = 300 min, respectively, to determine muscle protein synthesis rates during the early (0 120 min) and late ( min) postprandial phase. Blood samples were collected in EDTA-containing tubes and centrifuged at g for 10 min at 4 Cto obtain plasma. Aliquots of plasma were frozen in liquid nitrogen and stored at 280 C. Biopsies were collected from a separate incision from the middle region of the vastus lateralis, ;15 cm above the patella and 3 cm below entry through the fascia, with use of the percutaneous needle biopsy technique. Muscle samples were dissected and freed from any visible fat and connective tissue, immediately frozen in liquid nitrogen, and stored at 280 C until further analysis. For a schematic representation of the infusion protocol, see Supplemental Figure 2. Intrinsically labeled casein protein and milk serum. Details on the intrinsically L-[1-13 C]-phenylalanine and L-[1-13 C]-leucine labeled casein protein have been reported previously (30). Briefly, intrinsically L-[1-13 C]- phenylalanine and L-[1-13 C]-leucine labeled milk protein was obtained via infusion of L-[1-13 C]-phenylalanine (455 mmol/min) and L-[1-13 C]-leucine (200 mmol/min) into a lactating Holstein cow (30). The L-[1-13 C]- phenylalanine and L-[1-13 C]-leucine enrichments in the micellar casein were measured by gas chromatography mass spectrometry (GC-MS) (Agilent 6890N GC coupled with a 5973 inert mass selective detector) and averaged 38.7 mole percent excess (MPE) and 9.3 MPE, respectively (30). Milk serum, containing ;10.3% lactose, ;0.3% protein, ;0.06% fat, and ;1.1% minerals was obtained from FrieslandCampina (The Netherlands). The milk serumwasdilutedinwatertoobtain a lactose concentration of 5% typical of skimmed milk and provided an additional 30.9-g carbohydrate, 0.9-g protein, and 0.2-g fat as compared to water. Plasma and muscle analyses. Plasma glucose and insulin concentrations were analyzed with use of commercially available kits (GLUC3; Roche, Ref: , and Immunologic; Roche, Ref: , respectively). Plasma amino acid concentrations and enrichments were determined by GC-MS (Agilent 7890A GC/5975C; MSD). Myofibrillar protein-bound L-[ring- 2 H 5 ]-phenylalanine enrichments were determined by GC-MS analysis, whereas the L-[1-13 C]-phenylalanine and L-[1-13 C]-leucine enrichments were determined by GC-C-isotope ratio mass spectrometer analysis (GC-C-IRMS; Trace GC Ultra, IRMS model MAT 253; Thermo Scientific). For complete details, see the Supplemental Methods. Calculations. Ingestion of L-[1-13 C]-phenylalanine labeled casein protein, intravenous infusion of L-[ring- 2 H 5 ]-phenylalanine and L-[ring- 3,5-2 H 2 ]-tyrosine, and arterialized blood sampling were used to assess whole-body amino acid kinetics in nonsteady state conditions. Total, exogenous, and endogenous rate of appearance (R a ) and plasma availability of dietary phenylalanine (i.e., the fraction of dietary phenylalanine that appeared in the systemic circulation, Phe plasma ) were calculated with use of modified SteeleÕs equations (23, 31). Total rate of disappearance (R d ) of phenylalanine from the circulation was calculated by adding the rate of phenylalanine hydroxylation (first step in phenylalanine oxidation) to the rate of phenylalanine use for protein synthesis. Myofibrillar protein fractional synthetic rate (FSR) was 4 Abbreviations used: Cas, casein dissolved in water; Cas+Serum, casein dissolved in bovine milk serum; FSR, fractional synthetic rate; GC-MS, gas chromatography mass spectrometry; MPE, mole percent excess; R a, rate of appearance; R d, rate of disappearance Churchward-Venne et al. calculated with use of the standard precursor-product equation. For complete details, see the Supplemental Methods. Statistics. All data are expressed as means 6 SEMs. Baseline subject characteristics were compared with use of an unpaired t test. Plasma glucose, insulin, amino acid concentrations, and amino acid enrichments were analyzed with use of a 2-factor (treatment 3 time) repeated measures ANOVA. Changes in whole-body phenylalanine kinetics (exogenous R a, endogenous R a,totalr a,totalr d ) were analyzed by a 2-factor (treatment 3 time) repeated measures ANOVA. Peak exogenous R a, time-of-peak exogenous R a, and the amount of casein-derived phenylalanine that appeared in the circulation were compared with use of an unpaired t test. Changes in whole-body protein metabolism (synthesis, breakdown, oxidation, net-balance) were analyzed by a 2-factor (treatment 3 time) repeated measures ANOVA. Myofibrillar protein FSR was analyzed by a 2-factor (treatment 3 time) repeated measures ANOVA. L-[1-13 C]- phenylalanine enrichments (MPE) were analyzed by a 2-factor (treatment 3 time) repeated measures ANOVA. MauchleyÕs test was used to evaluate if the assumption of sphericity was violated. If a significant MauchleyÕs test was determined, the Greenhouse-Geisser correction factor was used to adjust the df accordingly. A Tukey post hoc analysis was performed whenever a significant F ratio was found to isolate specific differences. Statistical significance was set at P < All calculations were performed with use of IBM SPSS Statistics (version 21). Results Plasma analyses. Plasma glucose and insulin concentrations are shown in Supplemental Figure 3. Plasma glucose concentrations increased above baseline (t = 2240 min) values after Cas+ Serum ingestion during the postprandial period from t = 30 to 120 min, with no changes observed in the Cas treatment (treatment 3 time interaction; P < 0.001). Similarly, plasma insulin concentrations increased to a greater magnitude from 30 to 120 min after Cas+Serum ingestion when compared to Cas ingestion during the postprandial period (treatment 3 time interaction; P = 0.01). Plasma phenylalanine, tyrosine, and leucine concentrations are shown in Figure 1. Plasma amino acid concentrations increased after protein ingestion in all groups. Phenylalanine concentrations increased above baseline concentrations (main effect for time; P <0.001)fromt =15to300minin the postprandial period in both the Cas and Cas+Serum treatment with no differences between treatments. Tyrosine concentrations increased above baseline concentrations from t = 30 to 300 min and t =90to300mininthepostprandialperiodafterCasandCas+ Serum, respectively, and differed between treatments at t = 60 and 90 min (treatment 3 time interaction; P = 0.003). Leucine concentrations increased over time reaching concentrations significantly higher than postabsorptive concentrations from t = 15 to 300 min after Cas and Cas+Serum ingestion, with significant differences between treatments from t = 30 to 180 min(treatment 3 time interaction; P = 0.001). Figure 2 shows plasma L-[ring- 2 H 5 ]-phenylalanine (infused tracer), L-[1-13 C]-leucine (infused and ingested tracer), and L-[1-13 C]-phenylalanine (ingested tracer). Plasma L-[ring- 2 H 5 ]- phenylalanine enrichments demonstrated a treatment 3 time interaction (P = 0.02) whereby enrichments were higher in the Cas treatment when compared to the Cas+Serum treatment at t = 2120, 240, and 300 min. Plasma L-[1-13 C]-leucine enrichments demonstrated a treatment 3 time interaction (P < 0.001) whereby enrichments were higher in the Cas treatment when compared to the Cas+Serum treatment at t = 2120 min, but lower in the Cas treatment when compared to the Cas+Serum treatment at t = 150 min. Plasma L-[1-13 C]-phenylalanine enrichments increased rapidly after casein ingestion (treatment 3 time interaction; P = 0.01), with Cas achieving greater enrichments at

4 FIGURE 1 Plasma phenylalanine (A), tyrosine (B), and leucine (C) concentrations (micromoles per liter) during basal postabsorptive conditions and after ingestion of 25-g intrinsically labeled casein with or without milk serum in older men. Values are means 6 SEMs, n = 16. The asterisk indicates different from Cas+Serum, P, Cas, casein dissolved in water; Cas+Serum, casein dissolved in bovine milk serum. t = 45 and 60 min but Cas+Serum achieving greater enrichments at t =180min. Whole-body phenylalanine kinetics and metabolism. Whole-body phenylalanine kinetics are presented in Figure 3. Exogenous phenylalanine rates of appearance (i.e., the rate at which dietary protein derived phenylalanine enters the circulation) demonstrated a treatment 3 time interaction (P = 0.03) whereby Cas resulted in a more rapid rate of dietary protein derived phenylalanine appearance and disappearance when compared to Cas+Serum (Figure 3A). No differences in exogenous phenylalanine peak rates of appearance were observed between the Cas and Cas+Serum treatment ( vs mmol kg 21 min 21,respectively;P = 0.95). Furthermore, there were no differences in the time to reach exogenous phenylalanine peak rate of appearance between Cas and Cas+Serum ( vs min, respectively; P = 0.20). The amount of casein-derived phenylalanine that appeared in the circulation throughout the 300-min postprandial period did not differ between the Cas and Cas+Serum treatment (46 6 3vs %, respectively; P = 0.80). Endogenous phenylalanine rates of appearance (i.e., the rate at which phenylalanine derived from whole-body protein breakdown enters the circulation) were FIGURE 2 Plasma L-[ring- 2 H 5 ]-phenylalanine (A), L-[1-13 C]-leucine (B), and L-[1-13 C]-phenylalanine (C) enrichments (MPE) during basal postabsorptive conditions and after ingestion of 25-g intrinsically labeled casein with or without milk serum in older men. Values are means 6 SEMs, n = 16. The asterisk indicates different from Cas +Serum, P, Cas, casein dissolved in water; Cas+Serum, casein dissolved in bovine milk serum; MPE, mole percent excess. reduced from t = 45 to 300 min after protein ingestion with no differences between treatments (main effect for time; P < 0.001). Total phenylalanine rate of appearance was increased from t = 15 to 300 min after protein ingestion with no differences between treatments (main effect for time; P < 0.001). Total phenylalanine rate of disappearance was increased from t = 15 to 300 min after protein ingestion with no differences between treatments (main effect for time; P < 0.001). Postabsorptive and postprandial (0 300 min) whole-body protein metabolism are shown in Figure 4. Casein ingestion, irrespective of the treatment, reduced whole-body protein breakdown (main effect for time; P < 0.001), increased whole-body protein synthesis (main effect for time; P < 0.001), increased whole-body protein oxidation (main effect for time; P < 0.001), and increased whole-body net-protein balance (main effect for time; P < 0.001). There were no significant differences observed in whole-body protein metabolism between treatments. Muscle analyses. Myofibrillar protein FSR, calculated with use of L-[ring- 2 H 5 ]-phenylalanine and blood plasma as the precursor pool, is presented in Figure 5A. Casein ingestion, irrespective of treatment, resulted in a stimulation of myofibrillar protein synthesis rates in the postprandial period from t = Casein and muscle protein synthesis in older men 1441

5 FIGURE 3 Whole-body phenylalanine kinetics. Exogenous R a (A), endogenous R a (B), total R a (C), and total R d (D) (micromoles phenylalanine per kilogram per minute) during basal postabsorptive conditions and after ingestion of 25-g intrinsically labeled casein with or without milk serum in older men. Values are means 6 SEMs, n = 16. The asterisk indicates different from Cas+Serum, P, Cas, casein dissolved in water; Cas+Serum, casein dissolved in bovine milk serum; R a, rate of appearance; R d, rate of disappearance. 120 to 300 min that was significantly greater than both basal and early t = min postprandial rates of myofibrillar protein synthesis (main effect for time; P < 0.001). Similarly, myofibrillar protein FSR, calculated with use of L-[1-13 C]- leucine and blood plasma as the precursor pool, was stimulated in the postprandial period from t = 120 to 300 min after casein ingestion irrespective of treatment (Figure 5B). Myofibrillar 1442 Churchward-Venne et al. FIGURE 4 Whole-body protein metabolism. Breakdown, synthesis, oxidation, and net balance (micromoles phenylalanine per kilogram per hour) during basal postabsorptive conditions and after ingestion of 25-g intrinsically labeled casein with or without milk serum in older men. Values are means 6 SEMs, n = 16. The asterisk indicates different from basal conditions, P, Cas, casein dissolved in water; Cas+Serum, casein dissolved in bovine milk serum. protein FSR was significantly greater from t = 120 to 300 min in the postprandial period than both the basal myofibrillar protein FSR and early t = min postprandial myofibrillar protein FSR (main effect for time; P < 0.001). Myofibrillar L-[1-13 C]- phenylalanine enrichments (MPE), representing dietary protein derived phenylalanine incorporation into myofibrillar protein, are shown in Figure 6. Myofibrillar L-[1-13 C]-phenylalanine enrichments (MPE) had increased after casein ingestion at both t = 120 and 300 min in the postprandial period with greater enrichment at t = 300 vs. 120 min (main effect for time; P < 0.001). No differences were observed between treatments. Discussion Previous studies examining postprandial aminoacidemia and the subsequent muscle protein synthetic response after micellar casein ingestion have assessed the postprandial response to the ingestion of isolated casein extracted from the milk matrix (4, 6, 7, 21). In the current study, we hypothesized that the milk matrix may increase gastric ph and/or stimulate gastric emptying, thereby resulting in a more rapid casein digestion and amino acid absorption, a more rapid increase in plasma amino acid availability, and subsequently greater postprandial whole-body protein accretion as well as skeletal muscle protein synthesis when compared to the ingestion of isolated casein without a milk matrix (i.e., dissolved in water). However, in contrast to our hypothesis, we found that micellar casein in a milk matrix is more slowly digested and absorbed when compared to the ingestion of isolated micellar casein without the milk matrix. Despite these temporal differences in micellar casein derived plasma amino acid availability over the 300-min postprandial period, there were no differences in postprandial whole-body protein metabolism (Figure 4), myofibrillar protein synthesis rates (Figure 5A, B), or the use of dietary protein derived amino acids for incorporation into myofibrillar protein (Figure 6). In the present study, we used intrinsically L-[1-13 C]-leucine and L-[1-13 C]-phenylalanine labeled micellar casein protein, obtained by infusing a lactating Holstein cow with large quantities of L-[1-13 C]-leucine and L-[1-13 C]-phenylalanine, collecting milk, and purifying the casein fraction (30). The high [1-13 C]- phenylalanine enrichment concentration (38.7 MPE) of the casein allowed us to specifically assess the true digestibility, plasma bioavailability, and the subsequent incorporation of the dietary micellar casein derived amino acids into myofibrillar protein

6 FIGURE 5 Myofibrillar protein FSR calculated with use of L- [ring- 2 H 5 ]-phenylalanine (A) and L-[1-13 C]-leucine (B) during basal postabsorptive conditions from 0 to 120 min and from 120 to 300 min after ingestion of 25-g intrinsically labeled casein with or without milk serum in older men. Values are means 6 SEMs, n = 15. Times without a common letter are different from each other, P, Cas, casein dissolved in water; Cas+Serum, casein dissolved in bovine milk serum; FSR, fractional synthetic rate. when provided with or without the milk matrix. The casein was also lowly labeled with L-[1-13 C]-leucine (9.3 MPE) to match the expected target labeling in the plasma L-[1-13 C]-leucine precursor pool during a primed constant intravenous L-[1-13 C]-leucine infusion. This allowed us to calculate the FSR with use of the precursor-product equation after the ingestion of micellar casein while minimizing disturbances in the plasma L-[1-13 C]-leucine precursor pool as previously described (30). Protein digestion and amino acid absorption kinetics have previously been demonstrated to affect postprandial muscle protein synthetic rates after protein ingestion, possibly by modulating the amount and the rate at which amino acids enter the systemic circulation (4, 21 23). It is generally thought that a faster rate of protein digestion and absorption, resulting in a more rapid FIGURE 6 L-[1-13 C]-phenylalanine enrichment in myofibrillar protein at t = 0 min, t = 120 min, and t = 300 min after ingestion of 25-g intrinsically labeled casein with or without milk serum in older men. Values are means 6 SEMs, n = 15 (Cas) or n = 16 (Cas+Serum). Times without a common letter are different from each other, P, Cas, casein dissolved in water; Cas+Serum, casein dissolved in bovine milk serum; MPE, mole percent excess. hyperaminoacidemia/hyperleucinemia, results in a greater muscle anabolic response (4, 6, 27, 32). For example, we have previously shown that hydrolyzed casein, which is more rapidly digested and absorbed than intact micellar casein, tends to induce a greater FSR response ( vs %/h) than micellar casein (27). However, other studies have detected differences in protein digestion and absorption and/or aminoacidemia that did not result in a divergent muscle protein synthesis response (16, 33). Similarly, in the present study differences in the temporal profile of amino acids/leucine availability in response to the ingestion of casein in a milk matrix vs. isolated casein in water were not sufficient to attenuate the whole-body or skeletal muscle protein anabolic response. Specifically, we found that the exogenous phenylalanine R a tended to be delayed but more sustained after caseininamilkmatrixwhencomparedtoisolatedcaseininwater (Figure 3A). However, the amount of casein-derived phenylalanine that appeared in the circulation over the 300-min postprandial period was similar between treatments (Cas: vs. Cas+ Serum: %, respectively), indicating that the temporal pattern of aminoacidemia was not the critical factor defining the whole-body or skeletal muscle anabolic response because there were no differences in postprandial whole-body protein kinetics (Figure 4), postprandial myofibrillar protein rates (Figure 5A, B), or dietary protein derived amino acid deposition into myofibrillar protein (Figure 6). In the present study, casein ingestion, irrespective of whether it was ingested with a milk matrix or dissolved in water, served to increase whole-body protein synthesis, suppress whole-body protein breakdown, and subsequently facilitate a more positive whole-body net protein balance over the 300-min postprandial period (Figure 4). Furthermore, casein increased myofibrillar protein synthesis rates but only when assessed from 120 to 300 min in the postprandial period (Figure 5A, B). Lastly, the use of dietary protein derived amino acids for de novo myofibrillar protein was increased at both 120 and 300 min in the postprandial period (Figure 6). These findings demonstrate that at the 25-g dose provided, casein ingestion served as an anabolic stimulus at both the whole-body and skeletal muscle level in older men irrespective of whether casein was provided in isolated form dissolved in water or when ingested with a milk matrix. Previous studies have demonstrated that older subjects demonstrate higher splanchnic amino acid extraction (34), and we have shown a significant reduction in the amount of dietary protein derived amino acids that enter systemic circulation after protein ingestion in older vs. younger subjects (16), thus it is unclear whether similar findings would be apparent in younger subjects. Previous work from our laboratory has demonstrated that carbohydrate coingestion with protein delays dietary protein digestion and absorption, but does not reduce the postprandial muscle protein synthetic response (16). Of interest, this occurs despite a significantly greater insulin response after protein coingestion with carbohydrate vs. protein alone (16), suggesting that the insulin response elicited from protein alone may be a sufficient stimulus to facilitate the amino acid mediated stimulation of protein synthesis. In a similar manner, the carbohydrate (;30 g lactose) present within the milk matrix may have delayed protein digestion and amino acid absorption when compared to the ingestion of isolated casein dissolved in water. As shown in Figure 1C, casein ingested in a milk matrix resulted in a more attenuated rise in plasma leucine concentrations, implying that components present within the milk matrix restricted the rise in postprandial plasma amino acid appearance. Alternatively, the greater insulin response after casein in a milk matrix may have increased amino acid uptake from the circulation and/or reduced proteolysis, thereby reducing leucine efflux Casein and muscle protein synthesis in older men 1443

7 into the circulation. However, the combined use of intravenous stable isotope (L-[ring- 2 H 5 ]-phenylalanine, L-[ring-3,5-2 H 2 ]- tyrosine, and L-[1-13 C]-leucine) labeled amino acid infusion coupled with the ingestion of intrinsically labeled (L-[1-13 C]- phenylalanine and L-[1-13 C]-leucine) micellar casein protein confirms that the attenuated rise in the plasma amino acid concentrations after casein in a milk matrix was attributed to a delay in the digestion and absorption of casein as compared to isolated casein dissolved in water (Figures 2C and 3A). Therefore, the carbohydrate and/or greater energy intake after casein in a milk matrix may have delayed the digestion and absorption of casein, which was a stronger stimulus than any potential of the milk matrix to buffer gastric acid and therefore the extent of micellar casein coagulation within the stomach. In summary, casein ingestion in a milk matrix delays protein digestion and amino acid absorption but does not modulate postprandial whole-body protein accretion, or muscle protein synthesis, when compared to micellar casein provided independently of the normal milk matrix in healthy older men. These findings demonstrate that the anabolic response to micellar casein protein is not compromised or enhanced by the milk matrix. Acknowledgments We thank Joy Goessens for technical assistance and Stefan H Gorissen for helpful discussion. HMH and LJCvL designed the research; TAC-V, TS, and AMAL conducted the research; LJCvL provided essential reagents and materials; TAC-V, TS, and JvK analyzed the data; TAC-V performed the statistical analysis; and TAC-V and LJCvL wrote the paper and had primary responsibility for final content. All authors read and approved the final manuscript. References 1. Rosenberg IH. Sarcopenia: origins and clinical relevance. J Nutr 1997;127(Suppl 5):990S 1S. 2. 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