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P4, a specific combination of dairy proteins (whey and casein) and plant-based protein isolates (pea and soy), has been shown to provide a more balanced amino acid (AA) profile than its single constituent proteins; however, less is known about how this translates to muscle protein synthesis (MPS). The aim of this study was to investigate the effect of P4 compared to whey or casein against fasted control on MPS. C57BL/6J mice, aged 25 months, were fasted overnight, followed by oral gavage of either whey, P4, casein, or water as a fasted control. Thirty minutes after ingestion, puromycin (0.04 µmol∙g−1 bodyweight) was subcutaneously injected; 30-min thereafter, mice were sacrificed. MPS was measured by the SUnSET method, and signalling proteins were determined in the left-tibialis anterior (TA) muscle by the WES technique. AA composition was determined in plasma and right-TA muscle. Dried blood spots (DBS) were analysed for postprandial AA dynamics at 10, 20, 45, 60 min. MPS was 1.6-fold increased with whey (p = 0.006) and 1.5-fold with P4 compared to fasted (p = 0.008), while no change was seen with casein. This was confirmed by a significant increase of phosphorylated/total ratio of 4E-BP1 for both whey (p = 0.012) and P4 (p = 0.001). No changes were observed in p70S6K and mTOR phosphorylation/total ratio with whey or P4. Intramuscular leucine levels were lower for P4 (0.71 µmol∙g dry weight−1) compared to whey (0.97 µmol∙g dry weight−1) (p = 0.0007). Ten minutes postprandial, DBS showed significantly increased blood AA levels of BCAAs, histidine, lysine, threonine, arginine, and tyrosine for P4 versus fasted. In conclusion, a hybrid mix of dairy and plant-based proteins (P4) resulted in a MPS response that was similar to whey protein in aged mice after fasting. This suggests that other anabolic triggers beyond leucine or the well-balanced amino acid profile and bioavailability of the blend benefit stimulation of MPS.

Antioxidant (AOX) deficiencies are commonly observed in older adults and oxidative stress has been suggested to contribute to sarcopenia. Here we investigate if 1) low levels of dietary antioxidants had a negative impact on parameters of muscle mass, function and quality, and 2) to study if nutritional interventions with AOX and/or leucine-enriched whey protein could improve these muscle parameters in aged mice. 18-months-old mice were fed a casein-based antioxidant-deficient (lowox) diet or a casein-based control-diet (CTRL) for 7 months. During the last 3 months, lowox-mice were subjected to either: a) continued lowox, b) supplementation with vitamin A/E, Selenium and Zinc (AOX), c) substitution of casein with leucine-enriched whey protein (PROT) or d) a combination of both AOX and PROT (TOTAL). After 7 months lowox-mice displayed lower muscle strength and more muscle fatigue compared to CTRL. Compared to lowox-mice, PROT-mice showed improved muscle power, grip strength and less muscle fatigue. AOX-mice showed improved oxidative status, less muscle fatigue, improved grip strength and mitochondrial dynamics compared to lowox-mice. The TOTAL-mice showed the combined effects of both interventions compared to lowox-mice. In conclusion, nutritional intervention with AOX and/or leucine-enriched whey protein can play a role in improving muscle health in a AOX-deficient mouse model.

Background A chronic low-grade inflammatory profile (CLIP) is associated with sarcopenia in older adults. Protein and Vitamin (Vit)D have immune-modulatory potential, but evidence for effects of nutritional supplementation on CLIP is limited. Aim To investigate whether 13 weeks of nutritional supplementation of VitD and leucine-enriched whey protein affected CLIP in subjects enrolled in the PROVIDE-study, as a secondary analysis. Methods Sarcopenic adults (low skeletal muscle mass) aged ≥ 65 years with mobility limitations (Short Physical Performance Battery 4–9) and a body mass index of 20–30 kg/m2 were randomly allocated to two daily servings of active (n = 137, including 20 g of whey protein, 3 g of leucine and 800 IU VitD) or isocaloric control product (n = 151) for a double-blind period of 13 weeks. At baseline and after 13 weeks, circulating interleukin (IL)-8, IL-1 receptor antagonist (RA), soluble tumor-necrosis-factor receptor (sTNFR)1, IL-6, high-sensitivity C-reactive protein, pre-albumin and 25-hydroxyvitamin(OH)D were measured. Data-analysis included repeated measures analysis of covariance (corrected for dietary VitD intake) and linear regression. Results IL-6 and IL-1Ra serum levels showed overall increases after 13 weeks (p = 0.006 and p

Introduction In 2008, the NutriAction study showed that (risk of) malnutrition was highly prevalent (57%) among Belgian older people living in the community or in a nursing home. In 2013, this study was repeated to re-evaluate the occurrence of malnutrition, as well as mobility problems and dependence in activities of daily living (ADL). Methods Health care professionals (HCPs) associated with homecare organizations and nursing homes across Belgium were invited to screen their patients and complete an online questionnaire. Nutritional status, presence of pre-specified comorbidities, mobility, and ADL dependency were assessed. Results In total, 3299 older patients were analysed: 2480 (86.3 ± 6.3 years) nursing home (NH) residents and 819 (82.7 ± 6.1 years) community dwelling (CD). Overall, 12% was malnourished (MNA-SF score

Background & aims: It has been suggested that anabolic resistance, or a blunted protein synthetic response to anabolic stimuli, contributes to the failure of muscle mass maintenance in older adults. The amino acid leucine is one of the most prominent food-related anabolic stimuli. However, data on muscle protein synthesis (MPS) after administration of a single bolus of leucine in aged populations is lacking and long-term single leucine supplementation has not been shown to increase muscle mass. This study aimed to determine the MPS response to the administration of a single bolus of leucine or to leucine combined with whey protein, in aged mice.Methods: Overnight fasted C57/BL6RJ mice at 25-mo of age received an oral gavage with leucine or whey-protein enriched with leucine (0.75 g/kg bodyweight total leucine in both) or 0.5 mL water (fasted control). Subsequently, mice were s.c. injected with puromycin (0.04 mu mol/g bw at t = 30, 45 or 60 min) and were sacrificed 30 min thereafter. Amino acid concentrations were determined in plasma and right muscle tibialis anterior (TA). Left TA was used to analyse MPS by SUnSET method and phosphorylation rate of Akt, 4E-BP1 and p70S6k by western blot.Results: In aged mice, leucine administration failed to increase MPS, despite a 6-fold increase in plasma leucine and elevated muscle free leucine levels (P Conclusions: MPS is stimulated in aged mice by leucine-enriched whey protein but not by leucine administration only. Administration of other amino acids may be required for leucine administration to stimulate muscle protein synthesis in aged mice. (c) 2018 Nutricia Research. Published by Elsevier Ltd on behalf of European Society for Clinical Nutrition and Metabolism.

Background: A promising strategy to help older adults preserve or build muscle mass is to optimize muscle anabolism through providing an adequate amount of high-quality protein at each meal.Objective: This "proof of principle" study investigated the acute effect of supplementing breakfast with a vitamin D and leucine-enriched whey protein medical nutrition drink on postprandial muscle protein synthesis and longer-term effect on muscle mass in healthy older adults.Methods: A randomized, placebo-controlled, double-blind study was conducted in 24 healthy older men [mean ± SD: age 71 ± 4 y; body mass index (in kg/m2) 24.7 ± 2.8] between September 2012 and October 2013 at the Unit of Human Nutrition, University of Auvergne, Clermont-Ferrand, France. Participants received a medical nutrition drink [test group; 21 g leucine-enriched whey protein, 9 g carbohydrates, 3 g fat, 800 IU cholecalciferol (vitamin D3), and 628 kJ] or a noncaloric placebo (control group) before breakfast for 6 wk. Mixed muscle protein fractional synthesis rate (FSR) was measured at week 0 in the basal and postprandial state, after study product intake with a standardized breakfast with the use of l-[2H5]-phenylalanine tracer methodology. The longer-term effect of the medical nutrition drink was evaluated by measurement of appendicular lean mass, representing skeletal muscle mass at weeks 0 and 6, by dual-energy X-ray absorptiometry.Results: Postprandial FSR (0-240 min) was higher in the test group than in the control group [estimate of difference (ED): 0.022%/h; 95% CI: 0.010%/h, 0.035%/h; ANCOVA, P = 0.001]. The test group gained more appendicular lean mass than the control group after 6 wk (ED: 0.37 kg; 95% CI: 0.03, 0.72 kg; ANCOVA, P = 0.035), predominantly as leg lean mass (ED: 0.30 kg; 95% CI: 0.03, 0.57 kg; ANCOVA, P = 0.034).Conclusions: Supplementing breakfast with a vitamin D and leucine-enriched whey protein medical nutrition drink stimulated postprandial muscle protein synthesis and increased muscle mass after 6 wk of intervention in healthy older adults and may therefore be a way to support muscle preservation in older people. This trial was registered at www.trialregister.nl as NTR3471.

BACKGROUND & AIMS: The requirement of leucine and essential amino acids (EAA) to stimulate muscle protein synthesis increases with age. To target muscle anabolism it is suggested that higher postprandial blood levels of leucine and EAA are needed in older people. The aim was to evaluate the impact of oral nutritional supplements with distinct protein source and energy density, resembling mixed meals, on serum amino acid profiles and on gastrointestinal behaviour. METHODS: Four iso-nitrogenous protein (21 g) supplements were studied containing leucine-enriched whey protein with 150/320 kcal (W150/W320) or casein protein with 150/320 kcal (C150/C320); all products contained carbohydrates (10 or 32 g) and fat (3 or 12 g). Postprandial serum AA profiles were evaluated in twelve healthy, older subjects who participated in a randomized, controlled, single blind, cross-over study. Gastrointestinal behaviour was studied in vitro by looking at gastric coagulation and cumulative intestinal protein digestion over time. RESULTS: The peak serum leucine concentration was twofold higher for W150 vs. C150 (521 +/- 15 vs. 260 +/- 15 mumol/L, p < 0.001), higher for W320 vs. C320 (406 +/- 15 vs. 228 +/- 15 mumol/L, p < 0.001), and higher for low-caloric vs. high-caloric products (p < 0.001 for pooled analyses; p < 0.001 for interaction protein source*caloric density). Similar effects were observed for the peak concentrations of EAA and total AA (TAA). In vitro gastric coagulation was observed only for the casein protein supplements. Intestinal digestion for 90 min resulted in higher levels of free TAA, EAA, and leucine for W150 vs. C150, for W150 vs. W320, and for C150 vs. C320 (p < 0.0125). CONCLUSIONS: A low caloric leucine-enriched whey protein nutritional supplement provides a higher rise in serum levels of TAA, EAA and leucine compared to casein protein or high caloric products in healthy, elderly subjects. These differences appear to be mediated in part by the gastrointestinal behaviour of these products. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: NCT02013466.

Background: Studying the muscle protein synthetic response to food intake in elderly is important, as it aids the development of interventions to combat sarcopenia. Although sarcopenic elderly are the target group for many of these nutritional interventions, no studies have assessed basal or post-prandial muscle protein synthesis rates in this population.Objective: To assess the basal and post-prandial muscle protein synthesis rates between healthy and sarcopenic older men.Design: A total of 15 healthy (69 +/- 1 y) and 15 sarcopenic (81 +/- 1 y) older men ingested a leucine-enriched whey protein nutritional supplement containing 21 g of protein, 9 g of carbohydrate, and 3 g of fat. Stable isotope methodology combined with frequent collection of blood and muscle samples was applied to assess basal and post-prandial muscle protein fractional synthetic rates. Handgrip strength, muscle mass, and gait speed were assessed to identify sarcopenia, according to international criteria. Results: Basal mixed muscle protein fractional synthetic rates (FSR) averaged 0.040 +/- 0.005 and 0.032 +/- 0.003%/h (mean +/- SEM) in the sarcopenic and healthy group, respectively (P = 0.14). Following protein ingestion, FSR increased significantly to 0.055 +/- 0.004 and 0.053 +/- 0.004%/h in the post-prandial period in the sarcopenic (P = 0.003) and healthy groups (P Conclusions: Basal muscle protein synthesis rates are not lower in sarcopenic older men compared to healthy older men. The ingestion of 21 g of a leucine-enriched whey protein effectively increases muscle protein synthesis rates in both sarcopenic and healthy older men. (C) 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Context: An impaired muscle protein synthetic response to feeding likely contributes to muscle loss with aging. There are few data available on the effect of the macronutrient composition of clinical supplements on the postprandial muscle protein synthetic response in older subjects. Objective: The objective of the study was to determine the impact of the macronutrient composition of a nutritional supplement on the postprandial muscle protein synthetic response in older men. Methods: A total of 45 nonsarcopenic older men ( aged 69 ± 1 y; body mass index 25.7 ± 0.3 kg/m2 ) were randomly assigned to ingest 21 g of leucine-enriched whey protein with carbohydrate ( 9 g ) and fat ( 3 g ) ( Pro-En ), an isonitrogenous amount of 21 g of leucine-enriched whey protein without carbohydrate and fat ( Pro ), or an isocaloric mixture ( 628 kJ ) containing carbohydrate and fat only ( En ). Stable isotope tracer methodology was applied to assess the basal as well as the postprandial muscle protein synthesis rates in the three groups. Results: Ingestion of protein in the Pro-En and Pro groups significantly increased muscle protein synthesis rates when compared with the basal rates ( from 0.032 ± 0.003%/h to 0.05%/h 3 ± 0.004%/h and 0.040%/h ± 0.003%/h to 0.049%/h ± 0.003%/h, respectively; P < .05 ), whereas ingestion of carbohydrate and fat did not increase muscle protein synthesis rates in the En group ( from 0.039%/h ± 0.004%/h to 0.040%/h ± 0.003%/h; P = .60 ). Despite the greater postprandial rise in circulating insulin concentration in the Pro-En group, no significant differences were observed in postprandial muscle protein synthesis rates between the Pro-En and Pro groups ( P = .32 ). Postprandial muscle protein synthesis rates were higher in the Pro-En vs En group ( P = .01 ). Conclusion: The ingestion of a nutritional supplement containing 21 g of leucine-enriched whey protein significantly raises muscle protein synthesis rates in nonsarcopenic older men, but coingestion of carbohydrate and fat does not modulate the postprandial muscle protein synthetic response to protein ingestion in older men.

ScopeIn recent years, there has been a growing body of evidence pointing to an effect of vitamin D on muscle mass and function. Our aim was to investigate the combined effect of 1,25(OH)(2)-vitamin D-3 (1,25(OH)(2)D-3) with anabolic factors insulin and leucine on protein fractional synthesis rate (FSR) and regulation in the mouse C2C12 myotube. Methods and resultsAfter differentiation, myotubes were cultured in 1,25(OH)(2)D-3 solutions at 0, 1, or 10 nM for 72 h. Cells were treated by l-[1-C-13]valine and puromycin in presence or not of leucine and insulin, and protein FSR was determined by measuring tracer enrichments and puromycin incorporation in proteins, respectively. Protein expression and phosphorylation state of insulin receptor (IR), Akt, GSK3, mTOR, p70 S6 kinase, rpS6, and 4EBP1 were measured by Western blot. Transcript levels of IR and 1,25(OH)(2)D-3 receptor (VDR) were determined by qPCR. 1,25(OH)(2)D-3 (10 nM) with leucine and insulin increased protein FSR in C2C12 myotubes (14-16%). IR and VDR mRNA expression was increased with 1,25(OH)(2)D-3 treatment. The Akt/mTOR-dependent pathway was activated by insulin and leucine and further enhanced by 1,25(OH)(2)D-3. Conclusion1,25(OH)(2)D-3 sensitizes the Akt/mTOR-dependant pathway to the stimulating effect of leucine and insulin, resulting in a further activation of protein synthesis in murine C2C12 skeletal myotubes.

Immune function is compromised in many cancer patients, leading to an increased risk of (infectious) complications. Chemotherapy-induced neutropenia is a common cause of treatment-induced immune suppression. In the present study, the effect of a specific nutritional combination (SNC) on bacterial translocation was studied in a model of chemotherapy-induced neutropenia in C3H/HeN mice colonized with Pseudomonas aeruginosa PAO-1. Dietary intervention started after stable colonization with P. aeruginosa to compare the SNC containing high protein, l-leucine, fish oil, and specific oligosaccharides to an isoenergetic control diet. After 3 wk, the mice were treated with cyclophosphamide to induce neutropenia. This rendered the mice susceptible to Pseudomonas translocation, which was quantified 5 d later. Intervention with the SNC resulted in a reduced incidence and intensity of bacterial translocation to the liver (P0.05) and a similar trend in the lungs (P ≤ 0.057). In addition, the SNC reduced the fecal pH (P0.05) and decreased P. aeruginosa counts in fecal samples (P0.05). Moreover, plasma concentrations of proinflammatory cytokines were correlated with the reduced bacterial translocation to the liver (ρ0.78; P0.001). In conclusion, dietary intervention with the SNC significantly reduced the incidence and severity of P. aeruginosa translocation in a mouse model of chemotherapy-induced immune suppression. Several mechanisms might have played a role, including the modulation of the intestinal microbiota, an improved gut barrier function, immune function, and a reduced inflammatory state. These results suggest an opportunity to develop new applications in cancer patients, with the aim to reduce infectious and other complications.

Cancer cachexia, which is characterized by muscle wasting, is associated with increased morbidity and mortality. Because muscle protein synthesis may be increased and protein breakdown reduced by leucine supplementation, we used the C26 tumor-bearing cachectic mouse model to assess the effects of dietary supplementation with leucine on muscle weight and the markers of muscle protein breakdown (mRNA of atrogin and murf). Male CD2F1 mice were subcutaneously inoculated with tumor cells (tumor-bearing mice; TB) or were sham injected (control; C). They were fed standard diets or diets supplemented with leucine [1 gr (TB1Leu) or 8 gr (TB8Leu) supplemented leucine per kg feed]; TB and C received 8.7% Leu/g protein, TB1Leu received 9.6% Leu/g protein and TB8Leu received 14.6 Leu/g protein. After 21 days, the following were determined: body weights, plasma amino-acid concentrations, tumor size and muscle mass of the gastrocnemius (mG), tibialis anterior (mTA), extensor digitorum longus (mEDL) and soleus (mS) muscles. In tumor-bearing (TB) mice, carcass and skeletal muscle masses decreased, and levels of atrogin and murf mRNA in the mEDL increased. Muscle-mass loss was counteracted dose-dependently by leucine supplementation: relative to TB, the mass of the mG was +23% in TB8Leu, and +22% in mTA (p

Decreased ability of muscles to respond to anabolic stimuli is part of the underlying mechanism for muscle loss with aging. Previous studies suggest that substantial amounts of essential amino acids (EAA), whey protein and leucine are beneficial for stimulation of acute muscle protein synthesis in older adults. However, these studies supplied only proteins, and no bolus studies have been done with dairy products or supplements that contained also fat and carbohydrates besides proteins. The aim of this study was to evaluate whether a specifically designed nutritional supplement in older adults stimulates muscle protein synthesis acutely to a greater extent than a conventional dairy product. Moreover, the combined effect with resistance exercise was studied by using a unilateral resistance exercise protocol. Utilizing a randomized, controlled, double blind study design, healthy older adults received a single bolus of a high whey protein, leucine-enriched supplement (EXP: 20g whey protein, 3g total leucine, 150kcal; n = 9) or an iso-caloric milk protein control (Control: 6g milk protein; n = 10), immediately after unilateral resistance exercise. Postprandial mixed muscle protein fractional synthesis rate (FSR) was measured over 4h using a tracer infusion protocol with L-[ring-13C6]-phenylalanine and regular blood and muscle sampling. FSR was significantly higher overall after EXP (0.0780 ± 0.0070%/h) vs Control (0.0574 ± 0.0066%/h (EMM ± SE)) (p = 0.049). No interaction between treatment and exercise was observed (p = 0.519). Higher postprandial concentrations of EAA and leucine are possible mediating factors for the FSR response, while plasma insulin increase did not dictate the FSR response. Moreover, when the protein intake from the supplements was expressed per kg leg lean mass (LLM), a significant correlation was observed with resting postprandial FSR (r = 0.48, P = 0.038). Ingestion of a high whey protein, leucine-enriched supplement resulted in a larger overall postprandial muscle protein synthesis rate in healthy older subjects compared with a conventional dairy product. This acute effect is promising for long-term effects on parameters of muscle mass, strength and function in sarcopenic older people, which requires further study. This trial is registered in the Dutch Trial Register under number NTR1823 .