Protein Leucine Content Is a Determinant of Shorter- and Longer-Term

See corresponding editorial on page 143.

Protein leucine content is a determinant of shorter- and longer-term

muscle protein synthetic responses at rest and following resistance Downloaded from https://academic.oup.com/ajcn/article-abstract/107/2/217/4911436 by guest on 02 May 2020 exercise in healthy older women: a randomized, controlled trial

Michaela C Devries,1 Christopher McGlory,1 Douglas R Bolster,3 Alison Kamil,3 Maike Rahn,3 Laura Harkness,3 Steven K Baker,2 and Stuart M Phillips1

1Department of Kinesiology, and 2Division of Physical Medicine and Rehabilitation, McMaster University, Hamilton, ON, Canada and 3PepsiCo R&D Nutri- tion, Barrington, IL

ABSTRACT INTRODUCTION Background: Older women may not be consuming enough pro- Aging is associated with sarcopenia, which is defined as a protein to maintain muscle mass. Augmentation of protein intake with gressive loss of muscle mass, strength, and function (1). Sarcope- leucine may enhance the muscle protein synthetic response in older nia is estimated to affect >20% of adults aged >65 y and >50% women to aid in maintaining muscle mass. of those aged >80 y (2). Complications that can arise with sar- Objective: We measured the acute (hourly) and integrated (daily) copenia include an increased risk of falls (3), disability and frailty myofibrillar protein synthesis (myoPS) response to consumption of (4), type 2 diabetes (5), and inability to perform activities of daily a high-quality mixed protein beverage compared with an isonitroge- living (4), all of which have a negative impact on quality of life. nous protein beverage with added leucine. Older adults exhibit a decreased anabolic response to protein Design: In a parallel design, free-living, healthy older women (aged feeding (6), which is one mechanism underpinning sarcopenic 65–75 y, n = 11/group) consumed a fixed, weight-maintaining diet · –1 · –1 muscle mass loss. Compared with younger adults, older adults with protein at 1.0 g kg d and were randomly assigned to ∼ twice-daily consumption of either 15 g milk protein beverage con- required 67% more protein per eating occasion to maximally stimulate muscle protein synthesis (MPS; 0.40 g protein · kg–1 · taining 4.2 g leucine (LEU) or 15 g mixed protein (milk and soy) –1 beverage containing 1.3 g leucine (CON). Unilateral leg resistance meal ) and achieve equivalent rates (6). Consuming 3 meals/d at · –1 · –1 ∼ exercise allowed a determination of acute ([13C ]-phenylalanine in- 0.4 g protein kg meal would translate to 1.2 g protein 6 · –1 · –1 fusion, hourly rate) and integrated (deuterated water ingestion, daily kg d , which is in line with protein intake recommenda- rate) exercised and rested myoPS responses. tions for older adults (7, 8) and estimates of protein requirement Results: Acute myoPS increased in response to feeding in the rested (9–11). Also of concern is that 40% of older adults are not meet- (CON: 13% ± 4%; LEU: 53% ± 5%) and exercised (CON: 30% ± ing the Recommended Daily Allowance (RDA) for protein (0.8 · –1 · –1 4%; LEU: 87% ± 7%) leg in both groups, but the increase was greater g kg d ) and 10% of older women are not even meeting the –1 –1 in LEU (P < 0.001). Integrated myoPS increased during the supple- Estimated Average Requirement of 0.66 g protein · kg · d (12, mentation period in both legs (rested: 9% ±1%; exercised: 17% ± 13). 2%; P < 0.001) in LEU, but in the exercised leg only (7% ± 2%; One strategy to augment MPS is via supplemental protein P < 0.001) in CON. consumption. Nonetheless, the per-meal quantities of protein Conclusions: A 15-g protein-containing beverage with ∼4 g leucine needed to maximally stimulate MPS (6) would mean consuming induced greater increases in acute and integrated myoPS than did larger, potentially unfeasible, quantities of protein due to age- an isonitrogenous, isoenergetic mixed-protein beverage. Declines in related declines in appetite (14), protein-mediated satiety, poor muscle mass in older women may be attenuated with habitual twice- daily consumption of a protein beverage providing 15 g protein Supported by PepsiCo R&D. and higher (4.2 g/serving) amounts of leucine. This trial was reg- Address correspondence to SMP (e-mail: [email protected]). istered at clinicaltrials.gov as NCT02282566. Am J Clin Nutr Abbreviations: Cmax, maximum concentration; CON, control group; D2O, 2018;107:217–226. deuterated water; EAA, essential amino acid; GC, gas chromatography; LEU, leucine-supplemented group; MPS, muscle protein synthesis; MS, mass spectrometry; myoPS, myofibrillar protein synthesis; RM, repetition maximum; Keywords: muscle protein synthesis, aging, sarcopenia, leucine, Tmax, time to Cmax. protein quality Received April 25, 2017. Accepted for publication November 13, 2017. First published online February 26, 2018; doi: https://doi.org/10.1093/ ajcn/nqx028.

Am J Clin Nutr 2018;107:217–226. Printed in USA. © 2018 American Society for Nutrition. All rights reserved. 217 218 DEVRIES ET AL. dentition, or food choice. A potentially viable strategy to en- TABLE 1 1 hance the impact that a lower protein content supplement could Nutritional content of study beverages have on MPS is to increase leucine intake. Leucine enhances LEU CON the acute MPS response to lower doses of protein in younger Serving size, fl oz 8 8 (15) and older adults (16). We recently showed that the addition Energy, kcal 110 110 of leucine to the daily meals of older men enhanced daily inte- Total protein, g 15 15 grated rates of MPS in both rested and exercised muscle (17). The Amino acid profile aim of the current trial was to compare the effects of a protein- Leucine from peptide, g 3.1 − containing beverage with a meal-like quantity (15 g) of protein Inherent leucine, g 1.1 1.3 and that was similar to what is available on the commercial mar- Tryptophan, g 0.2 0.2 ket for older adults in terms of protein quality to a beverage with Cysteine, g 0.1 0.1 the same amount of protein but with higher leucine. Our main Methionine, g 0.3 0.4 Aspartic acid, g 1.0 1.4

outcomes were acute and integrated rates of myofibrillar protein Downloaded from https://academic.oup.com/ajcn/article-abstract/107/2/217/4911436 by guest on 02 May 2020 Threonine, g 0.6 0.8 synthesis (myoPS) in both rested and resistance exercised mus- Serine, g 0.7 0.8 cle. We chose to study older women, as they are relatively un- Glutamic acid, g 2.6 3.1 derstudied compared with men but do have elevated protein re- Proline, g 1.3 1.3 quirements (10, 11). Additionally, older women, compared with Glycine, g 0.2 0.4 older men, may be refractory to aminoacidemia-induced stimu- Alanine, g 0.4 0.6 lation of MPS (18). Nonetheless, women do exhibit stimulation Valine, g 0.9 0.9 of MPS, albeit to a lesser extent than that seen in men, with con- Isoleucine, g 0.7 0.8 Tyrosine, g 0.6 0.7 sumption of high-leucine-containing proteins (19). We hypoth- Phenylalanine, g 0.6 0.8 esized that both acute and integrated rates of myoPS would be Lysine, g 1.1 1.2 greater following consumption of a protein beverage with higher Histidine, g 0.3 0.4 leucine compared with an isonitrogenous protein-containing Arginine, g 0.4 0.7 beverage. Fat, g 2.8 2.8 Saturated fat, g 0.5 0.5 Carbohydrates, g 7 8 Calcium, mg 320 290 Vitamin D, IU 85 85 METHODS 1CON, control high-protein, lower leucine–containing Participants beverage; LEU, active treatment beverage. Twenty-two free-living, healthy older women between the ages of 65 and 75 y were recruited as participants through adver- tisements in local newspapers. The study was approved by the General study design Hamilton Integrated Research Ethics Board (HIREB 14-735) and Using a single-blind, randomized parallel-group design, the met all standards for the ethical engagement of human subjects in project coordinator block randomized women (in groups of 4) research set out in the Canadian tricouncil research policy (http:// using a computer-generated random-number method stratified www.pre.ethics.gc.ca/pdf/eng/tcps2/TCPS_2_FINAL_Web.pdf). based on age, BMI, and reported physical activity level to 1 Each participant was informed of the purpose of the study, ex- of 2 groups: leucine-supplemented group (LEU) (n = 11) or perimental procedures, and potential risks prior to providing control group (CON) (n = 11). The LEU beverage contained written consent. This study was registered at clinicaltrials.gov 15 g milk protein (80% casein, 20% whey) as a blend of con- as NCT02282566. Women were eligible to participate in the centrate (milk protein concentrate 95%) and isolate (milk pro- study if they were generally healthy, between the ages of 65 tein isolate 5%) containing 4.2 g total leucine. The CON bev- and 75 y, ≥5 y postmenopausal, tobacco nonusers, and were erage contained 15 g blended protein consisting of milk protein habitually taking between 3500 and 10,000 steps/d (as assessed concentrate (51.5%), milk protein isolate (2.6%), soy protein iso- by a pedometer prior to commencing the trial). Participants late (32.8%), and sodium caseinate (13.1%) containing 1.3 g total were excluded if they had cardiac, pulmonary, liver, or kidney leucine (n = 11). The CON beverage was formulated to mimic abnormalities, uncontrolled hypertension, rheumatoid arthritis, the blend of proteins typically found in nutritional support bever- diabetes, metabolic disorders, progressive degenerative neuro- ages targeted to this population. The LEU beverage was formu- logical disease (i.e., Parkinson disease, Alzheimer disease, or lated with a blend of leucine peptides (PepFormTM , Glanbia Nu- dementia), had taken any medications that would affect protein tritionals, Inc.), that facilitated leucine stability within the protein metabolism (i.e,. corticosteroids, nonsteroidal anti-inflammatory beverage to deliver increased amounts of leucine while keeping drugs), or were unable to consume dairy, soy, or sulfites. Sample total protein content the same as the CON beverage. The nutri- size was based on previously published data in older men from tional content of the study beverages is shown in Table 1.Al- our laboratory (20) and others (21). Employing an α of 0.05 and location was concealed from participants for the duration of the at 80% power we determined that we would be able to detect a study. Prior to commencing the trial, participants underwent as- 35% increase in myofibrillar fractional synthetic rate in response sessments of their level of habitual physical activity and dietary to feeding with 8 participants/group. In order to avoid any type intake, body composition, and single leg muscle strength. The II error, to be cautious, and to protect study power we recruited overall study design is shown in Figure 1A. The trial consisted 11 participants/group. of 2 assessments of myoPS. We used deuterated water (D2O) to LEUCINE AND MPS IN OLDER WOMEN 219 Downloaded from https://academic.oup.com/ajcn/article-abstract/107/2/217/4911436 by guest on 02 May 2020

FIGURE 1 Schematic of overall study protocol (A) and infusion trial (B). BW, body weight; D2O, deuterated water. determine integrated rates of myoPS prior to (2 d, baseline (HUR Health and Fitness Equipment). Within the trial, half of myoPS) and during (6 d, supplementation myoPS) the supple- the participants were randomly assigned to exercise their domi- 13 mentation period and we used an acute infusion of [ C6]-ring nant leg and the other half to exercise their nondominant leg. Prior phenylalanine to determine acute effects of the study beverage to testing, participants were familiarized with the knee extension on myoPS (Figure 1A). The study was conducted at McMaster machine by performing a single set of knee extensions with a University. nonfatiguing weight for ≥10 repetitions. Participants rested for 2 min and then started the 3–5 RM testing. Unilateral 3–5 RM values were determined for knee extension within 4 attempts, Preliminary testing with each attempt separated 1 min, and 1-RM values were estimated (www.exrx.net). 1-RM values were used to calculate the Prior to commencing the trial, participants wore a pedometer load corresponding to 50% and 60% 1 RM that were used for and accelerometer for 3 d (2 week days, 1 weekend day) so we the resistance exercise sessions during the acute protocol and on could obtain an estimate of habitual physical activity. Pedome- day 3 of supplementation. Participants also underwent assess- ters (StepsCount) were placed on the waistband of clothing at ment of body composition using dual-energy X-ray absorptiom- the level of the hip aligned vertically above the knee. The ac- etry (DXA, GE). celerometer (BodyMedia) was placed over the upper arm on the right triceps brachii. Over the same 3-d period, participants also recorded their dietary intake to enable determination of habitual energy and macronutrient consumption. Acute and integrated MPS At least 1 wk before starting the trial, participants reported to Two days prior to the infusion trial (acute myoPS) visit partici- the laboratory for assessment of single leg muscle strength and pants reported to the laboratory following an overnight fast. Rest- body composition. Single leg isotonic strength of the knee ex- ing blood and saliva samples were taken and participants con- tensors was determined by measuring 3–5 repetition maximum sumed 100 mL of D2O(D2O visit) to allow for determination (RM) for knee extension using air-resistance strength machines of integrated rates of myoPS prior to (day –2 to day 0: baseline 220 DEVRIES ET AL. period) and during (day 0 to day 6: supplementation period) the ditional food or drink energy-containing beverages. Participants nutritional supplementation period (Figure 1A). Participants were could consume water and noncaloric beverages ad libitum. given meals to consume for the following 2 d, and were instructed to refrain from strenuous exercise and alcohol consumption. Par- ticipants were also instructed on how to collect saliva samples Plasma and saliva analysis and were given a salivette to collect a fasted saliva sample the 13 following morning. Plasma [ C6]-phenylalanine enrichment was determined by gas chromatography (GC)-mass spectrometry (MS) (GC: 6890N, Two days after consuming the D2O, participants reported to the laboratory in the fasted state for assessment of fasted- and MS: 5973; Hewlett-Packard) as previously described (22). fed-state myoPS (acute visit: Figure 1B). Prior to commencing Plasma amino acid concentrations were determined using the TM the infusion, a saliva sample was collected. Catheters were in- Pico-Tag method as previously described (23). Plasma glu- serted into the antecubital veins—1 for blood sampling and 1 for cose and insulin concentrations were measured as described pre- 13 viously (22). D2O enrichment of saliva samples was used to de- infusion of [ C6]-ring phenylalanine. Prior to initiating the in- Downloaded from https://academic.oup.com/ajcn/article-abstract/107/2/217/4911436 by guest on 02 May 2020 2 fusion, a fasted blood sample was taken and a priming dose termine total body water H enrichment as a surrogate for plasma 13 alanine 2H labelling by cavity ring-down spectroscopy with a liq- (2 μmol/kg) of [ C6]-ring phenylalanine was admin- istered. Three hours after the initiation of the infusion uid water isotope analyzer, as previously described (24). The rate (0.05 μmol · kg−1 · min−1) participants had a muscle biopsy of decay was modelled and used to calculate MPS. taken from the vastus lateralis of 1 leg to assess fasted rates of myoPS. Upon completion of the biopsy, participants completed 4 sets of resistance exercise (2 sets at 50% and 2 sets at 60% Muscle analysis 1-RM) on a single leg (determined a priori at random) with 2 min of rest between sets. Upon completion of the exercise protocol a Muscle samples (∼30–50 mg) were homogenized to yield the blood sample was drawn and participants immediately consumed myofibrillar fraction. Briefly, the samples were homogenized on their study beverage. Study protocol indicated that participants ice in buffer [10 μL/mg 25 mM Tris 0.5% v/v Triton X-100 and had to drink their study beverage within 10 min of receiving it; protease/phosphatase inhibitor cocktail tablets (Complete Pro- however, all participants consumed the beverage within 3 min. tease inhibitor Mini-Tabs; Roche, and PhosSTOP; Roche Applied Blood samples were taken throughout the protocol as described Science)] and centrifuged at 15000 × g for 10 min at 4°Cand in Figure 1B. Four hours after the beverage consumption, partic- the pellet retained. The myofibrillar protein pellet was solubilized ipants had muscle biopsies taken from the vastus lateralis of both and centrifuged as previously described (25) and the supernatant legs to determine feeding and feeding plus exercise effects of the containing the myofibrillar proteins was collected. Myofibrillar study beverage. The study design for the acute visit is shown in proteins were precipitated in 1 mL of 1 M perchloric acid, the Figure 1B. supernatant discarded, and the fraction was washed twice with Upon completion of the acute infusion, participants were pro- 70% ethanol. The myofibrillar-protein-enriched pellets were hy- vided with food and study beverages (2 beverages/d, to be con- drolyzed in 0.5 M HCl at 110°C for 72 h to release their respec- 13 sumedinthemorningwithbreakfastand1hbeforebed)forthe tive amino acids. Myofibrillar [ C6]-phenylalanine enrichment remainder of the day and the next 5 d. Participants were also was determined using GC pyrolysis-isotope ratio MS (25). My- 2 instructed to refrain from consuming alcohol or participating in ofibrillar H-alanine enrichment was determined using Thermo strenuous exercise, and were given salivettes to collect saliva each Finnigan Delta V isotope ratio MS coupled with Thermo Trace morning upon waking. Participants returned to the laboratory on GC Ultra with GC pyrolysis interface III and Conflow IV as pre- the morning of day 3 and repeated the resistance exercise protocol viously described (24). that was performed on the acute infusion trial day on the same leg as in the previous 2 visits. On the morning of day 6, participants returned to the laboratory following an overnight fast, blood and Calculations saliva samples were collected, and muscle biopsies were taken The MPS rate was determined as %/h and %/d for [13C ]- from both legs to allow for determination of integrated rates of 6 phenylalanine and [2H]-alanine, respectively using the precursor- myoPS in the feeding and the feeding + exercise limb. product equation:

Diet provision MPS (%/h) = [ (E2b − E1b ) / Eprex t x 100 (1) Participants were provided with all food to be consumed throughout the trial beginning2dpriortotheacutetrial.Daily energy needs were determined using the Harris-Benedict equa- where E2b and E1b are the myofibrillar protein enrichments tion applying the appropriate activity factor (based on the Physi- at times 1 and 2, respectively, Epre is the average enrichment cal Activity Scale for the Elderly questionnaire). Diets consisted of the precursor over the course of the infusion (plasma for −1 −1 13 of 1.0 g protein · kg · d with the remaining energy com- [ C6]-phenylalanine and body water for D2O), and t is the ing from carbohydrates (∼55% of total) and fat (∼30% of to- tracer incorporation time in hours. The use of “tracer-naïve” sub- tal). During the intervention period, the study beverages were jects allowed us to use a preinfusion or ingestion blood sam- incorporated into the provided diet such that dietary protein pro- ple as the baseline enrichment (E1b) for calculation of fasting vision remained 1.0 g protein · kg−1 · d−1. Participants were in- MPS, an approach that has been previously validated in our structed to consume all of the provided food and to not eat any ad- laboratory (26). LEUCINE AND MPS IN OLDER WOMEN 221 Downloaded from https://academic.oup.com/ajcn/article-abstract/107/2/217/4911436 by guest on 02 May 2020

FIGURE 2 Participant flowchart. CON, control group;2 D O, deuterated water; LEU, leucine-supplemented group.

Statistics Table 2. With the exception of knee extension 1-RM, there was Baseline participant characteristics, and amino acid time to no difference in baseline characteristics between groups. 1-RM was greater in the CON compared with LEU group (P = 0.03). maximum concentration (Tmax), maximum concentration (Cmax), and AUC were compared using a nonpaired t test. Other data were compared using a 2-way mixed model ANOVA with be- Dietary intake tween (group) and within (condition: baseline or fasting, feeding, feeding + exercise) factors. Significant interaction terms from the There were no differences between groups in energy, protein, ANOVA resulted in using Tukey’s post hoc test. Correlational fat, or carbohydrate intake during the 2 d prior to the acute inter- analysis was performed to examine the relation between peak vention (baseline period) or during the supplementation period. leucine concentration and leucine AUC, and MPS. Significance Dietary intake data are given in Table 3. was set at P < 0.05. All statistical analysis was completed using SPSS (IBM SPSS Statistics for Windows, version 21; IBM Corp.). Data are presented as means ± SEM unless otherwise in- Plasma amino acids, glucose, and insulin dicated. Amino acid concentration data are shown in Figure 3 and summary data for the same are given in Table 4. Summed total amino acid concentration increased in response to feeding in both groups RESULTS (P < 0.0001) and returned to baseline by 240 min (Figure 3A). There were no differences in the total amino acid response be- Participants tween groups. Summed essential amino acid (EAA) concentra- Participants were recruited between December 2014 and tion increased in response to feeding in both groups (P < 0.001) November 2015. A flowchart showing the progress from recruit- and returned to baseline by 180 min with no difference between ment through study completion is shown in Figure 2. Twenty-two groups (data not shown). Leucine concentrations are shown in older women were recruited for this study, with 11 randomly as- Figure 3BandTable 4. Leucine concentration increased in re- signed to each group. All participants completed the trial. One sponse to feeding in both groups (P < 0.001), peaked at 51 min subject from CON was excluded from the integrated MPS as- (CON) and 52 min (LEU) and remained elevated above base- sessment due to insufficient muscle tissue available to conduct line at 240 min. Plasma leucine concentration was greater at the analysis. Baseline participant characteristics are shown in 20, 40, 60, and 120 min following ingestion of LEU compared 222 DEVRIES ET AL.

TABLE 2 Participant characteristics1

LEU (n = 11) CON (n = 11) P Age, y 69 ± 168± 10.66 Weight, kg 72.8 ± 3.9 72.1 ± 3.2 0.90 Height, m 1.65 ± 0.02 1.63 ± 0.02 0.61 BMI, kg/m2 26.4 ± 0.9 26.9 ± 0.9 0.73 Steps/d 6695 ± 1080 7544 ± 880 0.57 Daily PA >3 METs, min/d 72 ± 13 109 ± 19 0.13 Average METs 1.4 ± 0.1 1.3 ± 0.1 0.36 Energy expenditure >3 METs, kcal/d 318 ± 48 400 ± 55 0.30 Lean body mass, kg 40.5 ± 1.7 41.2 ± 2.0 0.81 Fat mass, kg 32.3 ± 3.0 27.9 ± 2.2 0.31

Body fat, % 42.2 ± 1.4 40.0 ± 1.6 0.35 Downloaded from https://academic.oup.com/ajcn/article-abstract/107/2/217/4911436 by guest on 02 May 2020 Knee extension strength, kg 22.8 ± 1.9* 29.5 ± 2.0 0.03 Systolic BP, mm Hg 131 ± 3 130 ± 40.81 Diastolic BP, mm Hg 72 ± 275± 10.39 Resting heart rate, bpm 69 ± 269± 40.92 Energy intake, kcal/d 1910 ± 97 1800 ± 123 0.51 Protein intake, g · kg–1 · d–1 1.1 ± 0.1 1.1 ± 0.1 0.44 Carbohydrate intake, g/d 236 ± 12 239 ± 19 0.90 Fat intake, g/d 72 ± 759± 50.16 Protein intake, % Ein 17 ± 117± 10.73 Carbohydrate intake, % Ein 50 ± 153± 20.22 Fat intake, % Ein 34 ± 230± 10.09 1Data are means ± SEMs. *Significantly different from CON (P < 0.05). Avg, average; bpm, beats per minute; CON, control group; Ein, energy intake; LEU, leucine-supplemented group; MET, metabolic equivalent; PA, physical activity; RM, repetition maximum. Analysis by nonpaired t test.

with CON (P < 0.001). Leucine AUC (P < 0.001) and Cmax ingestion (data not shown). There was no difference in glucose (P < 0.001) were greater following LEU compared with CON, AUC or Cmax;however,Tmax occurred earlier with LEU com- with no difference in Tmax. pared with CON (P = 0.03). Plasma insulin concentrations in- Plasma glucose concentrations are shown in Table 4. Plasma creased in response to feeding in both groups (Table 4; P < 0.001) glucose concentration increased in response to feeding in both and returned to baseline by 240 min after drink ingestion (data not groups (P < 0.001) and returned to baseline by 60 min after drink shown). Plasma insulin concentration was higher at 20 and 40 min following ingestion of LEU compared with CON (P = 0.01, data not shown). Cmax and insulin AUC were higher following LEU TABLE 3 compared with CON (P = 0.02 and P < 0.001, respectively). Dietary intake during the supplementation period for participants in the LEU and CON groups1

Treatment × Acute MPS = = LEU (n 11) CON (n 11) time, P Acute myoPS for LEU and CON are shown in Figure 4.There Energy intake, kcal/d 0.47 was an increase in myoPS in the rested and exercised legs in re- Baseline 1718 ± 57 1820 ± 54 sponse to feeding in both CON (rested: 13% ± 4%; exercised: Supplementation 1804 ± 51 1859 ± 40 30% ± 4%; P = 0.01) and LEU (rested: 53% ± 5%; exercised: Protein intake, g/d 0.69 87% ± 7%; P < 0.001). Postprandial myoPS was greater in the Baseline 67 ± 373± 3 rested and exercised legs in LEU compared with CON (P < Supplementation 75 ± 376± 3 0.001). Protein intake, g · kg–1 · d–1 0.52 Baseline 0.95 ± 0.05 1.01 ± 0.02 Integrated MPS Supplementation 1.05 ± 0.03 1.06 ± 0.02 Integrated myoPS for LEU and CON is shown in Figure 5. Fat intake, g/d 0.22 There was no difference in myoPS between CON and LEU during Baseline 66 ± 269± 3 ± ± the baseline period (i.e., presupplementation). Integrated myoPS Supplementation 69 2701 increased during the supplementation period in both the rested Carbohydrate intake, g/d 0.43 (9% ± 1%) and exercised (17% ± 2%) leg with LEU (both P < Baseline 221 ± 8 230 ± 5 0.001), but only in the exercised (7% ± 2%) leg with CON (P ± ± Supplementation 224 7 236 6 < 0.001). Integrated myoPS during the supplementation period 1Data are means ± SEM. Analysis by 2-way ANOVA. CON, control was greater in the LEU group than CON in both the rested and group; LEU, leucine-supplemented group. the exercised legs (P < 0.001). LEUCINE AND MPS IN OLDER WOMEN 223 Downloaded from https://academic.oup.com/ajcn/article-abstract/107/2/217/4911436 by guest on 02 May 2020 FIGURE 3 Concentrations of summed TAAs (A) and leucine (B). Data are reported for 11 participants/group. Analysis of means was by 2-way ANOVA. (A) Main effect of time (P < 0.001), time × treatment interaction (P = 0.62). (B) *Significantly different from CON at the same time point (time × treatment interaction, P < 0.001). CON, control group; LEU, leucine-supplemented group; TAA, total amino acid.

Correlational analyses lowing LEU than CON in both the rested and exercised state. In both the rested and exercised leg there was a correla- Further, we report that the LEU beverage, when integrated into, tion between acute MPS and leucine C (rested: r = 0.570, rather than being provided as supplement to, a daily diet pro- max · –1 · –1 P = 0.01; exercised: r = 0.564, P = 0.01) as well as between viding protein at 1.0 g kg d for 6 d resulted in increased acute MPS and leucine AUC (rested: r = 0.522, P = 0.02; exer- myoPS rates above baseline levels, despite providing the same cised: r = 0.534, P = 0.01). amount of protein as the presupplemented diet. The increased integrated myoPS response was augmented by exercise in the LEU group. Incorporation of the CON beverage, containing 15 g high- DISCUSSION quality protein but lower leucine, into the subjects’ habitual diet We showed that the addition of leucine to a mixed-protein did not increase myoPS rates above that of the baseline period beverage containing 15 g high-quality protein and a total of 4.2 in the rested leg. As we hypothesized, when exercise was per- g leucine/serving (∼1.1 g inherent and ∼3.1 g exogenous) en- formed, myoPS rates were increased above baseline rates with hanced the myoPS response to feeding compared with ingestion CON. We also show that the acute myoPS response to protein of an isoenergetic, isonitrogenous beverage containing ∼1.3 g feeding was related to the leucine Cmax following drink ingestion leucine. We also showed that both the LEU and CON beverages as well as leucine AUC in both the rested and exercised leg. To- increased acute myoPS rates above fasted levels in older women, gether the findings from this study support a role for leucine inen- with the increase being greater in a leg that performed resistance hancing the myoPS response to protein feeding in older women, exercise. Importantly, the acute myoPS response was greater fol- and when incorporated into the daily diet may assist in preserving

TABLE 4 Amino acid, glucose and insulin data1

LEU (n = 11) CON (n = 11) Statistical analysis, P Total amino acids Cmax, μM 5065 ± 213 4888 ± 234 0.61 Tmax,min 50 ± 351± 40.82 AUC, μmol · min/L 531,733 ± 24,022 513,044 ± 18,844 0.57 Leucine * Cmax, μM 890 ± 70 453 ± 38 <0.0001 Tmax,min 52 ± 351± 40.85 AUC, μmol · min/L 92330 ± 6596* 42989 ± 7388 <0.0001 Glucose Cmax,mM 6.5 ± 0.2 6.4 ± 0.2 0.60 * Tmax,min 26 ± 3 38 ± 40.03 AUC, mmol · min/L 1094 ± 130 1256 ± 21 0.22 Insulin * Cmax, μIU/mL 37.4 ± 1.9 31.2 ± 1.9 0.02 Tmax,min 50 ± 456± 30.18 AUC, μIU · min/mL 4361 ± 538* 4040 ± 99 0.0008 1Data are means ± SEM. AUC was calculated over the 240 min following study beverage ingestion. Analysis by nonpaired t test. *Significantly different from CON (P < 0.01). , sum of; Cmax, maximum concentration; Tmax, time of maximum concentration. 224 DEVRIES ET AL. Doses of whey protein isolate of 0.25 g protein/kg (∼2.4gof leucine), but not 0.12 g protein/kg (1.2 g of leucine), were able to increase rates of MPS above fasted levels (20). If the same protein dose we reported previously (6) is correct, then the participants in the current trial (∼72 kg) would have required a dose of protein of ∼29 g (∼3.5 g of leucine) to maximize MPS (assuming that there is no sex-based difference in the protein dosage needed to maximize MPS). The LEU and CON beverages provided total leucine doses of 4.2 and 1.3 g, respectively, and thus the LEU beverage should have been sufficient to maximize MPS in this population, whereas the CON beverage would not, which is a conclu- sion broadly supported by our findingsFigure ( 3). Interestingly,

however, when the increase in myoPS induced by the LEU bever- Downloaded from https://academic.oup.com/ajcn/article-abstract/107/2/217/4911436 by guest on 02 May 2020 age is compared with the maximal increase in MPS seen in men following protein ingestion, the increase appears blunted in older women. Greater fasted rates of MPS and an attenuated increase FIGURE 4 Acute myoPS responses in the fasted state and in response to protein feeding in an exercised and rested leg. The box plot shows the me- in MPS in response to a hyperinsulinemic-hyperaminoacidemic- dian (line) and mean (+), with the box representing the IQR and the whiskers euglycemic clamp have been reported in older women compared representing the maximum and minimum values. Data are reported for 11 par- with young women and older men (27). Comparing between ticipants/group. Analysis by 2-way ANOVA. Time × treatment interaction, < < studies, we report a smaller increase in myoPS in older women P 0.01. *Significantly different from fastedP ( 0.01). Significantly ∼ different from fed-rested (P < 0.01). †Significantly different from CON in ( 53%) compared with the increase induced by a protein and the same condition (P < 0.001). CON, control group; FSR, fractional syn- leucine dose known to maximize MPS in older men (∼107%), thetic rate; LEU, leucine-supplemented group; myoPS, myofibrillar protein suggesting that older women may be less responsive to protein synthesis. and leucine to stimulate MPS. We observed a significant increase in integrated rates of muscle mass. Our findings have implications for the design of di- myoPS of 9% with the LEU beverage in the rested leg whereas ets to support retention of muscle mass in older persons and also the CON beverage showed no effect. Even when exercise was provide data in support of the importance of considering protein superimposed upon the nutritional intervention, CON was only quality and an increased leucine requirement to optimize MPS able to increase rates of myoPS to a level comparable to LEU for older persons (9–11). alone (CON exercised: 1.61% ± 0.02%/d compared with LEU The findings from this trial show that both the LEU andCON non-exercised: 1.62% ± 0.03%/d). These findings suggest that beverages increased acute myoPS rates above fasted levels. Pre- inclusion of beverages containing 15 g protein without higher vious work from our group has shown that high-quality protein leucine amounts consumed 2 times/d integrated into the habitual doses of 0.4 g protein/kg (∼3–4 g leucine dependent on protein diet of older women would not be efficacious in offsetting muscle quality) are needed to maximally stimulate MPS in older men (6). loss. A limitation of the current trial is the relatively short length of the integrated MPS assessment (only 6 d). In order to confirm these findings, longer-term trials using sophisticated measures of MPS and muscle mass are required. Several trials have shown that leucine can enhance the stim- ulatory effect on MPS of a less-than-maximally effective quantity of protein in younger (15, 28, 29) and older men (16), with older adults requiring a greater quantity of leucine to augment the MPS response (16). The current results show that the acute myoPS response to a less-than-maximally effective (16) dose of protein is enhanced with added leucine. The role of leucine as the key driver of the MPS response is further evidenced by the significant correlation between leucine Cmax and leucine AUC and MPS in both the non-exercised and exercised limbs in the current study. Congruent with the present data, results from a recent trial in our lab (17) showed that co-ingestion of 5 g leucine (a more-than- sufficient dose) with normal daily meals increased integrated myoPS rates, compared with placebo, in older men consum- FIGURE 5 Integrated myoPS: Baseline, prior to supplementation; Rest, ing either protein intakes at the Recommended Daily Allowance during supplementation in the rested leg; and Exercise, during supplemen- · –1 · –1 tation in the exercised leg. The box plot shows the median (line) and mean (0.8 g kg d ) and at a level recommended (7, 8) for older (+), with the box representing the IQR and the whiskers representing the adults (1.2 g · kg–1 · d–1) and shown to be required (9–11). Our maximum and minimum values. Data are reported for 11 participants/group. findings broadly complement and extend the findings of the previ- × < Analysis by 2-way ANOVA. Time treatment interaction, P 0.01. ous trials by providing data to support a role for the integration of *Significantly different from baseline (P < 0.01). Significantly different from rest (P < 0.01). †Significantly different from CON in the same condi- leucine into the habitual diet, and not necessarily supplemented tion (P < 0.001). CON, control group; FSR, fractional synthetic rate; LEU, on top of the habitual diet, to enhance muscle anabolism in older leucine-supplemented group; myoPS, myofibrillar protein synthesis. women. LEUCINE AND MPS IN OLDER WOMEN 225

Longer-term trials (3-6 mo) assessing the effects of leucine PepsiCo, Inc. DRB, AK, MR, and LH are employees of PepsiCo, Inc., and supplementation on muscle mass in older adults have reported contributed to the writing of the paper but had no role in data analysis or no benefit of leucine30 ( –33). Based on the estimated myofib- interpretation; the remaining authors had no conflicts of interest to declare. rillar protein mass of the participants in the LEU group (from DXA), the increased myoPS during consumption of the LEU REFERENCES beverage into the habitual diet would result in the synthesis of ∼ ∼ 1. Cruz-Jentoft A, Baeyens J, Bauer J, Boirie Y, Cederholm T, Landi F, an extra 3and 5 g myofibrillar protein/d in the nonexercised Martin F, Michel J, Rolland Y, Schneider S, et al. Sarcopenia: European and exercised legs, respectively. Importantly, these findings are consensus on definition and diagnosis: report of the European Working in line with a recent study from our lab where the addition of Group on Sarcopenia in Older People. Age Ageing 2010;39:412–23. 5 g leucine to daily meals induced the synthesis of an additional 2. Iannuzzi-Sucich M, Prestwood KM, Kenny AM. Prevalence of ∼ ∼ sarcopenia and predictors of skeletal muscle mass in healthy, older men 3and 6 g myofibrillar protein/d in the nonexercised and ex- and women. J Gerontol A Biol Sci Med Sci 2002;57:M772–7. ercised legs, respectively, of older men (17). Differences in doses 3. Landi F, Liperoti R, Russo A, Giovannini S, Tosato M, Capoluongo ∼ E, Bernabei R, Onder G. Sarcopenia as a risk factor for falls in between this trial ( 4.2 g leucine/beverage), another trial con- Downloaded from https://academic.oup.com/ajcn/article-abstract/107/2/217/4911436 by guest on 02 May 2020 ducted by our lab (5 g leucine/meal), and 2 of the longer-term sup- elderly individuals: results from the il SIRENTE study. Clin Nutr 2012; 31:652–8. plementation trials [2.5 g leucine/meal (30, 32, 33)] may explain 4. da Silva Alexandre T, de Oliveira Duarte Y, Ferreira Santos J, Wong these divergent findings. Nonetheless, the dose of leucine (2.5 g) R, Lebrao M. Sarcopenia according to the European Working Group used in the 2 aforementioned leucine supplementation trials (30, on Sarcopenia in Older People (EWGSOP) versus dynapenia as a risk 32) is similar to the dose shown to be effective at increasing MPS factor for disability in the elderly. J Nutr Health Aging 2014;18:547–53. 5. Wolfe RR. The underappreciated role of muscle in health and disease. in older adults [2.8 g (16)]; however, it is important to note that Am J Clin Nutr 2006;84:475–82. the dose of leucine required to enhance MPS may be greater when 6. Moore DR, Churchward-Venne TA, Witard O, Breen L, Burd NA, added to a mixed-macronutrient meal compared with an isolated Tipton KD, Phillips SM. Protein ingestion to stimulate myofibrillar protein or EAA-containing beverage. As theorized (17), the ad- protein synthesis requires greater relative protein intakes in healthy older versus younger men. J Gerontol A Biol Sci Med Sci 2015;70:57– dition of 5 g crystalline leucine to mixed-macronutrient meals 62. induced a lower peak plasma leucine concentration (∼450 μM) 7. Bauer J, Biolo G, Cederholm T, Cesari M, Cruz-Jentoft A, Morley compared with that induced by the addition of 2.8 g leucine to J, Phillips S, Sieber C, Stehle P, Teta D, et al. Evidence-based an EAA-containing beverage [∼700 μM(16)], despite provid- recommendation for optimal dietary protein intake in older people: a position paper from the PROT-AGE Study Group. J Am Med Dir Assoc ing almost double the amount of leucine. As such, it may be that 2013;14:542–59. the addition of 2.5 g leucine to mixed-macronutrient meals is in- 8. Wolfe RR, Miller S, Miller K. Optimal protein intake in the elderly. Clin sufficient to enhance MPS in older adults, accounting for some Nutr 2008;27(5):675–84. findings30 ( , 32, 33). Lastly, limitations of the methods used to 9. Rafii M, Chapman K, Elango R, Campbell WW, Ball RO, Pencharz PB, Courtney-Martin G. Dietary protein requirement of men >65 years old assess changes in body composition in the longer-term leucine determined by the indicator amino acid oxidation technique is higher supplementation studies (DXA, skinfolds) resulting from high than the current estimated average requirement. J Nutr 2016;146:681– between-scan/measurement variability may have influenced the 7. ability to detect small changes in lean body mass, particularly in 10. Rafii M, Chapman K, Owens J, Elango R, Campbell WW,Ball RO, Pencharz PB, Courtney-Martin G. Dietary protein requirement the absence of exercise, in these studies (30–32). of female adults >65 years determined by the indicator amino acid In summary, the findings from the current trial show that the oxidation technique is higher than current recommendations. J Nutr addition of leucine to a less-than-maximally effective (to stimu- 2015;145:18–24. late MPS) dose of protein can enhance both acute and integrated 11. Tang M, McCabe GP, Elango R, Pencharz PB, Ball RO, Campbell WW. Assessment of protein requirement in octogenarian women with rates of myoPS. Importantly, the enhanced rates of integrated use of the indicator amino acid oxidation technique. Am J Clin Nutr myoPS were found when the protein-beverage was incorporated 2014;99:891–8. into, not supplemented on top of, the habitual diet, and therefore 12. Houston D, Nicklas B, Ding J, Harris T, Tylavsky F, Newman A, Lee this nutrition strategy may be effective in promoting muscle an- J, Sahyoun N, Visser M, Kritchevsky S, et al. Dietary protein intake is associated with lean mass change in older, community-dwelling adults: abolism in older adults with reduced appetites. The findings from the Health, Aging, and Body Composition (Health ABC) Study. Am J this study suggest that the incorporation of a protein-beverage for- Clin Nutr 2008;87:150–5. tified with leucine, but not an isoenergetic, isonitrogenous protein 13. Volpi E, Campbell WW, Dwyer JT, Johnson MA, Jensen GL, Morley beverage formulated to mimic commercially available protein- JE, Wolfe RR. Is the optimal level of protein intake for older adults greater than the recommended dietary allowance? J Gerontol A Biol nutrition beverages, into the habitual diet of older women may Sci Med Sci 2013;68677–81. attenuate the declines in muscle mass that naturally occur with 14. Morley J. Decreased food intake in the elderly. J Gerontol A Biol Sci increasing age. Med Sci 2001;56(Special Issue II):81–8. 15. Churchward-Venne T, Breen L, Di Donato D, Hector A, Mitchell C, Moore DR, Stellingwerff T, Breuille D, Offord E, Baker S, We thank Tracy Rerecich and Diane Tuerke for their technical and labo- et al. Leucine supplementation of a low-protein mixed macronutrient ratory assistance, and Shellen Goltz for coordination of study logistics and beverage enhances myofibrillar protein synthesis in young men:a liaison with the study site. double-blind, randomized trial. Am J Clin Nutr 2014;99:276–86. The authors’ responsibilities were as follows—MCD, DRB, AK, SMP, 16. Katsanos C, Kobayashi H, Sheffield-Moore M, Aarsland A, Wolfe RR. and MR: conceived and designed the study; MCD and SMP: drafted the A high proportion of leucine is required for optimal stimulation of the rate of muscle protein synthesis by essential amino acids in the elderly. manuscript; and all authors: participated in some aspect of data collection Am J Physiol Endocrinol Metab 2006;291:E381–7. and/or analysis, provided content and/or editorial corrections, and read and 17. Murphy CH, Saddler NI, Devries MC, McGlory C, Baker SK, Phillips approved the final version of the manuscript. SMP reports having received SM. Leucine supplementation enhances integrative myofibrillar protein competitive research funding, travel expenses, and honoraria for speaking synthesis in free-living older men consuming lower- and higher-protein from the US National Dairy Council. The views expressed in this report are diets: a parallel-group crossover study. Am J Clin Nutr 2016;104:1594– those of the authors and do not necessarily represent position or policy of 606. 226 DEVRIES ET AL.

18. Smith GI, Atherton P, Villareal DT, Frimel TN, Rankin D, Rennie MJ, 26. Burd NA, West D, Rerecich T, Prior T, Baker S, Phillips S. Validation Mittendorfer B. Differences in muscle protein synthesis and anabolic of a single biopsy approach and bolus protein feeding to determine signaling in the postabsorptive state and in response to food in 65– myofibrillar protein synthesis in stable isotope tracer studies in humans. 80 year old men and women. PLoS One 2008;3:e1875. Nutr Metab (Lond) 2011;8:15. 19. Bukhari SSI, Phillips BE, Wilkinson DJ, Limb MC, Rankin D, Mitchell 27. Smith GI, Reeds DN, Hall AM, Chambers KT, Finck BN, Mittendorfer WK, Kobayashi H, Greenhaff PL, Smith K, Atherton PJ. Intake of low- B. Sexually dimorphic effect of aging on skeletal muscle protein dose leucine-rich essential amino acids stimulates muscle anabolism synthesis. Biol Sex Differ 2012;3:11. equivalently to bolus whey protein in older women at rest and after 28. Churchward-Venne TA, Burd NA, Mitchell CJ, West DW, Philp A, exercise. Am J Physiol Endocrinol Metab 2015;308:E1056–65. Marcotte GR, Baker SK, Baar K, Phillips SM. Supplementation of a 20. Yang Y, Breen L, Burd N, Hector A, Churchward-Venne T, Josse A, suboptimal protein dose with leucine or essential amino acids: effects on Tarnopolsky M, Phillips S. Resistance exercise enhances myofibrillar myofibrillar protein synthesis at rest and following resistance exercise protein synthesis with graded intakes of whey protein in older men. Br in men. J Physiol 2012;590(Pt 11):2751–65. J Nutr 2012;108:1780–8. 29. Dreyer H, Drummond M, Pennings B, Fujita S, Glynn E, Chinkes 21. Pennings B, Groen B, de Lange A, Gijsen A, Zorenc A, Senden J, D, Volpi E, Rasmussen B. Leucine-enriched essential amino acids van Loon L. Amino acid absorption and subsequent muscle protein and carbohydrate ingestion following resistance exercise enhances

accretion following graded intakes of whey protein in elderly men. Am mTOR signaling and protein synthesis in human muscle. Am J Physiol Downloaded from https://academic.oup.com/ajcn/article-abstract/107/2/217/4911436 by guest on 02 May 2020 J Physiol Endocrinol Metab 2012;302:E992–9. Endocrinol Metab 2008;294:E392–400. 22. Glover E, Phillips S, Oates B, Tang J, Tarnopolsky M, Selby A, Smith 30. Leenders M, Verdijk LB, van der Hoeven L, van Kranenburg J, Hartgens K, Rennie M. Immobilization induces anabolic resistance in human F, Wodzig WKWH, Saris WHM, van Loon LJC. Prolonged leucine myofibrillar protein synthesis with low and high dose amino acid supplementation does not augment muscle mass or affect glycemic infusion. J Physiol 2008;586:6049–61. control in elderly type 2 diabetic men. J Nutr 2011;141:1070–6. 23. Bidlingmeyer B, Cohen S, Tarvin T. Rapid analysis of amino acids using 31. Trabal J, Forga M, Leyes P, Torres F, Rubio J, Prieto E, Farran-Codina pre-column derivatization. J Chromatogr 1984;336:93–104. A. Effects of free leucine supplementation and resistance training on 24. Bell KE, Seguin C, Parise G, Baker SK, Phillips SM. Day-to-day muscle strength and functional status in older adults: a randomized changes in muscle protein synthesis in recovery from resistance, controlled trial. Clin Interv Aging 2015;10:713–23. aerobic, and high-intensity interval exercise in older men. J Gerontol 32. Verhoeven S, Vanschoonbeek K, Verdijk LB, Koopman R, Wodzig WK, A Biol Sci Med Sci 2015;70:1024–9. Dendale P, van Loon LJ. Long-term leucine supplementation does not 25. Burd NA, Andrews RJ, West DWD, Little JP, Cochran AJR, Hector increase muscle mass or strength in healthy elderly men. Am J Clin Nutr AJ, Cashaback JGA, Gibala MJ, Potvin JR, Baker SK, et al. Muscle 2009;89:1468–75. time under tension during resistance exercise stimulates differential 33. Paddon-Jones D, Rasmussen BB. Dietary protein recommendations muscle protein sub-fractional synthetic responses in men. J Physiol and the prevention of sarcopenia. Curr Opin Clin Nutr Metab Care 2012;590:351–62. 2009;12:86–90.