Dose-response of myofibrillar protein synthesis to ingested whey protein during energy restriction in overweight postmenopausal women: a randomized, controlled trial

49 Background: Diet-induced weight loss is associated with a decline in lean body mass, as 50 mediated by an impaired response of muscle protein synthesis (MPS). The dose-response of 51 MPS to ingested protein, with or without resistance exercise, is well characterised during energy 52 balance but limited data exist under conditions of energy restriction in clinical populations. 53 Objective: To determine the dose-response of MPS to ingested whey protein following short54 term diet-induced energy restriction in overweight, postmenopausal, women at rest and post55 exercise. 56 Design: Forty middle-aged (58.6±0.4 years), overweight (BMI: 28.6±0.4), postmenopausal 57 women were randomised to one of four groups: Three groups underwent 5 days of energy 58 restriction (~800 kcal/d). On day 6, participants performed a unilateral leg resistance exercise 59 bout before ingesting either a bolus of 15g (ERW15, n=10), 35g (ERW35, n=10) or 60g 60 (ERW60, n=10) of whey protein. The fourth group (n=10) ingested a 35g whey protein bolus 61 after 5 days of an energy balanced diet (EBW35, n=10). Myofibrillar fractional synthetic rate 62 (FSR) was calculated under basal, fed (FED) and post-exercise (FED-EX) conditions by 63 combining an L-[ring-C6]phenylalanine tracer infusion with the collection of bilateral muscle 64 biopsies. 65 Results: Myofibrillar-FSR was greater in ERW35 (0.043±0.003%/h, P=0.013) and ERW60 66 (0.042±0.003%/h, P=0.026) than ERW15 (0.032±0.003%/h), with no differences between 67 ERW35 and ERW60 (P=1.000). Myofibrillar-FSR was greater in FED (0.044±0.003%/h, 68 P<0.001) and FED-EX (0.048±0.003%/h, P<0.001) than BASAL (0.027±0.003%/h), but no 69 differences were detected between FED and FED-EX (P=0.732) conditions. No differences in 70 myofibrillar FSR were observed between EBW35 (0.042±0.003%/h) and ERW35 71 (0.043±0.003%/h, P=0.744). 72 Jo urn al Pr e-p roo f

and then rested in a supine position for 3 h before two further muscle biopsies were obtained 215 from the exercised (FED-EX) and non-exercised (FED) leg. 216

Protein beverages 217
Whey protein beverages (Lacprodan® HYDRO.REBUILD, Arla Foods Ingredients Group P/S, 218 Viby J, DK) were administered immediately after collection of the second muscle biopsy 219 obtained after exercise ( Table 2)

Muscle biopsy and blood sampling 227
All blood samples were dispensed into pre-chilled coated (EDTA or lithium heparin) blood 228 collection tubes. Serum-separator tubes were allowed to clot for 30 min before centrifugation 229 (1,500 g for 15 min at 5°C). As described above, a total of four muscle biopsies (two from each 230 leg; ~250 mg) were obtained from the vastus lateralis (~12-15 cm proximal to patella) under 231 local anaesthesia (10 ml Xylocain® 10mg/ml, AstraZeneca, Sweden) using a 5 mm Bergström 232 needle with manual suction. Muscle samples were snap frozen and stored at -80°C until further 233 analysis. 234

Stable isotope analysis 243
Plasma phenylalanine enrichments were determined as described previously (41). To isolate 244 intramuscular free amino acids and myofibrillar proteins, muscle samples (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)  and then centrifuged at 10,000 g for 15 min at 4°C. This process was repeated with the 249 remaining pellet without the protease inhibitor tablet solubilized in the buffer. The two 250 supernatants (~2 mL) were transferred to vials with 2 mL ice cold 100% acidic acid. The free 251 amino acids were subsequently purified over columns with acidified cation exchange resin as 252 described previously (42). Next, 1 mL NaOH (0.3 M) was added to the pellet from the 253 homogenization process containing structural proteins, homogenized for 30 s and left in a 254 heating block (50°C) for 2 × 30 min (vortexed in between) and centrifuged (10,000 g, 10 min, 255 4°C). Supernatants were transferred to vials suitable for hydrolysis. This process was repeated 256 with the remaining pellet and supernatants merged. Perchloric acid (1 mL 2 M) was added to 257 the supernatants containing myofibrillar proteins. Vials were vortexed and left on ice for 20 258 min. After centrifugation (3,000 g, 10 min, 4°C), supernatants were discarded and the pellets 259 washed twice in EtOH (1 mL 70%), vortexed and centrifuged (3,000 g, 10 min, 4°C). The 260 remaining pellets were vortexed in a mix of 2 mL HCl and 1 mL Dowex resin (Bio-Rad 261 Laboratories, Hercules, CA), before overnight incubation (110°C). Subsequently, the 262 12 myofibrillar amino acids were purified over cation exchange resin columns using NaOH (2M) 263 for elution. Amino acids were derivatized with N-acetyl-propyl as described previously (42). 264 Finally, the derivatized samples were injected into a gas-chromatography combustion isotope 265 ratio mass spectrometer (ThermoFisher Scientific, Hemel Hempstead, UK). For practical 266 reasons, the muscle samples were analyzed at University of Birmingham and University of 267 Nottingham. The analyses used the same protocols for sample preparation. Data was inspected 268 visually and statistically to identify any effect of analysis-site.
Where ∆Eprotein is the difference in tracer enrichment in the myofibrillar protein fraction between 275 two biopsy samples, Eprecursor is the arterialized blood precursor defined as the area under the 276 curve (AUC) for plasma enrichments of labelled phenylalanine over the 3-h incorporation 277 periods. ∆time is the time interval between muscle biopsies. 278

Data presentation and statistics 279
A sample size of 32 (8 participants/group) was calculated a priori based on previous data from 280 comparable studies with similar participant characteristics investigating the dose-response of 281 myofibrillar FSR to ingested protein in older men (34,35). This calculation was based on the 282 assumption that the minimal detectable difference in FSR between protein dosages would be 283 0.01 %/h when the SD of the means was set to be 0.007 %/h. The 1-β error of probability was 284 set at 0.8 and an α-level of < 0.05. (insulin, urea, glucose, amino acid concentrations and phenylalanine enrichments) were 296 analysed using a similar mixed model with protein dose and time as fixed effects, and 297 participants as a random effect. Main effects (protein dose, time) and interactions, as well as 298 post hoc analyses, were performed as described above. One-way analyses of variance 299 (ANOVA) was used for data presented as incremental area under the curve (iAUC). iAUC was 300 calculated with the baseline set as timepoint 0. Normality and homogeneity of data were 301 checked by inspecting QQ-plots and plots of residuals versus the fitted values. Serum insulin 302 concentrations were deemed heteroskedastic from visual inspection and consequently log-303 transformed before statistical analyses. Data are presented as means ± SEM unless otherwise 304 stated. All statistical analyses were performed using STATA version 14.2 (StataCorp LP,  305 Collage Station, TX, USA) and significance was set at an α-level of < 0.05. 306

Diet, exercise and body weight 308
Total energy and macronutrient intakes were lower in the energy-restricted diet groups than the 309 energy balance diet group (all P < 0.05, Table 3  Serum insulin concentrations peaked 30 -60 min after protein ingestion (P < 0.01) and 337 returned to baseline levels at 3 h post protein ingestion in ERW15 and ERW35 (Figure 5a). 338 The iAUC of serum insulin concentration was higher in ERW35 and ERW60 than ERW15 (P 339 < 0.05) and higher in ERW60 than in ERW35 (P = 0.033, Figure 5b (Figure 7). Post hoc analysis revealed a greater response of myofibrillar 353 FSR in ERW35 (32%, +0.010 ± 0.003%/h, P = 0.013) and ERW60 (29%, +0.009 ± 0.003%/h, 354 P = 0.026) than ERW15, with no differences between ERW35 and ERW60 (P = 1.000). A main 355 effect of condition was observed for all groups combined (P < 0.001), with myofibrillar FSR 356 63% greater in FED (+0.017 ± 0.004%/h, P < 0.001) and 79% greater in FED-EX (+0.021 ± 357 0.004%/h, P < 0.001) than BASAL, but no differences were detected between the FED and 358 FED-EX (P=0.732) conditions. In addition, no protein dose × condition interaction was 359 detected (P = 0.744) (Figure 7). Moreover, no main effects of diet (energy restriction vs. energy 360 balance, P = 0.744) or diet × condition interaction (P = 0.996) were observed for myofibrillar 361 FSR when EBW35 and ERW35 groups only were included in the statistical model. However, 362 a main effect of condition (P < 0.001) was observed for this analysis as well (Figure 7).  (30,31,44). The opposing argument suggests 394 the anabolic response to ingested protein is not limited by the maximal stimulation of protein 395 synthesis (45). This viewpoint is evidenced by studies that conducted whole-body assessments 396 of protein synthesis, i.e., aggregate protein synthesis rates across all body tissues combined, 397 rather than tissue-specific (i.e., muscle) measurements of MPS (46,47). In the present study, women, our results suggest that energy restricted middle-aged, overweight, postmenopausal, 406 women respond similarly to protein feeding as their male counterparts in energy balance. 407 Hence, taken together these data suggest that following 5 d of energy restriction, 35 g of whey 408 protein is sufficient for the maximal stimulation of MPS in middle-aged, overweight 409 postmenopausal woman. 410 The interaction of exercise training and increased dietary protein intake during a period 411 of energy deficit represents an evidence-based strategy to mitigate the impaired response of 412 MPS, and potential subsequent decline in muscle mass, associated with diet-induced weight 413 loss in overweight women (48,49). Consistent with this notion, a longitudinal study by 414 Layman, et al. (5) demonstrated that the addition of a resistance-based exercise training 415 program (2 d/wk resistance training + 5 d/wk walking) to a high protein diet (1.6 g/kg BM/d) 416 promoted the loss of fat mass and retention of lean body mass in middle-aged women that 417 undertook a 4-month weight loss trial. In addition, the impairment in basal myofibrillar FSR 418 following 5 days of energy restriction in resistance-trained young adults was restored following 419 a single bout of resistance exercise to levels observed at rest in energy balance (15). These 420 authors also reported that protein ingestion increased MPS in a dose-dependent manner above 421 rates observed at rest during energy balance (15). However, in the present study, and refuting 422 our original hypothesis, we report no additive effect of resistance exercise on the postprandial 423 response of MPS. Whereas myofibrillar FSR was greater in FED and FED-EX than BASAL 424 19 across dose groups, no statistical difference in MPS was observed between FED and FED-EX 425 conditions. In contrast, previous dose-response studies, conducted under conditions of energy 426 balance and utilizing the same unilateral exercise model as the present study, have demonstrated 427 greater MPS rates in the exercised vs. rested leg in healthy young (31), middle-aged (34) and 428 older (35) adults. Hence, we may deduce that 5 days in energy deficit is sufficient to inhibit the 429 exercise-induced stimulation of MPS in middle-aged, postmenopausal woman that are less 430 responsive to resistance exercise as an anabolic stimulus compared with their resistance-trained 431 young adult counterparts (15,23). 432 An alternative factor that may underpin the lack of exercise-induced stimulation of MPS 433 may be the relatively short 3 h tracer incorporation period employed in the present study. The attenuated rate of MPS previously reported during energy restriction (14)(15)(16) has 443 been proposed to represent an adaptive mechanism to conserve energy during weight loss. This 444 notion is intuitive given that MPS is an energetically expensive metabolic process that requires 445 ~4 moles of ATP to initiate the translation elongation step of MPS (53). Accordingly, studies 446 in healthy, weight stable, young adults demonstrate an ∼25% decrease in basal rates of MPS 447 during the early (5-10 d) phase of an energy-restricted diet (13,15,16)     All values are means ± SD. Data were analysed using a one-factor ANOVA. *significant difference vs. energy restricted groups for corresponding measurements (P < 0.001). ER, energy restricted diet group; EB, energy balanced diet group; CHO, carbohydrate; PRO, protein.       Based on the CONSORT guidelines.

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