To determine whole-body protein turnover responses to high-protein diets during weight loss, 39 adults (age, 21±1 years; VO2peak, 48±1 ml kg−1 min−1; body mass index, 25±1 kg m2) were randomized to diets providing protein at the recommend dietary allowance (RDA), 2 × -RDA or 3 × -RDA. A 10-day weight maintenance period preceded a 21-day, 40% energy deficit. Postabsorptive (FASTED) and postprandial (FED) whole-body protein turnover was determined during weight maintenance (day 10) and energy deficit (day 31) using [1-13C]leucine. FASTED flux, synthesis and breakdown were lower (P<0.05) for energy deficit than weight maintenance. Protein flux and synthesis were higher (P<0.05) for FED than FASTED. Feeding attenuated (P<0.05) breakdown during weight maintenance but not energy deficit. Oxidation increased (P<0.05) between dietary protein levels and feeding stimulated oxidation, although oxidative responses to feeding were higher (P<0.05) for energy deficit than weight maintenance. FASTED net balance decreased between dietary protein levels, but in the FED state, net balance was lower for 3 × -RDA as compared with RDA and 2 × -RDA (diet-by-state, P<0.05). Consuming dietary protein at levels above the RDA, particularly 3 × -RDA, during short-term weight loss increases protein oxidation with concomitant reductions in net protein balance.
Energy intake is critical for optimizing whole-body protein utilization, as maintaining energy balance reduces the reliance on exogenous amino acids as substrates, and allows the body to utilize protein for non-energy yielding functions,1 such as the maintenance of lean body mass. However, during energy deficit (e.g., weight loss), protein utilization is degraded. Acute (⩽ days) energy deficit upregulates whole-body protein turnover, as proteolysis and oxidation are typically increased to provide amino acids to sustain protein synthesis and hepatic gluconeogenesis.2, 3 Subsequently, as the duration of energy deficit is extended (⩾3 weeks), whole-body protein breakdown and synthesis are reduced, conserving energy and endogenous protein reserves.4
Increasing dietary protein intake upregulates whole-body protein turnover and improves net protein utilization during energy balance.5 Therefore, consuming dietary protein at levels exceeding the recommended dietary allowance (RDA, >0.8 g kg−1 per day) may offset energy deficit-induced downregulations in whole-body protein turnover. Numerous studies in overweight and obese adults have demonstrated protein conservation secondary to energy deficit, and suggest a whole-body protein metabolic advantage through the consumption of dietary protein at levels approaching two times the RDA.6, 7, 8, 9, 10, 11, 12 Whether high-protein intakes confer similar benefits in physically active adults, whose protein requirements necessary for the optimization of whole-body protein balance during energy deficit may exceed two times the RDA, is not well described.
Our group recently demonstrated that consuming two times and three times the RDA for protein during weight loss enhanced nitrogen retention, spared lean mass and conserved muscle protein anabolic sensitivity to feeding.13 In the same study, consuming a protein-containing mixed-meal during short-term energy deficit attenuated intramuscular protein degradation independent of dietary protein level.14 Perhaps of greatest importance, consuming dietary protein beyond two times the RDA failed to provide further skeletal muscle protection during short-term energy deficit, thereby suggesting that consuming dietary protein above 1.6 g kg−1 per day may be unnecessary during short-term weight loss. However, because the relative contribution of skeletal muscle to whole-body protein utilization is low (∼30–40%),15 whether consuming dietary protein beyond two times the RDA enhances whole-body protein balance, and therefore may be considered metabolically necessary during short-term weight loss, has not been determined.
The objective of this study was to characterize postabsorptive (FASTED) and postprandial (FED) whole-body protein turnover responses to dietary protein intakes spanning the acceptable macronutrient distribution range (AMDR; 1-3 × -RDA) during short-term energy deficit. We hypothesized that consuming high-protein diets would upregulate whole-body protein turnover in a dose-dependent manner and attenuate energy deficit-induced reductions in protein synthesis, thereby enhancing protein balance.
Materials and methods
After providing informed, written consent, 39 weight stable adults were assigned to diets providing protein at 0.8 (RDA, n=13; 11 men, 2 women), 1.6 (2 × -RDA, n=14; 11 men, 3 women) and 2.4 (3 × -RDA, n=12; 10 men, 2 women) g kg−1 per day for 31 days. A 10-day weight maintenance period was followed by a 21-day, 40% energy deficit. Volunteers resided on the metabolic ward at the USDA Grand Forks Human Nutrition Research center to ensure experimental control.
Diet and physical activity
Details regarding the controlled diet and physical activity interventions have been reported previously.13 Energy requirements were individualized and adjusted to ensure weight maintenance (days 1–10). Protein intake remained constant throughout the intervention, and was provided as mixed, high-quality proteins. Fat accounted for ⩽30% of total energy, and carbohydrate provided the remainder of the prescribed energy. At the conclusion of weight maintenance, volunteers consumed the 21-day energy deficit diet, which reduced energy intake by 30%. Research dietitians prepared 3-day menus and administered and supervised meals to ensure compliance.
Volunteers performed resistance exercise 3 days per week (single-joint movements for 3 × 15 repetitions) and daily endurance exercise (40–60% VO2peak,) at prescribed levels comparable to those reported at baseline to maintain prestudy fitness. Exercise was highly controlled and monitored for accuracy. Resistance exercise prescriptions remained constant but the duration of endurance exercise during energy deficit was increased to cause participants to expend 10% more energy than predicted weight maintenance energy requirements, such that a 40% energy deficit was achieved by combining increased exercise-induced energy expenditure with the 30% reduction in energy intake. As reported, body mass remained steady during weight maintenance, and mean body mass loss in response to the 21-day energy deficit was 3.2±0.2 kg, regardless of dietary protein group (RDA, 3.5±0.3 kg; 2 × -RDA, 2.7±0.2 kg; and 3 × -RDA, 3.3±0.3 kg).13
Whole-body protein turnover
FASTED and FED whole-body protein turnover was determined following an overnight fast at the conclusion of weight maintenance (day 10) and energy deficit (day 31) using bolus injections of [13C]bicarbonate (2.35 μmol kg−1) and primed, continuous infusions of L-[l-13C]leucine (7.6 μmol kg−1; 7.6 μmol kg−1 min−1) for 480 min. At 300 min, volunteers consumed a nutrition supplement (Boost Nestle HealthCare Nutrition, Florham Park, NJ, USA) providing 480 kcal, 20 g of protein, 8 g of fat and 82 g carbohydrate.
Plasma [13C]α-ketoisocaproate and breath 13CO2 enrichments were determined from samples collected at isotopic plateau in 15 min intervals for FASTED (210–300 min) and FED (390–480 min) using gas chromatography and isotope-ratio mass spectroscopy, respectively. Whole-body protein turnover (flux) was determined using methods described previously.16 Breakdown was calculated as flux minus tracer infusion rate for FASTED, and flux minus tracer and leucine intake during FED (leucine intake: weight maintenance, 23.3 μmol kg−1 h−1 and energy deficit, 24.2 μmol kg−1 h−1). Oxidation was calculated from the 13CO2 excretion rate using the reciprocal pool model with a fractional bicarbonate retention factor of 0.7 for FASTED and 0.82 for FED.17 Synthesis was calculated as flux minus oxidation. Net balance was calculated as the difference between total leucine intake (including the tracers) and oxidation.
Volunteers were block randomized and homogeneity was confirmed (overall mean age, 21±1 years; VO2peak, 48±1 ml kg−1 min−1; body mass index, 25±1 kg m−2).13 Turnover was analyzed using a mixed-model repeated-measures analysis of variance including within-subjects factors of energy (weight maintenance and energy deficit) and state (FASTED and FED), and a between-subjects factor of diet (RDA, 2 × -RDA and 3 × -RDA). Compound symmetry was determined as the appropriate covariance model, and Bonferroni adjustments were used for multiple comparisons when significant interactions were observed. Significance was defined as P<0.05. Data were analyzed using the Proc Glimmix in SAS, V.9.3 (SAS Institute Inc., Cary, NC, USA) and are expressed as means±s.e.m.
Energy deficit attenuated whole-body protein turnover independent of dietary protein level, as FASTED flux, synthesis and breakdown were lower for energy deficit than weight maintenance (energy-by-state, P<0.05; Table 1). Feeding upregulated protein turnover regardless of dietary protein level, as flux and synthesis were higher for FED than FASTED, whereas feeding attenuated breakdown during weight maintenance but not energy deficit (energy-by-state, P<0.05).
Oxidation increased (P<0.05) progressively between dietary protein levels independent of energy status, and feeding stimulated oxidation regardless of protein intake, although oxidative responses to feeding were higher for energy deficit than weight maintenance (energy-by-state, P<0.05; Figure 1a). FASTED net balance decreased (P<0.05) between dietary protein levels regardless of energy status. However, in the FED state, net balance was lower for 3 × -RDA as compared with RDA and 2 × -RDA (diet-by-state, P<0.05; Figure 1b).
The major finding from this randomized, controlled trial was that consuming dietary protein at 2 × and 3 × -RDA had no effect on the downregulation in whole-body protein turnover observed in response to short-term energy deficit. Increasing dietary protein near the upper limit of the AMDR (35% total energy intake) may have diminished protein utilization during weight loss, as increased oxidation and decreased net balance suggest an increased reliance on exogenous amino acids for non-protein yielding functions (for example, energy production). Coupled with previous findings indicating that consuming protein at levels 3 × -RDA failed to confer further lean mass protection as compared with 2 × -RDA in response to short-term weight loss,13 the current findings suggest that consuming dietary protein beyond 2 × -RDA, at the expense of carbohydrate, is likely unnecessary.
The effects of acute and sustained energy deficit on basal whole-body protein metabolism are well documented.18 Whole-body protein turnover is initially upregulated in response to acute energy deprivation, as evidenced by increased proteolysis, oxidation and circulating amino-acid concentrations.3, 19 However, prolonged energy deficit elicits a downregulation in whole-body protein turnover, representing a protein metabolic adaptation to conserve endogenous protein stores.1, 4, 10, 11, 12 For example, Stein et al.4 demonstrated a 20% reduction in whole-body protein turnover in overweight adults in response to a 4-week, 40% energy deficit. Others confirmed the energy deficit-induced downregulation in whole-body protein turnover in obese adults following severe energy deficit.11, 12 Findings from the current study demonstrate this adaptive response in normal-weight individuals, as protein flux, synthesis and proteolysis were reduced by approximately 10% in response to the 21-day, 40% energy deficit. However, based on previous studies,20 we hypothesized that dietary protein intake in excess of the RDA during weight loss would attenuate energy-induced reductions in whole-body protein synthesis, further reduce proteolysis and enhance net protein balance. Interestingly, dietary protein had no effect on whole-body protein turnover, which is consistent with findings reported by Pikosky et al.,21 who demonstrated no differences in whole-body protein turnover responses to exercise-induced energy deficit (1000 kcal per day) when volunteers consumed protein at either of 1.0 or 1.8 g kg−1 per day.
Similar to Friedlander et al.,22 energy deficit had no effect on postprandial whole-body protein turnover. Habitual dietary protein intake also had no effect on postprandial turnover responses to a protein-containing meal. These findings appear to conflict with Phillips,23 who hypothesized that metabolic advantages gained from consuming dietary protein at levels above the RDA during weight loss are potentially attributed to protein-induced modulations in postprandial protein turnover. Although we hypothesized that habitual consumption of high-protein diets would expand extracellular amino-acid pools and alter postprandial responses to fixed protein loads,24 we recognize that standardizing the nutritional supplement used to assess postprandial protein turnover does not reflect the average protein load consumed by volunteers assigned to the 2 × - and 3 × -RDA diets. Nevertheless, we previously reported that extracellular amino-acid concentrations increased progressively between diets and muscle protein synthetic responses to fixed protein consumption were enhanced for 2 × - and 3 × -RDA compared with RDA.
The optimal level of dietary protein necessary to prevent deficiencies and promote metabolic heath, including the maintenance of lean body mass following weight loss, has been heavily debated. Recently, the POUNDS LOST study failed to observe the preservation of lean body mass in response to a high-protein (25% total energy intake) weight loss diet.25 This finding conflicts with our previous report13 as well as others,9, 26 which demonstrate that consuming dietary protein at levels at least two times the RDA during weight loss spares lean body mass and promotes the loss of body fat. It is important to recognize that in the POUNDS LOST study, de Souza et al.25 manipulated dietary protein as a percentage of total energy intake (15 vs 25%), and not on an absolute (g per day) or relative (g kg−1 per day) basis. As such, it is not surprising that lean mass was conserved with higher protein diets in our study but not in the POUNDS LOST study, as actual protein intake differed by as much as 100 g per day between the investigations.13, 25 In fact, the de Souza et al.25 study provided 75 and 86 g of protein for those assigned to 15% and 25% protein diets, respectively, whereas the current study provided 64 g (RDA), 123 g (2 × -RDA) and 186 g (3 × -RDA). Our findings indicate that protein intake at levels beyond two times the RDA may be excessive, as oxidation and net balance are negatively impacted, with no further benefits on lean mass. As such, it may be appropriate to recommend protein intakes at levels comparable to the 2 × -RDA diet during weight loss, whether based on body weight or as percentage of total energy intake, as Americans habitually consume protein at comparable levels (∼1.2–1.4 g kg−1 per day).27
Overconsumption of dietary protein, especially during energy balance when metabolic requirements for energy and protein are met, is concerning given concerns regarding renal dysfunction28, 29 and cardiometabolic risk (for example, insulin resistance).30 However, in healthy adults, there is no direct evidence that consuming high-protein diets is detrimental to renal function,31 and furthermore, cross-sectional, longitudinal studies in healthy adults consuming a high-protein diet reported healthy cardiometabolic profiles.32, 33, 34 Although we cannot predict long-term health-related outcomes in the current study, our results, which indicate an inefficient utilization of dietary protein when intake exceeds two times the RDA, do not support the overconsumption of dietary protein (>1.6 g kg−1 per day), regardless of energy status.
Although effective for maintaining lean body mass,13 consuming dietary protein at levels in excess of the RDA during short-term weight loss has no apparent benefit on whole-body protein utilization. Most importantly, data from the current study suggest that dietary protein at levels three times the RDA may be excessive, as consuming protein at 3 × -RDA resulted in a marked increase in protein oxidation and diminished net protein balance during weight loss. These findings support the contention that adults exposed to energy deficit may gain metabolic advantages by increasing dietary protein intake to two times, but not three times the RDA.
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This work was supported by the US Army Medical Research and Material Command and the US Department of Agriculture, Agricultural Research Service, under agreement no. 58-1950-7707.
The authors declare no conflict of interest.
The investigators adhered to the policies for protection of human subjects as prescribed in Army Regulation 70-25, and the research was conducted in adherence with the provisions of 32 CFR part 219. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. Any citations of commercial organizations and trade names in this report do not constitute an official Department of the Army endorsement of approval of the products or services of these organizations. Any opinions, findings, conclusion or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the US Department of Agriculture.
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Pasiakos, S., Margolis, L., McClung, J. et al. Whole-body protein turnover response to short-term high-protein diets during weight loss: a randomized controlled trial. Int J Obes 38, 1015–1018 (2014). https://doi.org/10.1038/ijo.2013.197
- weight loss
- protein turnover
- net balance
- leucine oxidation
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