Stefan M. Pasiakos

Effects of high-protein diets on fat-free mass and muscle protein synthesis following weight loss: a randomized controlled trial

The purpose of this work was to determine the effects of varying levels of dietary protein on body composition and muscle protein synthesis during energy deficit (ED). A randomized controlled trial of 39 adults assigned the subjects diets providing protein at 0.8 (recommended dietary allowance; RDA), 1.6 (2X‐RDA), and 2.4 (3X‐RDA) g kg–1 d–1 for 31 d. A 10‐d weight‐maintenance (WM) period was followed by a 21 d, 40% ED. Body composition and postabsorptive and postprandial muscle protein synthesis were assessed during WM (d 9‐10) and ED (d 30‐31). Volunteers lost (P<0.05) 3.2 ± 0.2 kg body weight during ED regardless of dietary protein. The proportion of weight loss due to reductions in fat‐free mass was lower (P<0.05) and the loss of fat mass was higher (P<0.05) in those receiving 2X‐RDA and 3X‐RDA compared to RDA. The anabolic muscle response to a protein‐rich meal during ED was not different (P>0.05) from WM for 2X‐RDA and 3X‐RDA, but was lower during ED than WM for those consuming RDA levels of protein (energy × protein interaction, P<0.05). To assess muscle protein metabolic responses to varied protein intakes during ED, RDA served as the study control. In summary, we determined that consuming dietary protein at levels exceeding the RDA may protect fat‐free mass during short‐term weight loss.—Pasiakos, S. M., Cao, J. J., Margolis, L. M., Sauter, E. R., Whigham, L. D., McClung, J. P., Rood, J. C., Carbone, J. W., Combs, G. F., Jr., Young, A. J. Effects of high‐protein diets on fat‐free mass and muscle protein synthesis following weight loss: a randomized controlled trial. FASEB J. 27, 3837–3847 (2013). www.fasebj.org

Acute Energy Deprivation Affects Skeletal Muscle Protein Synthesis and Associated Intracellular Signaling Proteins in Physically Active Adults

To date, few studies have characterized the influence of energy deprivation on direct measures of skeletal muscle protein turnover. In this investigation, we characterized the effect of an acute, moderate energy deficit (10 d) on mixed muscle fractional synthetic rate (FSR) and associated intracellular signaling proteins in physically active adults. Eight men and 4 women participated in a 20-d, 2-phase diet intervention study: weight maintenance (WM) and energy deficient (ED; approximately 80% of estimated energy requirements). Dietary protein (1.5 g x kg(-1) x d(-1)) and fat (approximately 30% of total energy) were constant for WM and ED. FSR and intracellular signaling proteins were measured on d 10 of both interventions using a primed, constant infusion of [(2)H(5)]-phenylalanine and Western blotting techniques, respectively. Participants lost approximately 1 kg body weight during ED (P < 0.0001). FSR was reduced approximately 19% (P < 0.05) for ED (0.06 +/- 0.01%/h) compared with WM (0.074 +/- 0.01%/h). Protein kinase B and eukaryotic initiation factor 4E binding protein 1 phosphorylation were lower (P < 0.05) during ED compared with WM. AMP activated protein kinase phosphorylation decreased (P < 0.05) over time regardless of energy status. These findings show that FSR and associated synthetic intracellular signaling proteins are downregulated in response to an acute, moderate energy deficit in physically active adults and provide a basis for future studies assessing the impact of prolonged, and perhaps more severe, energy restriction on skeletal muscle protein turnover.

Leucine-enriched essential amino acid supplementation during moderate steady state exercise enhances postexercise muscle protein synthesis

BACKGROUND The effects of essential amino acid (EAA) supplementation during moderate steady state (ie, endurance) exercise on postexercise skeletal muscle metabolism are not well described, and the potential role of supplemental leucine on muscle protein synthesis (MPS) and associated molecular responses remains to be elucidated. OBJECTIVE This randomized crossover study examined whether EAA supplementation with 2 different concentrations of leucine affected post-steady state exercise MPS, whole-body protein turnover, and mammalian target of rapamycin 1 (mTORC1) intracellular signaling. DESIGN Eight adults completed 2 separate bouts of cycle ergometry [60 min, 60% VO(2)peak (peak oxygen uptake)]. Isonitrogenous (10 g EAA) drinks with different leucine contents [leucine-enriched (l)-EAA, 3.5 g leucine; EAA, 1.87 g leucine] were consumed during exercise. MPS and whole-body protein turnover were determined by using primed continuous infusions of [(2)H(5)]phenylalanine and [1-(13)C]leucine. Multiplex and immunoblot analyses were used to quantify mTORC1 signaling. RESULTS MPS was 33% greater (P < 0.05) after consumption of L-EAA (0.08 ± 0.01%/h) than after consumption of EAA (0.06 ± 0.01%/h). Whole-body protein breakdown and synthesis were lower (P < 0.05) and oxidation was greater (P < 0.05) after consumption of L-EAA than after consumption of EAA. Regardless of dietary treatment, multiplex analysis indicated that Akt and mammalian target of rapamycin phosphorylation were increased (P < 0.05) 30 min after exercise. Immunoblot analysis indicated that phosphorylation of ribosomal protein S6 and extracellular-signal regulated protein kinase increased (P < 0.05) and phosphorylation of eukaryotic elongation factor 2 decreased (P < 0.05) after exercise but was not affected by dietary treatment. CONCLUSION These findings suggest that increasing the concentration of leucine in an EAA supplement consumed during steady state exercise elicits a greater MPS response during recovery. This trial is registered at clinicaltrials.gov as NCT01366924.

Skeletal Muscle Responses to Negative Energy Balance: Effects of Dietary Protein

Sustained periods of negative energy balance decrease body mass due to losses of both fat and skeletal muscle mass. Decreases in skeletal muscle mass are associated with a myriad of negative consequences, including suppressed basal metabolic rate, decreased protein turnover, decreased physical performance, and increased risk of injury. Decreases in skeletal muscle mass in response to negative energy balance are due to imbalanced rates of muscle protein synthesis and degradation. However, the underlying physiological mechanisms contributing to the loss of skeletal muscle during energy deprivation are not well described. Recent studies have demonstrated that consuming dietary protein at levels above the current recommended dietary allowance (0.8 g · kg(-1) · d(-1)) may attenuate the loss of skeletal muscle mass by affecting the intracellular regulation of muscle anabolism and proteolysis. However, the specific mechanism by which increased dietary protein spares skeletal muscle through enhanced molecular control of muscle protein metabolism has not been elucidated. This article reviews the available literature related to the effects of negative energy balance on skeletal muscle mass, highlighting investigations that assessed the influence of varying levels of dietary protein on skeletal muscle protein metabolism. Further, the molecular mechanisms that may contribute to the regulation of skeletal muscle mass in response to negative energy balance and alterations in dietary protein level are described.

Chocolate Milk and Endurance Exercise Recovery Protein Balance, Glycogen and Performance

PURPOSE This study examined effects of fat-free chocolate milk (MILK) consumption on kinetic and cellular markers of protein turnover, muscle glycogen, and performance during recovery from endurance exercise. METHODS Male runners participated in two trials separated by 1 wk and consumed either MILK or a nonnitrogenous isocaloric carbohydrate (CHO) control beverage (CON) after a 45-min run at 65% of V˙O(2peak). Postexercise muscle protein fractional synthetic rate (FSR) and whole-body protein turnover were determined during 3 h of recovery using muscle biopsies and primed constant infusions of L-[ring-²H₅]phenylalanine and L-[1-¹³C]leucine, respectively. Phosphorylation of translational signaling proteins and activity of proteolytic molecules were determined using Western blotting and enzymatic activity assays. Muscle glycogen was quantified, and treadmill time to exhaustion was determined after the recovery period. RESULTS Consuming MILK after exercise resulted in higher mixed muscle FSR with lower whole-body proteolysis and synthesis compared with CON (P ≤ 0.05). Phosphorylation of eIF4E-BP1 and FOXO3a was higher for MILK (P < 0.01), whereas Akt phosphorylation was lower during recovery regardless of dietary treatment (P < 0.05). Enzymatic activity assays indicated lower caspase-3 activity during recovery for MILK (P < 0.01) and higher 26S proteasome activity for CON (P < 0.01). Muscle glycogen was not affected by either dietary treatment; however, time to exhaustion was greater for MILK than for CON (P < 0.05). CONCLUSIONS The effects of consumption of MILK after endurance exercise on FSR, signaling molecules of skeletal muscle protein turnover, leucine kinetics, and performance measures suggest unique benefits of milk compared with a CHO-only beverage.

Exercise and Amino Acid Anabolic Cell Signaling and the Regulation of Skeletal Muscle Mass

A series of complex intracellular networks influence the regulation of skeletal muscle protein turnover. In recent years, studies have examined how cellular regulators of muscle protein turnover modulate metabolic mechanisms contributing to the loss, gain, or conservation of skeletal muscle mass. Exercise and amino acids both stimulate anabolic signaling potentially through several intracellular pathways including the mammalian target of rapamycin complex 1 and the mitogen activated protein kinase cell signaling cascades. As novel molecular regulators of muscle integrity continue to be explored, a contemporary analysis of the literature is required to understand the metabolic mechanisms by which contractile forces and amino acids affect cellular process that contribute to long-term adaptations and preservation of muscle mass. This article reviews the literature related to how exercise and amino acid availability affect cellular regulators of skeletal muscle mass, especially highlighting recent investigations that have identified mechanisms by which contractile forces and amino acids modulate muscle health. Furthermore, this review will explore integrated exercise and nutrition strategies that promote the maintenance of muscle health by optimizing exercise, and amino acid-induced cell signaling in aging adults susceptible to muscle loss.

Supplemental dietary leucine and the skeletal muscle anabolic response to essential amino acids

Skeletal muscle protein synthesis (MPS) is regulated by a number of dietary factors, to include essential amino acids (EAAs). Leucine, a branched-chain amino acid, has been identified as a stimulator of MPS in many cell culture and animal studies. However, whether supplemental leucine exerts a unique stimulatory effect, as compared to other EAAs, on muscle anabolism in humans has not been clearly demonstrated. A recent study found no improvement in resting MPS in adults who consumed a 10 g EAA supplement providing added leucine (3.5 g leucine) when compared to a control 10 g EAA supplement (1.8 g leucine). These findings suggest that added leucine is unnecessary for the stimulation of MPS when sufficient EAAs are provided; however, the study of supplemental leucine during conditions such as endurance exercise, caloric deprivation, and ageing may be warranted.

Changes in intestinal microbiota composition and metabolism coincide with increased intestinal permeability in young adults under prolonged physiological stress

The magnitude, temporal dynamics, and physiological effects of intestinal microbiome responses to physiological stress are poorly characterized. This study used a systems biology approach and a multiple-stressor military training environment to determine the effects of physiological stress on intestinal microbiota composition and metabolic activity, as well as intestinal permeability (IP). Soldiers (n = 73) were provided three rations per day with or without protein- or carbohydrate-based supplements during a 4-day cross-country ski-march (STRESS). IP was measured before and during STRESS. Blood and stool samples were collected before and after STRESS to measure inflammation, stool microbiota, and stool and plasma global metabolite profiles. IP increased 62 ± 57% (mean ± SD, P < 0.001) during STRESS independent of diet group and was associated with increased inflammation. Intestinal microbiota responses were characterized by increased α-diversity and changes in the relative abundance of >50% of identified genera, including increased abundance of less dominant taxa at the expense of more dominant taxa such as Bacteroides Changes in intestinal microbiota composition were linked to 23% of metabolites that were significantly altered in stool after STRESS. Together, pre-STRESS Actinobacteria relative abundance and changes in serum IL-6 and stool cysteine concentrations accounted for 84% of the variability in the change in IP. Findings demonstrate that a multiple-stressor military training environment induced increases in IP that were associated with alterations in markers of inflammation and with intestinal microbiota composition and metabolism. Associations between IP, the pre-STRESS microbiota, and microbiota metabolites suggest that targeting the intestinal microbiota could provide novel strategies for preserving IP during physiological stress.NEW & NOTEWORTHY Military training, a unique model for studying temporal dynamics of intestinal barrier and intestinal microbiota responses to stress, resulted in increased intestinal permeability concomitant with changes in intestinal microbiota composition and metabolism. Prestress intestinal microbiota composition and changes in fecal concentrations of metabolites linked to the microbiota were associated with increased intestinal permeability. Findings suggest that targeting the intestinal microbiota could provide novel strategies for mitigating increases in intestinal permeability during stress.

Effects of winter military training on energy balance, whole-body protein balance, muscle damage, soreness, and physical performance

Physiological consequences of winter military operations are not well described. This study examined Norwegian soldiers (n = 21 males) participating in a physically demanding winter training program to evaluate whether short-term military training alters energy and whole-body protein balance, muscle damage, soreness, and performance. Energy expenditure (D2(18)O) and intake were measured daily, and postabsorptive whole-body protein turnover ([(15)N]-glycine), muscle damage, soreness, and performance (vertical jump) were assessed at baseline, following a 4-day, military task training phase (MTT) and after a 3-day, 54-km ski march (SKI). Energy intake (kcal·day(-1)) increased (P < 0.01) from (mean ± SD (95% confidence interval)) 3098 ± 236 (2985, 3212) during MTT to 3461 ± 586 (3178, 3743) during SKI, while protein (g·kg(-1)·day(-1)) intake remained constant (MTT, 1.59 ± 0.33 (1.51, 1.66); and SKI, 1.71 ± 0.55 (1.58, 1.85)). Energy expenditure increased (P < 0.05) during SKI (6851 ± 562 (6580, 7122)) compared with MTT (5480 ± 389 (5293, 5668)) and exceeded energy intake. Protein flux, synthesis, and breakdown were all increased (P < 0.05) 24%, 18%, and 27%, respectively, during SKI compared with baseline and MTT. Whole-body protein balance was lower (P < 0.05) during SKI (-1.41 ± 1.11 (-1.98, -0.84) g·kg(-1)·10 h) than MTT and baseline. Muscle damage and soreness increased and performance decreased progressively (P < 0.05). The physiological consequences observed during short-term winter military training provide the basis for future studies to evaluate nutritional strategies that attenuate protein loss and sustain performance during severe energy deficits.

Calcium and vitamin D supplementation maintains parathyroid hormone and improves bone density during initial military training: A randomized, double-blind, placebo controlled trial

Calcium and vitamin D are essential nutrients for bone health. Periods of activity with repetitive mechanical loading, such as military training, may result in increases in parathyroid hormone (PTH), a key regulator of Ca metabolism, and may be linked to the development of stress fractures. Previous studies indicate that consumption of a Ca and vitamin D supplement may reduce stress fracture risk in female military personnel during initial military training, but circulating markers of Ca and bone metabolism and measures of bone density and strength have not been determined. This randomized, double-blind, placebo-controlled trial sought to determine the effects of providing supplemental Ca and vitamin D (Ca+Vit D, 2000mg and 1000IU/d, respectively), delivered as 2 snack bars per day throughout 9weeks of Army initial military training (or basic combat training, BCT) on PTH, vitamin D status, and measures of bone density and strength in personnel undergoing BCT, as well as independent effects of BCT on bone parameters. A total of 156 men and 87 women enrolled in Army BCT (Fort Sill, OK; 34.7°N latitude) volunteered for this study. Anthropometric, biochemical, and dietary intake data were collected pre- and post-BCT. In addition, peripheral quantitative computed tomography was utilized to assess tibia bone density and strength in a subset of volunteers (n=46). Consumption of supplemental Ca+Vit D increased circulating ionized Ca (group-by-time, P=0.022), maintained PTH (group-by-time, P=0.032), and increased the osteoprotegerin:RANKL ratio (group-by-time, P=0.006). Consistent with the biochemical markers, Ca+Vit D improved vBMD (group-by-time, P=0.024) at the 4% site and cortical BMC (group-by-time, P=0.028) and thickness (group-by-time, P=0.013) at the 14% site compared to placebo. These data demonstrate the benefit of supplemental Ca and vitamin D for maintaining bone health during periods of elevated bone turnover, such as initial military training. This trial was registered with ClincialTrials.gov, NCT01617109.