Lee M. Margolis

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

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.

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.

Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis

This study was undertaken to characterize the ubiquitin proteasome system (UPS) response to varied dietary protein intake, energy deficit (ED), and consumption of a mixed meal. A randomized, controlled trial of 39 adults consuming protein at 0.8 (recommended dietary allowance [RDA]), 1.6 (2×‐RDA), or 2.4 (3×‐RDA) g · kg–1·· d–1 for 31 d. A 10‐d weight maintenance (WM) period was followed by 21 d of 40% ED. Ubiquitin (Ub)‐mediated proteolysis and associated gene expression were assessed in the postabsorptive (fasted) and postprandial (fed; 480 kcal, 20 g protein) states after WM and ED by using muscle biopsies, fluorescence‐based assays, immunoblot analysis, and real‐time qRT‐PCR In the assessment of UPS responses to varied protein intakes, ED, and feeding, the RDA, WM, and fasted measures served as appropriate controls. ED resulted in the up‐regulation of UPS‐associated gene expression, as mRNA expression of the atrogenes muscle RING finger‐1 (MuRF1) and atrogin‐1 were 1.2‐ and 1.3‐fold higher (P<0.05) for ED than for WM. However, mixed‐meal consumption attenuated UPS‐mediated proteolysis, independent of energy status or dietary protein, as the activities of the 26S proteasome subunits β1, β2, and β5 were lower (P<0.05) for fed than for fasted. Muscle protein ubiquitylation was also 45% lower (P<0.05) for fed than for fasted, regardless of dietary protein and energy manipulations. Independent of habitual protein intake and despite increased MuRF1 and atrogin‐1 mRNA expression during ED, consuming a protein‐containing mixed meal attenuates Ub‐mediated proteolysis.—Carbone, J. W., Margolis, L. M., McClung, J. P., Cao, J. J., Murphy, N. E., Sauter, E. R., Combs, G. F., Jr., Young, A. J., Pasiakos, S. M., Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis. FASEB J. 27, 5104–5111 (2013). www.fasebj.org

Calcium homeostasis and bone metabolic responses to high-protein diets during energy deficit in healthy young adults: a randomized controlled trial

BACKGROUND Although consuming dietary protein above current recommendations during energy deficit (ED) preserves lean body mass, concerns have been raised regarding the effects of high-protein diets on bone health. OBJECTIVE The objective was to determine whether calcium homeostasis and bone turnover are affected by high-protein diets during weight maintenance (WM) and ED. DESIGN In a randomized, parallel-design, controlled trial of 32 men and 7 women, volunteers were assigned diets providing protein at 0.8 [Recommended Dietary Allowance (RDA)], 1.6 (2 × RDA), or 2.4 (3 × RDA) g · kg(-1) · d(-1) for 31 d. Ten days of WM preceded 21 d of ED, during which total daily ED was 40%, achieved by reduced dietary energy intake (∼30%) and increased physical activity (∼10%). The macronutrient composition (protein g · kg(-1) · d(-1) and % fat) was held constant from WM to ED. Calcium absorption (ratio of (44)Ca to (42)Ca) and circulating indexes of bone turnover were determined at day 8 (WM) and day 29 (ED). RESULTS Regardless of energy state, mean (±SEM) urinary pH was lower (P < 0.05) at 2 × RDA (6.28 ± 0.05) and 3 × RDA (6.23 ± 0.06) than at the RDA (6.54 ± 0.06). However, protein had no effect on either urinary calcium excretion (P > 0.05) or the amount of calcium retained (P > 0.05). ED decreased serum insulin-like growth factor I concentrations and increased serum tartrate-resistant acid phosphatase and 25-hydroxyvitamin D concentrations (P < 0.01). Remaining markers of bone turnover and whole-body bone mineral density and content were not affected by either the protein level or ED (P > 0.05). CONCLUSION These data demonstrate that short-term consumption of high-protein diets does not disrupt calcium homeostasis and is not detrimental to skeletal integrity. This trial was registered at www.clinicaltrials.gov as NCT01292395.

Optimized dietary strategies to protect skeletal muscle mass during periods of unavoidable energy deficit

Interactions between dietary protein and energy balance on the regulation of human skeletal muscle protein turnover are not well described. A dietary protein intake above the recommended dietary allowance during energy balance typically enhances nitrogen retention and up‐regulates muscle protein synthesis, which in turn may promote positive protein balance and skeletal muscle accretion. Recent studies show that during energy deficit, muscle protein synthesis is down‐regulated with concomitant increases in ubiquitin proteasome‐mediated muscle proteolysis and nitrogen excretion, reflecting the loss of skeletal muscle mass. However, consuming high‐protein diets (1.6‐2.4 g/kg per day), or high‐quality, protein‐based meals (15‐30 g whey) during energy deficit attenuates intracellular proteolysis, restores muscle protein synthesis, and mitigates skeletal muscle loss. These findings are particularly important for physically active, normal‐weight individuals because attenuating the extent to which skeletal muscle mass is lost during energy deficit could prevent decrements in performance, reduce injury risk, and facilitate recovery. This article reviews the relationship between energy status, protein intake, and muscle protein turnover, and explores future research directives designed to protect skeletal muscle mass in physically active, normal‐weight adults.—Pasiakos, S. M., Margolis, L. M., and Orr, J. S. Optimized dietary strategies to protect skeletal muscle mass during periods of unavoidable energy deficit. FASEB J. 29, 1136‐1142 (2015). www.fasebj.org

Effects of supplemental energy on protein balance during 4-d arctic military training

UNLABELLED Soldiers often experience negative energy balance during military operations that diminish whole-body protein retention, even when dietary protein is consumed within recommended levels (1.5-2.0 g·kg·d). PURPOSE The objective of this study is to determine whether providing supplemental nutrition spares whole-body protein by attenuating the level of negative energy balance induced by military training and to assess whether protein balance is differentially influenced by the macronutrient source. METHODS Soldiers participating in 4-d arctic military training (AMT) (51-km ski march) were randomized to receive three combat rations (CON) (n = 18), three combat rations plus four 250-kcal protein-based bars (PRO, 20 g protein) (n = 28), or three combat rations plus four 250-kcal carbohydrate-based bars daily (CHO, 48 g carbohydrate) (n = 27). Energy expenditure (D2O) and energy intake were measured daily. Nitrogen balance (NBAL) and protein turnover were determined at baseline (BL) and day 3 of AMT using 24-h urine and [N]-glycine. RESULTS Protein and carbohydrate intakes were highest (P < 0.05) for PRO (mean ± SD, 2.0 ± 0.3 g·kg·d) and CHO (5.8 ± 1.3 g·kg·d), but only CHO increased (P < 0.05) energy intake above CON. Energy expenditure (6155 ± 515 kcal·d), energy balance (-3313 ± 776 kcal·d), net protein balance (NET) (-0.24 ± 0.60 g·d), and NBAL (-68.5 ± 94.6 mg·kg·d) during AMT were similar between groups. In the combined cohort, energy intake was associated (P < 0.05) with NET (r = 0.56) and NBAL (r = 0.69), and soldiers with the highest energy intake (3723 ± 359 kcal·d, 2.11 ± 0.45 g protein·kg·d, 6.654 ± 1.16 g carbohydrate·kg·d) achieved net protein balance and NBAL during AMT. CONCLUSION These data reinforce the importance of consuming sufficient energy during periods of high energy expenditure to mitigate the consequences of negative energy balance and attenuate whole-body protein loss.

Thermal-Work Strain During Marine Rifle Squad Operations in Afghanistan

The physiological burden created by heat strain and physical exercise, also called thermal-work strain, was quantified for 10 male Marines (age 21.9 ± 2.3 years, height 180.3 ± 5.2 cm, and weight 85.2 ± 10.8 kg) during three dismounted missions in Helmand Province, Afghanistan. Heart rate (HR) and core body temperature (T core) were recorded every 15 seconds (Equivital EQ-01; Hidalgo, Cambridge, United Kingdom) during periods of light, moderate, and heavy work and used to estimate metabolic rate. Meteorological measures, clothing characteristics, anthropometrics, and estimated metabolic rates were used to predict T core for the same missions during March (spring) and July (summer) conditions. Thermal-work strain was quantified from HR and T core values using the Physiological Strain Index (PSI) developed by Moran et al. July PSI and T core values were predicted and not observed due to lack of access to in-theater warfighters at that time. Our methods quantify and compare the predicted and observed thermal-work strain resulting from environment and worn or carried equipment and illustrate that a small increase in ambient temperature and solar load might result in increased thermal-work strain.

Effects of exercise mode, energy, and macronutrient interventions on inflammation during military training

Load carriage (LC) exercise may exacerbate inflammation during training. Nutritional supplementation may mitigate this response by sparing endogenous carbohydrate stores, enhancing glycogen repletion, and attenuating negative energy balance. Two studies were conducted to assess inflammatory responses to acute LC and training, with or without nutritional supplementation. Study 1: 40 adults fed eucaloric diets performed 90‐min of either LC (treadmill, mean ± SD 24 ± 3 kg LC) or cycle ergometry (CE) matched for intensity (2.2 ± 0.1 VO2peak L min−1) during which combined 10 g protein/46 g carbohydrate (223 kcal) or non‐nutritive (22 kcal) control drinks were consumed. Study 2: 73 Soldiers received either combat rations alone or supplemented with 1000 kcal day−1 from 20 g protein‐ or 48 g carbohydrate‐based bars during a 4‐day, 51 km ski march (~45 kg LC, energy expenditure 6155 ± 515 kcal day−1 and intake 2866 ± 616 kcal day−1). IL‐6, hepcidin, and ferritin were measured at baseline, 3‐h post exercise (PE), 24‐h PE, 48‐h PE, and 72‐h PE in study 1, and before (PRE) and after (POST) the 4‐d ski march in study 2. Study 1: IL‐6 was higher 3‐h and 24‐h post exercise (PE) for CE only (mode × time, P < 0.05), hepcidin increased 3‐h PE and recovered by 48‐h, and ferritin peaked 24‐h and remained elevated 72‐h PE (P < 0.05), regardless of mode and diet. Study 2: IL‐6, hepcidin and ferritin were higher (P < 0.05) after training, regardless of group assignment. Energy expenditure (r = 0.40), intake (r = −0.26), and balance (r = −0.43) were associated (P < 0.05) with hepcidin after training. Inflammation after acute LC and CE was similar and not affected by supplemental nutrition during energy balance. The magnitude of hepcidin response was inversely related to energy balance suggesting that eating enough to balance energy expenditure might attenuate the inflammatory response to military training.