Nancy E. Murphy

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

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.

Dietary protein level and source differentially affect bone metabolism, strength, and intestinal calcium transporter expression during ad libitum and food-restricted conditions in male rats.

High-protein (HP) diets may attenuate bone loss during energy restriction. The objective of the current study was to determine whether HP diets suppress bone turnover and improve bone quality in male rats during food restriction and whether dietary protein source affects this relation. Eighty 12-wk-old male Sprague Dawley rats were randomly assigned to consume 1 of 4 study diets under ad libitum (AL) control or restricted conditions [40% food restriction (FR)]: 1) 10% [normal-protein (NP)] milk protein; 2) 32% (HP) milk protein; 3) 10% (NP) soy protein; or 4) 32% (HP) soy protein. After 16 wk, markers of bone turnover, volumetric bone mineral density (vBMD), microarchitecture, strength, and expression of duodenal calcium channels were assessed. FR increased bone turnover and resulted in lower femoral trabecular bone volume (P < 0.05), higher cortical bone surface (P < 0.001), and reduced femur length (P < 0.01), bending moment (P < 0.05), and moment of inertia (P = 0.001) compared with AL. HP intake reduced bone turnover and tended to suppress parathyroid hormone (PTH) (P = 0.06) and increase trabecular vBMD (P < 0.05) compared with NP but did not affect bone strength. Compared with milk, soy suppressed PTH (P < 0.05) and increased cortical vBMD (P < 0.05) and calcium content of the femur (P < 0.01) but did not affect strength variables. During AL conditions, transient receptor potential cation channel, subfamily V, member 6 was higher for soy than milk (P < 0.05) and HP compared with NP (P < 0.05). These data demonstrate that both HP and soy diets suppress PTH, and HP attenuates bone turnover and increases vBMD regardless of FR, although these differences do not affect bone strength. The effects of HP and soy may be due in part to enhanced intestinal calcium transporter expression.

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.

Cardiometabolic Risk in US Army Recruits and the Effects of Basic Combat Training

Background Cardiometabolic disease risk in US military recruits and the effects of military training have not been determined. This study examined lifestyle factors and biomarkers associated with cardiometabolic risk in US Army recruits (209; 118 male, 91 female, 23±5 yr) before, during, and after basic combat training (BCT). Methodology/Principal Findings Anthropometrics; fasting total (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol; triglycerides (TG); glucose; and insulin were measured at baseline and every 3 wks during the 10 wk BCT course. At baseline, 14% of recruits were obese (BMI>30 kg/m2), 27% were cigarette smokers, 37% were sedentary, and 34% reported a family history of cardiometabolic disease. TC was above recommended levels in 8%, LDL in 39%, TG in 5%, and glucose in 8% of recruits, and HDL was below recommended levels in 33% of recruits at baseline. By week 9, TC decreased 8%, LDL 10%, TG 13%, glucose 6% and homeostasis model assessment of insulin resistance (HOMA-IR) 40% in men (P<0.05). In women, TC, LDL, glucose and HOMA-IR were decreased from baseline at weeks 3 and 6 (P<0.05), but were not different from baseline levels at week 9. During BCT, body weight declined in men but not women, while body fat percentage declined in both men and women (P<0.05). Conclusions/Significance At the start of military service, the prevalence of cardiometabolic risk in US military recruits is comparable to that reported in similar, college-aged populations. Military training appears to be an effective strategy that may mitigate risk in young people through improvements in lipid profiles and glycemic control.

Human Muscle Protein Synthetic Responses during Weight-Bearing and Non-Weight-Bearing Exercise: A Comparative Study of Exercise Modes and Recovery Nutrition

Effects of conventional endurance (CE) exercise and essential amino acid (EAA) supplementation on protein turnover are well described. Protein turnover responses to weighted endurance exercise (i.e., load carriage, LC) and EAA may differ from CE, because the mechanical forces and contractile properties of LC and CE likely differ. This study examined muscle protein synthesis (MPS) and whole-body protein turnover in response to LC and CE, with and without EAA supplementation, using stable isotope amino acid tracer infusions. Forty adults (mean ± SD, 22 ± 4 y, 80 ± 10 kg, VO2peak 4.0 ± 0.5 L∙min-1) were randomly assigned to perform 90 min, absolute intensity-matched (2.2 ± 0.1 VO2 L∙m-1) LC (performed on a treadmill wearing a vest equal to 30% of individual body mass, mean ± SD load carried 24 ± 3 kg) or CE (cycle ergometry performed at the same absolute VO2 as LC) exercise, during which EAA (10 g EAA, 3.6 g leucine) or control (CON, non-nutritive) drinks were consumed. Mixed-muscle and myofibrillar MPS were higher during exercise for LC than CE (mode main effect, P < 0.05), independent of dietary treatment. EAA enhanced mixed-muscle and sarcoplasmic MPS during exercise, regardless of mode (drink main effect, P < 0.05). Mixed-muscle and sarcoplasmic MPS were higher in recovery for LC than CE (mode main effect, P < 0.05). No other differences or interactions (mode x drink) were observed. However, EAA attenuated whole-body protein breakdown, increased amino acid oxidation, and enhanced net protein balance in recovery compared to CON, regardless of exercise mode (P < 0.05). These data show that, although whole-body protein turnover responses to absolute VO2-matched LC and CE are the same, LC elicited a greater muscle protein synthetic response than CE.

Military training elicits marked increases in plasma metabolomic signatures of energy metabolism, lipolysis, fatty acid oxidation, and ketogenesis

Military training studies provide unique insight into metabolic responses to extreme physiologic stress induced by multiple stressor environments, and the impacts of nutrition in mediating these responses. Advances in metabolomics have provided new approaches for extending current understanding of factors modulating dynamic metabolic responses in these environments. In this study, whole‐body metabolic responses to strenuous military training were explored in relation to energy balance and macronutrient intake by performing nontargeted global metabolite profiling on plasma collected from 25 male soldiers before and after completing a 4‐day, 51‐km cross‐country ski march that produced high total daily energy expenditures (25.4 MJ/day [SD 2.3]) and severe energy deficits (13.6 MJ/day [SD 2.5]). Of 737 identified metabolites, 478 changed during the training. Increases in 88% of the free fatty acids and 91% of the acylcarnitines, and decreases in 88% of the mono‐ and diacylglycerols detected within lipid metabolism pathways were observed. Smaller increases in 75% of the tricarboxylic acid cycle intermediates, and 50% of the branched‐chain amino acid metabolites detected were also observed. Changes in multiple metabolites related to lipid metabolism were correlated with body mass loss and energy balance, but not with energy and macronutrient intakes or energy expenditure. These findings are consistent with an increase in energy metabolism, lipolysis, fatty acid oxidation, ketogenesis, and branched‐chain amino acid catabolism during strenuous military training. The magnitude of the energy deficit induced by undereating relative to high energy expenditure, rather than macronutrient intake, appeared to drive these changes, particularly within lipid metabolism pathways.

Severe energy deficit at high altitude inhibits skeletal muscle mTORC1-mediated anabolic signaling without increased ubiquitin proteasome activity

Muscle loss at high altitude (HA) is attributable to energy deficit and a potential dysregulation of anabolic signaling. Exercise and protein ingestion can attenuate the effects of energy deficit on muscle at sea level (SL). Whether these effects are observed when energy deficit occurs at HA is unknown. To address this, muscle obtained from lowlanders (n = 8 males) at SL, acute HA (3 h, 4300 m), and chronic HA (21 d, ‐1766 kcal/d energy balance) before [baseline (Base)] and after 80 min of aerobic exercise followed by a 2‐mile time trial [postexercise (Post)] and 3 h into recovery (Rec) after ingesting whey protein (25 g) were analyzed using standard molecular techniques. At SL, Post, and REC, p‐mechanistic target of rapamycin (mTOR)Ser2448, p‐p70 ribosomal protein S6 kinase (p70S6K)Ser424/421, and p‐ribosomal protein S6 (rpS6)Ser235/236 were similar and higher (P < 0.05) than Base. At acute HA, Post p‐mTORSer2448 and Post and REC p‐p70S6KSer424/421 were not different from Base and lower than SL (P < 0.05). At chronic HA, Post and Rec p‐mTORSer2448 and p‐p70S6KSer424/421 were not different from Base and lower than SL, and, independent of time, p‐rpS6Ser235/236 was lower than SL (P < 0.05). Post proteasome activity was lower (P < 0.05) than Base and Rec, independent of phase. Our findings suggest that HA exposure induces muscle anabolic resistance that is exacerbated by energy deficit during acclimatization, with no change in proteolysis.—Margolis, L. M., Carbone, J. W., Berryman, C. E., Carrigan, C. T., Murphy, N. E., Ferrando, A. A., Young, A. J., Pasiakos, S. M. Severe energy deficit at high altitude inhibits skeletal muscle mTORC1‐mediated anabolic signaling without increased ubiquitin proteasome activity. FASEB J. 32, 5955–5966 (2018). www.fasebj.org