Early time-restricted eating improves markers of cardiometabolic health but has no impact on intestinal nutrient absorption in healthy adults.

Early time-restricted eating (eTRE) improves aspects of cardiometabolic health. Although the circadian system appears to regulate nutrient absorption, little is known about the effects of eTRE on intestinal absorption. In this randomized crossover trial, 16 healthy adults follow a controlled, weight maintenance diet for 9 days, consuming all calories between 0800 and 1400 (eTRE schedule) or 0800 and 2000 (control schedule). We measure the energy content of the diet, stool, and urine with bomb calorimetry and calculate intestinal energy absorption. The eTRE schedule is more effective than the control eating schedule for improving markers of cardiometabolic health, including 24-h mean glucose concentrations and glycemic variability, assessed as the mean amplitude of glycemic excursions. However, eTRE has no effect on intestinal energy and macronutrient absorption, gastrointestinal transit time, colonic hydrogen gas production, or stool microbial composition, suggesting eTRE does not impact gastrointestinal function. This trial is registered (ClinicalTrials.gov: NCT04877262).

Effects of testosterone enanthate on aggression, risk-taking, competition, mood, and other cognitive domains during 28 days of severe energy deprivation.

RATIONALE: Behavioral effects of testosterone depend on dose, acute versus sustained formulation, duration of administration, personality, genetics, and endogenous levels of testosterone. There are also considerable differences between effects of endogenous and exogenous testosterone. OBJECTIVES: This study was the secondary behavioral arm of a registered clinical trial designed to determine if testosterone protects against loss of lean body mass and lower-body muscle function induced by a severe energy deficit typical of sustained military operations. METHODS: Behavioral effects of repeated doses of testosterone on healthy young men whose testosterone was reduced by severe energy deficit were examined. This was a double-blind, placebo-controlled, between-group study. Effects of four weekly intramuscular injections of testosterone enanthate (200 mg/week, N = 24) or matching placebo (N = 26) were evaluated. Determination of sample size was based on changes in lean body mass. Tasks assessing aggression, risk-taking, competition, social cognition, vigilance, memory, executive function, and mood were repeatedly administered. RESULTS: During a period of artificially induced, low testosterone levels, consistent behavioral effects of administration of exogenous testosterone were not observed. CONCLUSIONS: Exogeneous testosterone enanthate (200 mg/week) during severe energy restriction did not reliably alter the measures of cognition. Study limitations include the relatively small sample size compared to many studies of acute testosterone administration. The findings are specific to healthy males experiencing severe energy deficit and should not be generalized to effects of other doses, formulations, or acute administration of endogenous testosterone or studies conducted with larger samples using tests of cognitive function designed to detect specific effects of testosterone.

Replacement of dietary carbohydrate with protein increases fat mass and reduces hepatic triglyceride synthesis and content in female obese Zucker rats.

Previous studies have demonstrated both energy restriction (ER) and higher protein (HP), lower carbohydrate (LC) diets downregulate hepatic de novo lipogenesis. Little is known about the independent and combined impact of ER and HP/LC diets on tissue-specific lipid kinetics in leptin receptor-deficient, obese rodents. This study investigated the effects of ER and dietary macronutrient content on body composition; hepatic, subcutaneous adipose tissue (SAT), and visceral AT (VAT) lipid metabolic flux (2 H2 O-labeling); and blood and liver measures of cardiometabolic health in six-week-old female obese Zucker rats (Leprfa+/fa+ ). Animals were randomized to a 10-week feeding intervention: ad libitum (AL)-HC/LP (76% carbohydrate/15% protein), AL-HP/LC (35% protein/56% carbohydrate), ER-HC/LP, or ER-HP/LC. ER groups consumed 60% of the feed consumed by AL. AL gained more fat mass than ER (P-energy = 0.012) and HP/LC gained more fat mass than HC/LP (P-diet = 0.025). Hepatic triglyceride (TG) concentrations (P-interaction = 0.0091) and absolute hepatic TG synthesis (P-interaction = 0.012) were lower in ER-HP/LC versus ER-HC/LP. ER had increased hepatic, SAT, and VAT de novo cholesterol fractional synthesis, absolute hepatic cholesterol synthesis, and serum cholesterol (P-energy≤0.0035). A HP/LC diet, independent of energy intake, led to greater gains in fat mass. A HP/LC diet, in the context of ER, led to reductions in absolute hepatic TG synthesis and TG content. However, ER worsened cholesterol metabolism. Increased adipose tissue TG retention with the HP/LC diet may reflect improved lipid storage capacity and be beneficial in this genetic model of obesity.

SIRT1 induction in the skeletal muscle of male mice partially attenuates changes to whole-body metabolism in response to androgen deprivation.

In males, androgens regulate whole body metabolism. The components in androgen target organs contributing to whole-body metabolic function remain ill defined. Sirtuin1 (SIRT1) protein levels are lower in the limb muscle of male mice subjected to androgen deprivation. Because SIRT1 can influence whole-body metabolism, the purpose was to assess whether muscle specific SIRT1 induction attenuated changes to whole-body metabolism in response to androgen deprivation. Physically mature male mice containing an inducible muscle specific SIRT1 transgene (SIRT1) were subjected to a sham or castration surgery and compared to sham and castrated male mice where the SIRT1 transgene was not induced (WT). The respiratory exchange ratio (RER), energy expenditure, and carbohydrate and fat oxidation rates were determined using metabolic cages. Castration lowered RER in WT mice and the lower RER coincided with lower energy expenditure, lower carbohydrate oxidation rates, and higher fat oxidation rates. SIRT1 induction attenuated the castration-induced changes to RER and fat oxidation rates. Changes to energy expenditure and glucose oxidation rates were not affected by SIRT1. Decreases in muscle SIRT1 protein in males may partially contribute to the dysregulation of whole-body metabolism in response to androgen deprivation.

Amino Acid Intake and Conformance With the Dietary Reference Intakes in the United States: Analysis of the National Health and Nutrition Examination Survey, 2001-2018.

BACKGROUND: The lack of complete amino acid composition data in food composition databases has made determining population-wide amino acid intake difficult. OBJECTIVES: This cross-sectional study characterizes habitual intakes of each amino acid and adherence to dietary requirements for each essential amino acid (EAA) in the US population. METHODS: Food and Nutrient Database for Dietary Studies ingredient codes with missing amino acid composition data were matched to similar ingredients with available data so that amino acid composition could be determined for 100% of foods reported in the dietary intake assessment component of NHANES. Amino acid intakes during NHANES 2001-2018 (n = 72,831; ≥2 y) were calculated as relative (mg·kg of ideal body weight-1·d-1) intakes. Data from NHANES 2011-2018 were used to determine the percentage of population consuming less than that recommended by the DRIs for each EAA by age, sex, and race/ethnicity. RESULTS: Relative intakes of EAAs and NEAAs were greatest in those 2-3 y and lowest in older individuals (≥71 y or ≥80 y). In females aged 2-18 y, relative intakes of EAAs were lowest in non-Hispanic White (NHW; histidine, lysine, threonine, methionine, and cysteine) and non-Hispanic Black (NHB; valine, isoleucine, leucine, phenylalanine, tryptophan, and tyrosine) populations and highest in the Asian population. In females aged ≥19 y, relative intakes were lowest in NHW (lysine and methionine only) and NHB populations and highest in the Asian population. In males aged 2-18 y, relative intakes of EAAs were lowest in the NHB population and highest in the Asian population. In males ≥19 y, relative intakes were lowest in NHB and NHW (lysine only) populations and highest in the Hispanic population. Less than 1% of individuals aged ≥19 y did not meet the Estimated Average Requirements for each EAA. CONCLUSIONS: EAA intakes in the US population exceed recommended minimum population requirements. Future studies can use the method described here to quantify amino acid intake and examine relationships with health and disease.

Metabolic Adaptations and Substrate Oxidation are Unaffected by Exogenous Testosterone Administration during Energy Deficit in Men.

INTRODUCTION/PURPOSE: The effects of testosterone on energy and substrate metabolism during energy deficit are unknown. The objective of this study was to determine the effects of weekly testosterone enanthate (TEST; 200 mg·wk -1 ) injections on energy expenditure, energy substrate oxidation, and related gene expression during 28 d of energy deficit compared with placebo (PLA). METHODS: After a 14-d energy balance phase, healthy men were randomly assigned to TEST ( n = 24) or PLA ( n = 26) for a 28-d controlled diet- and exercise-induced energy deficit (55% below total energy needs by reducing energy intake and increasing physical activity). Whole-room indirect calorimetry and 24-h urine collections were used to measure energy expenditure and energy substrate oxidation during balance and deficit. Transcriptional regulation of energy and substrate metabolism was assessed using quantitative reverse transcription-polymerase chain reaction from rested/fasted muscle biopsy samples collected during balance and deficit. RESULTS: Per protocol design, 24-h energy expenditure increased ( P < 0.05) and energy intake decreased ( P < 0.05) in TEST and PLA during deficit compared with balance. Carbohydrate oxidation decreased ( P < 0.05), whereas protein and fat oxidation increased ( P < 0.05) in TEST and PLA during deficit compared with balance. Change (∆; deficit minus balance) in 24-h energy expenditure was associated with ∆activity factor ( r = 0.595), but not ∆fat-free mass ( r = 0.147). Energy sensing (PRKAB1 and TP53), mitochondria (TFAM and COXIV), fatty acid metabolism (CD36/FAT, FABP, CPT1b, and ACOX1) and storage (FASN), and amino acid metabolism (BCAT2 and BCKHDA) genes were increased ( P < 0.05) during deficit compared with balance, independent of treatment. CONCLUSIONS: These data demonstrate that increased physical activity and not exogenous testosterone administration is the primary determinate of whole-body and skeletal muscle metabolic adaptations during diet- and exercise-induced energy deficit.

Effect of exogenous testosterone in the context of energy deficit on risky choice: Behavioural and neural evidence from males.

Previous research has shown greater risk aversion when people make choices about lives than cash. We tested the hypothesis that compared to placebo, exogenous testosterone administration would lead to riskier choices about cash than lives, given testosterone's association with financial risk-taking and reward sensitivity. A double-blind, placebo-controlled, randomized trial was conducted to test this hypothesis (Clinical Trials Registry: NCT02734238, www.clinicaltrials.gov). We collected functional magnetic resonance imaging (fMRI) data from 50 non-obese males before and shortly after 28 days of severe exercise-and-diet-induced energy deficit, during which testosterone (200 mg testosterone enanthate per week in sesame oil) or placebo (sesame seed oil only) was administered. Because we expected circulating testosterone levels to be reduced due to severe energy deficit, testosterone administration served a restorative function to mitigate the impact of energy deficit on testosterone levels. The fMRI task involved making choices under uncertainty for lives and cash. We also manipulated whether the outcomes were presented as gains or losses. Consistent with prospect theory, we observed the reflection effect such that participants were more risk averse when outcomes were presented as gains than losses. Brain activation in the thalamus covaried with individual differences in exhibiting the reflection effect. Testosterone did not impact choice, but it increased sensitivity to negative feedback following risky choices. These results suggest that exogenous testosterone administration in the context of energy deficit can impact some aspects of risky choice, and that individual differences in the reflection effect engage a brain structure involved in processing emotion, reward and risk.

Metabolomics of testosterone enanthate administration during severe-energy deficit.

INTRODUCTION: Testosterone administration attenuates reductions in total body mass and lean mass during severe energy deficit (SED). OBJECTIVES: This study examined the effects of testosterone administration on the serum metabolome during SED. METHODS: In a double-blind, placebo-controlled clinical trial, non-obese men were randomized to receive 200-mg testosterone enanthate/wk (TEST) (n = 24) or placebo (PLA) (n = 26) during a 28-d inpatient, severe exercise- and diet-induced energy deficit. This study consisted of three consecutive phases. Participants were free-living and provided a eucaloric diet for 14-d during Phase 1. During Phase 2, participants were admitted to an inpatient unit, randomized to receive testosterone or placebo, and underwent SED for 28-d. During Phase 3, participants returned to their pre-study diet and physical activity habits. Untargeted metabolite profiling was conducted on serum samples collected during each phase. Body composition was measured using dual-energy X-ray absorptiometry after 11-d of Phase 1 and after 25-d of Phase 2 to determine changes in fat and lean mass. RESULTS: TEST had higher (Benjamini-Hochberg adjusted, q < 0.05) androgenic steroid and acylcarnitine, and lower (q < 0.05) amino acid metabolites after SED compared to PLA. Metabolomic differences were reversed by Phase 3. Changes in lean mass were associated (Bonferroni-adjusted, p < 0.05) with changes in androgenic steroid metabolites (r = 0.42-0.70), acylcarnitines (r = 0.37-0.44), and amino acid metabolites (r = - 0.36-- 0.37). Changes in fat mass were associated (p < 0.05) with changes in acylcarnitines (r = - 0.46-- 0.49) and changes in urea cycle metabolites (r = 0.60-0.62). CONCLUSION: Testosterone administration altered androgenic steroid, acylcarnitine, and amino acid metabolites, which were associated with changes in body composition during SED.

Testosterone status following short-term, severe energy deficit is associated with fat-free mass loss in U.S. Marines.

The objective of this study was to determine metabolic and physiological differences between males with low testosterone (LT) versus those with normal testosterone (NT) following a period of severe energy deficit. In this secondary analysis, 68 male US Marines (mean ± SD, 24.6 ± 2.4 y) were dichotomized by testosterone concentration (< or ≥ 10.5 nmol/L as determined from a single blood sample collected between 0600-0630 after an 8-10 h overnight fast by automated immunoassay) following 7 days of near complete starvation (~300 kcal consumed/d, ~85% energy deficit) during Survival, Evasion, Resistance, and Escape (SERE) training. Dietary intake was assessed before (PRE) SERE. Body composition (dual-energy x-ray absorptiometry and peripheral quantitative computed tomography) and whole-body protein turnover (15 N alanine) were assessed before (PRE) and after (POST) SERE. Mean testosterone concentrations decreased PRE (17.5 ± 4.7 nmol/L) to POST (9.8 ± 4.0 nmol/L, p < 0.0001). When volunteers were dichotomized by POST testosterone concentrations [NT (n = 24) 14.1 ± 3.4 vs. LT (n = 44): 7.5 ± 1.8 nmol/L, p < 0.0001], PRE BMI, total fat mass, trunk fat mass, and testosterone were greater and the diet quality score and total carbohydrate intake were lower in NT compared to LT (p ≤ 0.05). LT lost more fat-free mass and less fat mass, particularly in the trunk region, compared to NT following SERE (p-interaction≤0.044). Whole-body protein synthesis, net balance, and flux decreased and whole-body protein breakdown increased from PRE to POST in both groups (p-time ≤0.025). Following short-term, severe energy deficit, Marines who exhibited low testosterone had greater fat-free mass loss than those who maintained normal testosterone concentrations. Altering body composition and dietary strategies prior to physical training that elicits severe energy deficit may provide an opportunity to attenuate post-training decrements in testosterone and its associated effects (e.g., loss of lean mass, performance declines, fatigue).

A Prolonged Bout of Running Increases Hepcidin and Decreases Dietary Iron Absorption in Trained Female and Male Runners.

BACKGROUND: Declines in iron status are frequently reported in those who regularly engage in strenuous physical activity. A possible reason is increases in the iron regulatory hormone hepcidin, which functions to inhibit dietary iron absorption and can be induced by the inflammatory cytokine interleukin-6 (IL-6). OBJECTIVES: The current study aimed to determine the impact of a prolonged bout of running on hepcidin and dietary iron absorption in trained female and male runners. METHODS: Trained female and male collegiate cross country runners (n = 28, age: 19.7 ± 1.2 y, maximal oxygen uptake: 66.1 ± 6.1 mL $\cdot$ kg -1$\cdot$ min-2, serum ferritin: 21.9 ± 13.3 ng/mL) performed a prolonged run (98.8 ± 14.7 min, 21.2 ± 3.8 km, 4.7 ± 0.3 min/km) during a team practice. Participants consumed a stable iron isotope with a standardized meal 2 h postrun and blood was collected 1 h later. The protocol was repeated 2 wk later except participants abstained from exercise (rest). RBCs were collected 15 d after exercise and rest to determine isotope enrichment. Differences between exercise and rest were assessed by paired t tests and Wilcoxon matched-pairs signed rank tests. Data are means ± SDs. RESULTS: Plasma hepcidin increased 51% after exercise (45.8 ± 34.4 ng/mL) compared with rest (30.3 ± 27.2 ng/mL, P = 0.0010). Fractional iron absorption was reduced by 36% after exercise (11.8 ± 14.6 %) compared with rest (18.5 ± 14.4 %, P = 0.025). Plasma IL-6 was greater after exercise (0.660 ± 0.354 pg/mL) than after rest (0.457 ± 0.212 pg/mL, P < 0.0001). Exploratory analyses revealed that the increase in hepcidin with exercise may be driven by a response in males but not females. CONCLUSIONS: A prolonged bout of running increases hepcidin and decreases dietary iron absorption compared with rest in trained runners with low iron stores. The current study supports that IL-6 contributes to the increase in hepcidin with prolonged physical activity, although future studies should explore potential sex differences in the hepcidin response.This trial was registered at Clinicaltrials.gov as NCT04079322.

Effects of Testosterone on Mixed-Muscle Protein Synthesis and Proteome Dynamics During Energy Deficit.

CONTEXT: Effects of testosterone on integrated muscle protein metabolism and muscle mass during energy deficit are undetermined. OBJECTIVE: The objective was to determine the effects of testosterone on mixed-muscle protein synthesis (MPS), proteome-wide fractional synthesis rates (FSR), and skeletal muscle mass during energy deficit. DESIGN: This was a randomized, double-blind, placebo-controlled trial. SETTING: The study was conducted at Pennington Biomedical Research Center. PARTICIPANTS: Fifty healthy men. INTERVENTION: The study consisted of 14 days of weight maintenance, followed by a 28-day 55% energy deficit with 200 mg testosterone enanthate (TEST, n = 24) or placebo (PLA, n = 26) weekly, and up to 42 days of ad libitum recovery feeding. MAIN OUTCOME MEASURES: Mixed-MPS and proteome-wide FSR before (Pre), during (Mid), and after (Post) the energy deficit were determined using heavy water (days 1-42) and muscle biopsies. Muscle mass was determined using the D3-creatine dilution method. RESULTS: Mixed-MPS was lower than Pre at Mid and Post (P < 0.0005), with no difference between TEST and PLA. The proportion of individual proteins with numerically higher FSR in TEST than PLA was significant by 2-tailed binomial test at Post (52/67; P < 0.05), but not Mid (32/67; P > 0.05). Muscle mass was unchanged during energy deficit but was greater in TEST than PLA during recovery (P < 0.05). CONCLUSIONS: The high proportion of individual proteins with greater FSR in TEST than PLA at Post suggests exogenous testosterone exerted a delayed but broad stimulatory effect on synthesis rates across the muscle proteome during energy deficit, resulting in muscle mass accretion during subsequent recovery.

Stress and the gut-brain axis: Cognitive performance, mood state, and biomarkers of blood-brain barrier and intestinal permeability following severe physical and psychological stress.

BACKGROUND: Physical and psychological stress alter gut-brain axis activity, potentially causing intestinal barrier dysfunction that may, in turn, induce cognitive and mood impairments through exacerbated inflammation and blood brain barrier (BBB) permeability. These interactions are commonly studied in animals or artificial laboratory environments. However, military survival training provides an alternative and unique human model for studying the impacts of severe physical and psychological stress on the gut-brain axis in a realistic environment. PURPOSE: To determine changes in intestinal barrier and BBB permeability during stressful military survival training and identify relationships between those changes and markers of stress, inflammation, cognitive performance, and mood state. MATERIALS AND METHODS: Seventy-one male U.S. Marines (25.2 ± 2.6 years) were studied during Survival, Evasion, Resistance, and Escape (SERE) training. Measurements were conducted on day 2 of the 10-day classroom phase of training (PRE), following completion of the 7.5-day field-based simulation phase of the training (POST), and following a 27-day recovery period (REC). Fat-free mass (FFM) was measured to assess the overall physiologic impact of the training. Biomarkers of intestinal permeability (liposaccharide-binding protein [LBP]) and BBB permeability (S100 calcium-binding protein B [S100B]), stress (cortisol, dehydroepiandrosterone sulfate [DHEA-S] epinephrine, norepinephrine) and inflammation (interleukin-6 [IL-6], high-sensitivity C-reactive protein [hsCRP]) were measured in blood. Cognitive performance was assessed by psychomotor vigilance (PVT) and grammatical reasoning (GR) tests, and mood state by the Profile of Mood States (total mood disturbance; TMD), General Anxiety Disorder-7 (GAD-7), and Patient Health (PHQ-9) questionnaires. RESULTS: FFM, psychomotor vigilance, and LBP decreased from PRE to POST, while TMD, anxiety, and depression scores, and S100B, DHEA-S, IL-6, norepinephrine, and epinephrine concentrations all increased (all p ≤ 0.01). Increases in DHEA-S were associated with decreases in body mass (p = 0.015). Decreases in FFM were associated with decreases in LBP concentrations (p = 0.015), and both decreases in FFM and LBP were associated with increases in TMD and depression scores (all p < 0.05) but not with changes in cognitive performance. Conversely, increases in S100B concentrations were associated with decreases in psychomotor vigilance (p < 0.05) but not with changes in mood state or LBP concentrations. CONCLUSIONS: Evidence of increased intestinal permeability was not observed in this military survival training-based model of severe physical and psychological stress. However, increased BBB permeability was associated with stress and cognitive decline, while FFM loss was associated with mood disturbance, suggesting that distinct mechanisms may contribute to decrements in cognitive performance and mood state during the severe physical and psychological stress experienced during military survival training.

Urinary Metabolites as Predictors of Acute Mountain Sickness Severity.

Individuals sojourning at high altitude (≥2,500m) often develop acute mountain sickness (AMS). However, substantial unexplained inter-individual variability in AMS severity exists. Untargeted metabolomics assays are increasingly used to identify novel biomarkers of susceptibility to illness, and to elucidate biological pathways linking environmental exposures to health outcomes. This study used untargeted nuclear magnetic resonance (NMR)-based metabolomics to identify urine metabolites associated with AMS severity during high altitude sojourn. Following a 21-day stay at sea level (SL; 55m), 17 healthy males were transported to high altitude (HA; 4,300m) for a 22-day sojourn. AMS symptoms measured twice daily during the first 5days at HA were used to dichotomize participants according to AMS severity: moderate/severe AMS (AMS; n=11) or no/mild AMS (NoAMS; n=6). Urine samples collected on SL day 12 and HA days 1 and 18 were analyzed using proton NMR tools and the data were subjected to multivariate analyses. The SL urinary metabolite profiles were significantly different (p≤0.05) between AMS vs. NoAMS individuals prior to high altitude exposure. Differentially expressed metabolites included elevated levels of creatine and acetylcarnitine, and decreased levels of hypoxanthine and taurine in the AMS vs. NoAMS group. In addition, the levels of two amino acid derivatives (4-hydroxyphenylpyruvate and N-methylhistidine) and two unidentified metabolites (doublet peaks at 3.33ppm and a singlet at 8.20ppm) were significantly different between groups at SL. By HA day 18, the differences in urinary metabolites between AMS and NoAMS participants had largely resolved. Pathway analysis of these differentially expressed metabolites indicated that they directly or indirectly play a role in energy metabolism. These observations suggest that alterations in energy metabolism before high altitude exposure may contribute to AMS susceptibility at altitude. If validated in larger cohorts, these markers could inform development of a non-invasive assay to screen individuals for AMS susceptibility prior to high altitude sojourn.

Effects of testosterone administration on fMRI responses to executive function, aggressive behavior, and emotion processing tasks during severe exercise- and diet-induced energy deficit.

BACKGROUND: Clinical administration of testosterone is widely used due to a variety of claimed physical and cognitive benefits. Testosterone administration is associated with enhanced brain and cognitive function, as well as mood, in energy-balanced males, although such relationships are controversial. However, the effects of testosterone administration on the brains of energy-deficient males, whose testosterone concentrations are likely to be well below normal, have not been investigated. METHODS: This study collected functional magnetic resonance imaging (fMRI) data from 50 non-obese young men before (PRE) and shortly after (POST) 28 days of severe exercise-and-diet-induced energy deficit during which testosterone (200 mg testosterone enanthate per week in sesame oil, TEST) or placebo (sesame seed oil only, PLA) were administered. Scans were also collected after a post-energy-deficit weight regain period (REC). Participants completed five fMRI tasks that assessed aspects of: 1) executive function (Attention Network Task or ANT; Multi-Source Interference Task or MSIT; AXE Continuous Processing Task or AXCPT); 2) aggressive behavior (Provoked Aggression Task or AGG); and 3) latent emotion processing (Emotional Face Processing or EMO). RESULTS: Changes over time in task-related fMRI activation in a priori defined task-critical brain regions during performance of 2 out of 5 tasks were significantly different between TEST and PLA, with TEST showing greater levels of activation during ANT in the right anterior cingulate gyrus at POST and during MSIT in several brain regions at REC. Changes over time in objective task performance were not statistically significant; testosterone-treated volunteers had greater self-reported anger during AGG at POST. CONCLUSIONS: Testosterone administration can alter some aspects of brain function during severe energy deficit and increase levels of anger.

Energy Restriction Suppresses Muscle Protein Synthesis, and High Protein Diets Extend Protein Half-Lives Across the Muscle Proteome in Obese Female Zucker Rats.

BACKGROUND: Effects of high protein (HP) diets and prolonged energy restriction (ER) on integrated muscle protein kinetics have not been determined. OBJECTIVE: The objective of this study was to measure protein kinetics in response to prolonged ER and HP on muscle protein synthesis (MPS; absolute rates of synthesis) and muscle protein breakdown (MPB; half-lives) for proteins across the muscle proteome. METHODS: Female 6-wk-old obese Zucker rats (Leprfa+/fa+, n = 48) were randomly assigned to one of four diets for 10 wk: ad libitum-standard protein (AL-SP; 15% kcal from protein), AL-HP (35% kcal from protein), ER-SP, and ER-HP (both fed 60% feed consumed by AL-SP). During week 10, heavy/deuterated water (2H2O) was administered by intraperitoneal injection, and isotopic steady-state was maintained via 2H2O in drinking water. Rats were euthanized after 1 wk, and mixed-MPS as well as fractional replacement rate (FRR), relative concentrations, and half-lives of individual muscle proteins were quantified in the gastrocnemius. Data were analyzed using 2-factor (energy × protein) ANOVAs and 2-tailed t-tests or binomial tests as appropriate. RESULTS: Absolute MPS was lower in ER than AL for mixed-MPS (-29.6%; P < 0.001) and MPS of most proteins measured [23/26 myofibrillar, 48/60 cytoplasmic, and 46/60 mitochondrial (P < 0.05)], corresponding with lower gastrocnemius mass in ER compared with AL (-29.4%; P < 0.001). Although mixed-muscle protein half-life was not different between groups, prolonged half-lives were observed for most individual proteins in HP compared with SP in ER and AL (P < 0.001), corresponding with greater gastrocnemius mass in HP than SP (+5.3%; P = 0.043). CONCLUSIONS: ER decreased absolute bulk MPS and most individual MPS rates compared with AL, and HP prolonged half-lives of most proteins across the proteome. These data suggest that HP, independent of energy intake, may reduce MPB, and reductions in MPS may contribute to lower gastrocnemius mass during ER by reducing protein deposition in obese female Zucker rats.

Greater protein intake at breakfast or as snacks and less at dinner is associated with cardiometabolic health in adults.

BACKGROUND & AIMS: Protein intake is inversely associated with waist circumference and positively associated with HDL-cholesterol concentrations. However, the relationship between protein intake during specific eating occasions and cardiometabolic health is not well documented. This cross-sectional study measured protein intake at meals and combined snacking occasions and evaluated associations between protein intake at meals or snacking occasions and markers of cardiometabolic health in adults. METHODS: Deciles of individual usual intake (IUI) for protein at meals and combined snacking occasions were calculated using NHANES 2013-2016 data (n = 10,112; ≥19 y). Associations between protein intake at meals or snacks and markers of cardiometabolic health were determined using regression analysis. Covariates included age, age2, gender, ethnicity, physical activity level, poverty income ratio, protein IUI at other eating occasions, and total energy IUI at the eating occasion being analyzed (model 1). P < 0.0042 was considered statistically significant. Registered as ISRCTN11120152. RESULTS: Protein intake ranged (10th to 90th percentiles) 5.9 ± 0.1 to 22.6 ± 0.3 g/d at breakfast, 14.0 ± 0.1 to 34.6 ± 0.4 g/d at lunch, 24.3 ± 0.3 to 46.8 ± 0.2 g/d at dinner, and 4.9 ± 0.1 to 16.5 ± 0.2 g/d at combined snacking occasions. Protein intake at breakfast was inversely associated with diastolic (-0.39 ± 0.10, P = 0.0003) and systolic (-0.40 ± 0.13 mmHg per decile, P = 0.0038) blood pressure. Protein intake at breakfast was positively related to HDL-cholesterol (0.75 ± 0.16 mg/dL per decile, P = 0.0001). Protein intake at dinner was positively associated with insulin concentrations (0.77 ± 0.23 uU/mL per decile, P = 0.0025) and the homeostatic model assessment of insulin resistance (HOMA-IR, 0.32 ± 0.09 per decile, P = 0.0017). Protein intake from snacks was inversely associated with diastolic blood pressure (-0.41 ± 0.09 mmHg per decile, P < 0.0001) and CVD risk score (-0.0018 ± 0.0004 per decile, P = 0.0001). Protein intakes at meals and snacks were not associated with BMI, waist circumference, glucose, total cholesterol, LDL-cholesterol, or triglycerides. CONCLUSIONS: In adults, protein consumption at breakfast is inversely associated with systolic and diastolic blood pressure and positively associated with HDL-cholesterol, while protein consumption at dinner is positively associated with HOMA-IR and insulin concentrations.

Sensitivity and reliability of zinc transporter and metallothionein gene expression in peripheral blood mononuclear cells as indicators of zinc status: responses to ex vivo zinc exposure and habitual zinc intake in humans.

Zn is an essential nutrient for humans; however, a sensitive biomarker to assess Zn status has not been identified. The objective of this study was to determine the reliability and sensitivity of Zn transporter and metallothionein (MT) genes in peripheral blood mononuclear cells (PBMCs) to Zn exposure ex vivo and to habitual Zn intake in human subjects. In study 1, human PBMCs were cultured for 24 h with 0-50 µm ZnSO4 with or without 5 µm N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and mRNA expression of SLC30A1-10, SLC39A1-14, MT1 subtypes (A, B, E, F, G, H, L, M and X), MT2A, MT3 and MT4 mRNA was determined. In study 2, fifty-four healthy male and female volunteers (31·9 (sd 13·8) years, BMI 25·7 (sd 2·9) kg/m2) completed a FFQ, blood was collected, PBMCs were isolated and mRNA expression of selected Zn transporters and MT isoforms was determined. Study 1: MT1E, MT1F, MT1G, MT1H, MT1L, MT1M, MT1X, MT2A and SLC30A1 increased with increasing concentrations of Zn and declined with the addition of TPEN. Study 2: Average daily Zn intake was 16·0 (sd 5·3) mg/d (range: 9-31 mg/d), and plasma Zn concentrations were 15·5 (SD 2·8) µmol/l (range 11-23 µmol/l). PBMC MT2A was positively correlated with dietary Zn intake (r 0·306, P = 0·03) and total Zn intake (r 0·382, P < 0·01), whereas plasma Zn was not (P > 0·05 for both). Findings suggest that MT2A mRNA in PBMCs reflects dietary Zn intake in healthy adults and may be a component in determining Zn status.

Testosterone supplementation upregulates androgen receptor expression and translational capacity during severe energy deficit.

Testosterone supplementation during energy deficit promotes whole body lean mass accretion, but the mechanisms underlying that effect remain unclear. To elucidate those mechanisms, skeletal muscle molecular adaptations were assessed from muscle biopsies collected before, 1 h, and 6 h after exercise and a mixed meal (40 g protein, 1 h postexercise) following 14 days of weight maintenance (WM) and 28 days of an exercise- and diet-induced 55% energy deficit (ED) in 50 physically active nonobese men treated with 200 mg testosterone enanthate/wk (TEST) or placebo (PLA) during the ED. Participants (n = 10/group) exhibiting substantial increases in leg lean mass and total testosterone (TEST) were compared with those exhibiting decreases in both of these measures (PLA). Resting androgen receptor (AR) protein content was higher and fibroblast growth factor-inducible 14 (Fn14), IL-6 receptor (IL-6R), and muscle ring-finger protein-1 gene expression was lower in TEST vs. PLA during ED relative to WM (P < 0.05). Changes in inflammatory, myogenic, and proteolytic gene expression did not differ between groups after exercise and recovery feeding. Mechanistic target of rapamycin signaling (i.e., translational efficiency) was also similar between groups at rest and after exercise and the mixed meal. Muscle total RNA content (i.e., translational capacity) increased more during ED in TEST than PLA (P < 0.05). These findings indicate that attenuated proteolysis at rest, possibly downstream of AR, Fn14, and IL-6R signaling, and increased translational capacity, not efficiency, may drive lean mass accretion with testosterone administration during energy deficit.

High-Altitude Acclimatization Suppresses Hepcidin Expression During Severe Energy Deficit.

Hennigar, Stephen R., Claire E. Berryman, Alyssa M. Kelley, Bradley J. Anderson, Andrew J. Young, James P. McClung, and Stefan M. Pasiakos. High-altitude acclimatization suppresses hepcidin expression during severe energy deficit. High Alt Med Biol. 21:232-236, 2020. Background: The erythropoietic cells in the bone marrow require iron to synthesize heme for incorporation into hemoglobin. Exposure to hypoxic conditions, such as extended sojourns to high altitude (HA), results in increased erythropoiesis and an increased physiological requirement for iron. In addition to increasing iron requirements, hypoxic conditions suppress appetite and often lead to decreased energy intake. The objective of this study was to determine the combined effects of severe energy deficit and hypoxia on hepcidin and measures of iron status in lowlanders sojourning to HA. Methods: Iron status indicators and hepcidin were determined in 17 healthy male volunteers (mean ± standard deviation, age 23 ± 6 years, body mass index 27 ± 4 kg/m2) fed a controlled diet (12 ± 1.2 mg iron/day) during a 20-day sojourn to 4300 m above sea level. Results: Chronic exposure to HA during severe energy deficit increased hematocrit by 12% (p < 0.01) and decreased serum hepcidin by 37% (p < 0.01) compared with baseline. Ferritin declined by 18% (p = 0.02) and transferrin saturation and soluble transferrin receptor increased by 55% and 83%, respectively (p < 0.01 for both) compared with baseline. Conclusions: HA acclimatization suppresses hepcidin expression to increase iron availability during severe energy deficit. Registered at ClinicalTrials.gov as NCT02731066.