Declines of skeletal muscle mass and functions are implicated in the progression of various clinical conditions such as cancers, obesity, insulin resistance, diabetes, and osteoporosis. While no effective and safe drugs against muscle wasting, such as sarcopenia and disease-associated cachexia, have been discovered, it is well documented that dietary essential amino acids (EAAs) or high-quality protein work synergistically to enhance the anabolic effect of resistance exercise training (RT), leading to gains in muscle mass, strength, and muscle quality. Dietary EAAs serve as precursors and signaling molecules for the synthesis of new muscle proteins (both contractile and mitochondrial) and stimulate neuromuscular junction remodeling. Furthermore, EAAs consumed in the post-absorptive state improve endurance capacity via stimulation of mitochondrial biogenesis (independent of PGC1-α) and mitochondrial dynamics (mitochondrial protein synthesis and fission). Here, we discuss (1) traditional molecular mechanisms regulating the muscle proteome through constant turnover (synthesis and breakdown), (2) novel mechanisms by which dietary supplementation of EAAs during RT simultaneously improves muscle strength and endurance, (3) stable isotope tracer methodologies that enable understanding of the dynamic muscle proteome and accurate assessment of functional muscle mass, and finally, (4) clinical implications of combined EAA and RT interventions in the context of muscle and metabolic dysfunction, including sarcopenia, cachexia, obesity, and chronic disease. Collectively, current evidence underscores the potential of balanced EAAs, particularly when combined with resistance training, as a safe, effective, and translationally relevant nutritional strategy to preserve and enhance muscle and metabolic health across healthy and clinical populations.

A growing body of evidence has demonstrated the existence of metabolic dysfunction in neurodegenerative diseases, including Alzheimer’s disease (AD), suggesting that deprivation of energy substrates impairs cellular dynamics. As the glucose utilization declines in AD patients, the need for alternative energy sources becomes crucial to sustain neuronal activities and prevent cell death induced by neurotoxic proteins, such as amyloid beta (A) aggregates. In this context, lactate has been investigated as a potential alternative brain energy substrate in several studies, yet its impact on neuronal cells under A-induced toxicity remains unclear. We confirmed significant suppression of energy production-related biological pathways by analyzing brain transcriptomic data of AD patients. In subsequent in vitro studies, exogenous lactate treatment ameliorated neuron-like cell death caused by A aggregates. Using a 13 C stable isotope tracer, we verified cellular lactate uptake and its incorporation into TCA cycle in neurons under the neurotoxic condition. ¹³C metabolic flux analysis further supported these findings by revealing that lactate treatment restored Aβ-suppressed mitochondrial TCA cycle fluxes. These metabolic improvements were accompanied by increased expression of mitochondrial proteins. These findings support lactate shuttling as a mechanism for supplying lactate-derived carbon to mitochondrial energy metabolism, which may improve neuronal resilience under Aβ-induced metabolic stress.

Background: Consensus concerning human dietary protein and indispensable amino acid (IAA) recommendations is lacking. A factorial model capable of predicting the minimum metabolic demand (MMD) for protein and each IAA and elucidating underlying processes would provide valuable mechanistic insight. Objectives: The study aimed to provide an understanding of the basal metabolic demand for protein and each IAA in the adult human using a factorial approach. Methods: A factorial model for the MMD for each AA and protein was developed by summing obligatory AA losses. Oxidative and gut endogenous losses have been given previously. Urinary free amino acids and peptides, hair, skin, nail, and miscellaneous losses, and losses because of functional roles and irreversible modification were estimated from the literature. MMD values were compared with current Food and Agriculture Organization/World Health Organization recommendations and used as reference patterns to calculate protein quality by the Digestible Indispensable Amino Acid Score. Results: The protein MMD was 634 mg/kgBW/d, close to the current Estimated Average Requirement (660 mg/kgBW/d). Isoleucine and leucine MMD values were also close to current recommendations (21 and 40 mg/kgBW/d, respectively), whereas other MMDs were increased by 15%-150%. Oxidative losses were the largest contributor to the MMD (54%-72%) followed by endogenous gut losses, particularly for threonine (33%). The MMD for total IAAs as a proportion of protein (41%) was close to that of body protein (42%) and almost 50% greater than current recommendations (28%). MMD-based protein quality scores were reduced compared with those based on current recommendations. Conclusions: A factorial model for estimation of the MMD for protein and each IAA provides a mechanistic understanding of requirements and their sensitivity to metabolic processes. Each parameter is affected by variations in protein quantity and quality, and different physiological states. Additional data, when available, will enable model refinement.

Introduction:Age-related anabolic resistance can lead to reduced muscle mass but can be costly and timely to diagnose. Multi-frequency bioelectrical impedance analysis could potentially be used as a non-invasive tool for the assessment of anabolic resistance as changes in frequency-dependent impedance values may reflect fluid shifts occurring with nutrient uptake. This exploratory pilot study evaluated the effects of acute essential amino acid (EAA) intake on raw bioimpedance and fluid shifts in healthy young adults (YA, n=5) and older adults (OA, n=7). Methods:Participants completed a five-hour protocol with multifrequency bioelectrical impedance analysis (MF-BIA) at baseline and every 30 minutes post-consumption of a 10 g EAA beverage. Whole-body and segmental values for impedance (Z), resistance (R), reactance (Xc), phase angle (PhA), and body water compartments were assessed. Results:YA demonstrated significantly higher Z, R, Xc, and PhA values compared to OA (p<0.05), particularly in the leg segments and at 50 kHz. Time effects revealed declines in R (p=0.013) and Xc (p=0.002) following EAA ingestion, consistent with postprandial fluid shifts. Fluid analysis showed significant group differences only for ECW/ICW ratio (p=0.001-0.004) with OA > YA and increases in TBW, ICW, and ECW over time. Conclusion:Raw bioimpedance values distinguished between age groups and reflected acute responses to nutrient intake. These findings suggest MF-BIA may be sensitive to short-term physiological changes and, with further validation, could support assessments of muscle quality and nutritional responsiveness.

There is an increased need to establish the protein requirements for body weight maintenance and optimal health in humans. Different methods were developed in the past to assess protein requirements in which known amounts of protein/amino acids were provided. The purpose of this paper is to propose a new concept of establishing protein requirements in healthy and diseased conditions using a novel stable isotope approach.In the past years, we consistently found that when using a novel stable isotope pulse approach the intracellular production of amino acids (i.e., phenylalanine and tyrosine) is more than double the plasma rate of appearance, as measured by the commonly used primed constant infusion approach, leading to a net protein breakdown that is more than twice than estimated in the past. Net protein breakdown in the fasted state may provide a good estimation of the actual net protein loss that would take place during the day and thus can be used to estimate daily protein requirements. Our recent study found that a net protein breakdown ∼1 g protein/kg body weight/day was equal to the habitual protein intake, suggesting that there is a relation between habitual protein intake and protein requirements. As net protein breakdown is lower with advanced aging and in patient populations with comorbidities, a lower protein requirement for body weight maintenance is suggested.We propose a new concept to establish actual protein requirements of healthy and disease conditions, using a pulse tracer administration and to consider individual habitual protein intake and health conditions.

Dietary protein quality refers to the capacity of a food to meet the human metabolic needs for essential amino acids (EAAs) and nitrogen. This is critical in low- and middle-income countries, where severe protein malnutrition occurs, and relevant in higher-income countries, where increasing dietary EAA intake may improve health and function. There are several methods to assess protein quality, each with different objectives. Chemical scoring metrics, like the digestible indispensable amino acid score, describe the EAA composition and digestibility of a protein source. However, these methods do not capture the metabolic activity of food-derived amino acids. Overreliance on a single metric leads to generic dietary recommendations lacking individual context. This review draws on chemical score and stable isotope methods to provide a comprehensive assessment of dietary protein quality. We translate these findings into practical recommendations for improving protein quality in the context of whole diets. High-quality protein sources are characterized by high EAA density (%EAAs/kcals), digestibility, bioavailability, and the capacity to stimulate protein synthesis. Practically, protein quality improves when using processing and cooking methods that reduce antinutrients, denature proteins, and reduce food particle size and structure. Conversely, protein quality decreases when exposing foods to prolonged storage, heat sterilization, and high surface temperatures. Diet modeling studies show that EAA density and protein quality are higher in omnivorous and lacto-ovo-vegetarian diets, and diets high in whole food plant-derived proteins may require greater total protein and energy intakes to compensate for lower protein quality. For incomplete plant-derived proteins, consuming complementary proteins may be beneficial. Considerations for dietary protein quality in older adults include chewing efficiency, food particle size, and higher EAA density and leucine intakes to maximize muscle protein synthesis. Recognizing dietary protein quality as a multifaceted, modifiable metric is essential to improving dietary recommendations and public health outcomes.

BACKGROUND:The etiology of exercise intolerance in heart failure with preserved ejection fraction (HFpEF) is multifactorial. Several contributing pathways may be improved by ketone ester (KE). OBJECTIVES:This study aims to determine whether KE improves exercise tolerance in HFpEF. METHODS:KETO-HFpEF (Ketogenic Exogenous Therapies in HFpEF) is a randomized, crossover, placebo-controlled trial of acute KE dosing in 20 symptomatic HFpEF participants. Coprimary endpoints include peak oxygen consumption (VO2) during incremental cardiopulmonary exercise testing and time to exhaustion during an additional constant-intensity exercise (75% peak workload) bout. RESULTS:The average age was 71 ± 8 years, 60% were women, and 65% were White. KE did not improve peak VO2 (KE: 10.4 ± 3.6 vs placebo: 10.5 ± 4.0 mL/kg/min; P = 0.75). At rest, heart rate, biventricular systolic function, and cardiac output (0.6 L/min [95% CI: 0.3-1.0 L/min]) were greater with KE vs placebo, whereas total peripheral resistance (-3.2 WU [95% CI: -5.2 to -1.2 WU]) and the arteriovenous oxygen content difference (-0.7 mL of O2/dL blood [95% CI: -1.2 to -0.2 mL]) were lower. These differences mostly disappeared during incremental exercise. KE did not improve exercise endurance during the constant-intensity protocol (9.7 ± 7.3 minutes vs 8.7 ± 4.4 minutes; P = 0.51). In 6 participants receiving 6,6-2H2-glucose infusions during constant-intensity exercise, plasma glucose appearance rate before and during exercise was lower with KE (-0.24 mg/kg/min; P < 0.001). During both exercise protocols, KE lowered: 1) respiratory exchange ratios, demonstrating decreased systemic carbohydrate use; 2) nonesterified fatty acids and glucose; and 3) estimated left ventricular filling pressures (E/e'). CONCLUSIONS:Despite robust ketosis, shifting substrate use away from carbohydrates, and decreasing estimated left ventricular filling pressures, acute KE supplementation did not improve peak VO2 or constant-intensity exercise in HFpEF. (Ketogenic Exogenous Therapies in HFpEF [KETO-HFpEF]; NCT04633460).

Background: Soy-based meat alternatives (SBMA) are becoming increasingly popular, but it is unclear if they have the same anabolic effect on skeletal muscle as animal meat. Objectives: We aimed to compare the stimulation of skeletal muscle protein synthesis by consumption of 1 or two 4 oz patties of SBMA with 4 oz (80% protein/20% fat) beef. Methods: The study design was a randomized controlled trial. Participants were aged 18-40 y of age and in good general health with a body mass index (kg/m2) between 20 and 32. Stable isotope tracer methods were used (L-[ring-2H5] phenylalanine, [U- 13 C 9-15N]- tyrosine, and L-[ring-2H4] tyrosine) to quantify the response of muscle protein fractional synthetic rate (FSR) to consumption of a single beef (4 oz), single SBMA (4 oz), or two 4 oz SBMA patties (8 oz). Whole-body rates of protein synthesis, breakdown, and net balance, as well as plasma essential amino acid concentrations, were also measured. Results: The increase above basal in muscle protein FSR following consumption of the 4 oz beef patty (0.020 f 0.016%/h) was significantly greater than the increase following consumption of 4 oz SBMA (P = 0.021; 0.003 f 0.010%/h) but not 8 oz SBMA (P = 0.454; 0.013 f 0.016%/h). The maximal essential amino acid concentration was significantly correlated (P = 0.046; r = 0.411) with the change in muscle FSR from the basal to the postprandial period. In addition, the change in muscle FSR from the basal to postprandial period was significantly correlated (P = 0.046; r = 0.412) with the corresponding change in whole-body protein synthesis. Conclusions: Consumption of a 4 oz beef patty stimulates muscle and whole-body protein synthesis > 4 oz SBMA patty and similarly to 8 oz of SBMA. This trial was registered at clinicaltrials.gov as NCT05197140.

Essential amino acid (EAA)-based compositions have been shown to be effective stimulators of muscle protein synthesis, but the lower limit of effective dosage is not clear. We have used stable isotope tracer methodology to quantify the response of muscle protein fractional synthetic rate (FSR) to a dose of 3.6 g of a high-leucine composition of EAAs plus arginine in older subjects. Muscle protein FSR increased 0.058%/hour over 3 h following consumption. When account was taken of the total muscle mass, this increase in muscle protein FSR represented approximately 80% of ingested EAAs. We conclude that a low dose of an EAA-based composition can effectively stimulate muscle protein synthesis.

BACKGROUND:Consensus regarding the required intake of indispensable amino acids (IDAAs) and protein [representing total amino acids (AAs)] in the adult is lacking. Oxidation is a major, although not exclusive, source of IDAA loss in humans body and a primary factor determining requirements; a quantitative understanding of oxidative IDAA losses is required. OBJECTIVES:This study aimed to develop a factorial diurnal model of total oxidative IDAA and protein losses in the adult human. METHODS:A factorial diurnal model of oxidative losses of protein and each IDAA at maintenance was developed by estimating the magnitude and variability of sources of oxidative loss from existing literature: inevitable catabolism (constitutive oxidation of each absorbed dietary AA), and protein turnover in the postprandial and postabsorptive states. Total oxidative losses were calculated by summing individual losses, validated against published independent nitrogen balance data and compared with current IDAA requirements. RESULTS:The factorial model predicted minimum oxidative total AA losses of 390 ± 60 mg/kg BW/d, 59% of the estimated average requirement for protein. Inevitable AA oxidation and oxidation associated with postabsorptive protein turnover were the major sources of the oxidative loss for protein, at 40% and 44%, respectively. Summed oxidative IDAA losses ranged from 64% (isoleucine) to 91% (tryptophan) of current requirements. Total oxidative losses predicted by the model were significant predictors of actual experimental oxidative losses obtained by nitrogen balance (R2 = 0.66; P = 0.049). CONCLUSIONS:The use of a factorial model for estimation of minimum IDAA and protein oxidative losses in the adult human provides an essential starting point for an updated understanding of protein and IDAA requirements. Further iterations of the model will estimate total protein and IDAA requirements, and account for variations in dietary protein quantity and quality, as well as different populations and physiologic states. Additional data, especially for inevitable oxidation in humans, and particularly with respect to individual IDAAs, are needed.

Objective: Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, insulin resistance, and hepatic steatosis (HS). Because dietary essential amino acid (EAA) supplementation has been shown to decrease HS in various populations, this study's objective was to determine whether supplementation would decrease HS in PCOS. Methods: A randomized, double-blind, crossover, placebo-controlled trial was conducted in 21 adolescents with PCOS (BMI 37.3 +/- 6.5 kg/m(2), age 15.6 +/- 1.3 years). Liver fat, very low-density lipoprotein (VLDL) lipogenesis, and triacylglycerol (TG) metabolism were measured following each 28-day phase of placebo or EAA. Results: Compared to placebo, EAA was associated with no difference in body weight (p = 0.673). Two markers of liver health improved: HS was lower (-0.8% absolute, -7.5% relative reduction, p = 0.013), as was plasma aspartate aminotransferase (AST) (-8%, p = 0.004). Plasma TG (-9%, p = 0.015) and VLDL-TG (-21%, p = 0.031) were reduced as well. VLDL-TG palmitate derived from lipogenesis was not different between the phases, nor was insulin sensitivity (p > 0.400 for both). Surprisingly, during the EAA phase, participants reported consuming fewer carbohydrates (p = 0.038) and total sugars (p = 0.046). Conclusions: Similar to studies in older adults, short-term EAA supplementation in adolescents resulted in significantly lower liver fat, AST, and plasma lipids and thus may prove to be an effective treatment in this population. Additional research is needed to elucidate the mechanisms for these effects.

In wealthy countries, the protein intake of adults is usually considered to be adequate, and considerations of protein quality are often deemed irrelevant. The objective was to examine dietary protein intakes of adults in developed countries in the context of dietary protein quality. An analysis of NHANES population data on actual protein intakes in the United States (a developed country) demonstrated that for a with utilizable protein intakes potentially falling below the EAR increased as the assumed DIAAS declined. Analysis of the NHANES data and several other datasets also indicated that total protein intakes can be limiting or close to limiting for the elderly and some vegetarians and vegans. Here, lower dietary protein quality can potentially lead to inadequate utilizable protein intakes. For many people in specific physiological states (e.g., weight loss, endurance sports, resistance exercise) attempting to meet higher dietary protein targets often with accompanying lowered energy intakes, low dietary protein quality can lead to protein calories expressed as a proportion of total calories, falling outside what may be acceptable limits (maximum of 30% protein calories from total calories). In general, individuals within the adult with lower protein quality (DIAAS <100%). Our analysis shows that dietary protein quality is relevant in mid- to high-income countries.

Scope : Dietary proteins and essential amino acids (EAAs) are the major nutritional supplements that support the growth and activity of gut microbes contributing to the wellbeing of their host. This study hypothesizes that daily supplementation of the diet with either EAAs or whey protein for 12 weeks would improve the gut microbiome of older adults. Methods and results : The stool samples are processed and subjected to Illumina-based 16S ribosomal ribonucleic acid (rRNA) gene amplicon sequencing. In both groups, the most abundant families are found in order of relative abundance included: Bacteroidaceae , Lachnospiraceae , Ruminococcaceae , Prevotellaceae , Rikenellaceae , Enterobacteriaceae , Oscillospiraceae , Tannerellaceae , and Akkermansiaceae , which indicate that these subjects are able to maintain a same healthy microbial diversity in their guts. A significant finding is a reduction of proinflammatory cytokine, interleukin-18 (IL-18) in the EAAs group. It also uses the standard 6-min walking test (6MWT) as a measure of cardiopulmonary fitness. At the end of the study, the subjects in the EAAs group perform significantly better in the 6MWT as compared to the whey group. Conclusion : It seems plausible that the improved physical performance and reduced proinflammatory cytokine, IL-18 seen in the EAAs group, are independent of changes in gut microbiota.

OBJECTIVES/GOALS: We endeavor to investigated the hypothesis that muscle protein synthesis (MPS) is stimulated more after consumption of a 4-ounce beef patty as compared to 4- and 8-ounces of a soy protein based meat alternative (SPBMA) and if a greater stimulation is related to differences in the responses of plasma essential amino acid (EAA) concentrations. METHODS/STUDY POPULATION: Participants were aged 18 to 40 years of age with a BMI between 20 and 32 kg/m2. Written informed consent was obtained from all participants, and approved by UAMS IRB. Participants were assigned to one of three intervention groups via a single-blinded permuted block randomization, stratified for sex: 4 oz beef patty; 4 oz SPBMA; 2 x 4 oz (8oz) SPBMA. The impossible burgerTM was selected as it is primarily soy protein, a high-quality plant protein, and specifically designed to mimic a beef burger. Stable isotope were infused to assess protein metabolism. Appropriate muscle and blood samples were obtained. Enrichment and plasma EAA concentrations were measured with mass spectrometry. ANOVA’s on the change from basal to postprandial were used to identify group difference, significance was accepted at p < 0.05. RESULTS/ANTICIPATED RESULTS: The MPS increase from basal to postprandial indicated a significant main effect of group (p = 0.026), with the beef group (0.020 ± 0.016%/hour) being significantly greater than the 4oz SPBMA (0.003 ± 0.010%/hour; p = 0.021) but not the 8oz PBMA group (0.013 ± 0.016%/hour; p = 0.454). Similar results were observed for whole-body protein synthesis, where the beef group (p = 0.042) and 8oz SPBMA (p = 0.033) were significantly greater than the 4oz SPBMA (p = 0.021). Whole-body protein balance was significantly greater in the 8oz SPBMA as compared to 4oz of beef and SPBMA. Lastly, we observed a significantly relationship (p = 0.046; r = 0.411) between the maximal plasma EAA concentration and change in MPS, indicating the greater rate of MPS following 4oz of beef is mediated by an higher increase in plasma EAA concentrations. DISCUSSION/SIGNIFICANCE: In conclusion, 4oz of beef stimulates muscle protein FSR more than 4oz of a SPBMA. A common SPBMA can stimulate increase in protein metabolism, however, greater amounts are required as compared to beef protein. Further, the change in the muscle protein FSR response was significantly correlated with the maximal EAA concentration.

BACKGROUND:Reduced meal frequency patterns have become popular for weight loss, maintenance, and improving cardiometabolic health. The extended fasting windows with these dietary patterns could lead to greater protein breakdown, which is a concern for middle-aged and older adults who may need higher protein intakes to maintain or increase net protein balance. OBJECTIVES:This study aimed to quantify muscle and whole-body protein kinetic responses to 3 different daily protein intakes within a 2-meal eating pattern. METHODS:Thirty participants (age: 61 ± 6 y, BMI: 26.5 ± 4.8 kg/m2) participated in this 24-h metabolic study using oral stable isotope tracer techniques and were randomized to 1 of 3 protein intakes: 1) recommended dietary allowance (RDA): 0.8 g/kg/d; 2) habitual United States intake: 1.1 g/kg/d; or 3) ≈2RDA: 1.5 g/kg/d distributed across 2 meals, consumed within a 9-h window. RESULTS:Whole-body net protein balance was significantly higher for 1.5 g/kg/d compared with 0.8 g/kg/d [mean difference: 0.55 g/kg; lean body mass (LBM)/d; 95% confidence interval (CI): 0.17, 0.93 g/kg LBM/d; P = 0.004] and 1.1 g/kg/d (mean difference: 0.6 g/kg LBM/d; 95%CI: 0.23, 0.97 g/kg LBM/d; P = 0.001), with no difference between 0.8 and 1.1 g/kg/d (mean difference: 0.05 g/kg LBM/d; 95%CI: -0.31, 0.40 g/kg LBM/d; P = 0.936). Muscle protein synthesis was not significantly different between any groups (P = 0.388). CONCLUSION:s: Within a 2-meal eating pattern, a protein intake of 1.5 g/kg/d led to a more positive whole-body net protein balance than intakes of 0.8 and 1.1 g/kg/d in middle-aged and older adults. This trial was registered at clinicaltrials.gov as NCT04830514.