BACKGROUND:Studies implicating dysfunctional mitochondrial respiration in metabolic tissues in the development of insulin resistance in obesity have only included adults. Peripheral blood mononuclear cells (PBMCs) and platelets have been found to reflect systemic mitochondrial fitness and bioenergetic health. We sought to identify bioenergetic differences in PBMCs and platelets from children with obesity and insulin resistance and determine associations with whole-body metabolism and/or biomarkers of metabolic health and inflammation. METHODS:We stratified prepubertal children (ages 5-10 years) into three groups: normal weight insulin sensitive (N-IS; n = 20), overweight/obese insulin sensitive (O-IS; n = 28) and overweight/obese insulin resistant (O-IR; n = 17). We measured oxygen consumption rate and proton efflux rate in PBMCs and platelets. We estimated whole-body resting metabolic rate by bioimpedance and dietary fatty acid oxidation by oral deuterated palmitate and quantifying recovery of D2O in urine. We used ANOVA for comparisons among groups and Spearman correlations for associations between circulating cell bioenergetics and whole-body metabolism and biomarkers. RESULTS:O-IS and O-IR PBMCs exhibited increased maximal mitochondrial respiration and spare respiratory capacity compared to N-IS. Bioenergetics shifted towards glycolysis in O-IS PBMCs as compared to both N-IS and O-IR PBMCs. In platelets, glycolysis and ATP production rates were decreased in O-IR compared to O-IS children. PBMC respiration positively correlated with BMIz, HOMA-IR and fasting glucose and insulin, but negatively correlated with inflammatory cytokines. Dietary fatty acid oxidation was higher in O-IS compared to N-IS children and positively correlated with PBMC spare respiratory capacity. Resting metabolic rate correlated positively with several parameters of PBMC mitochondrial respiration. CONCLUSIONS:PBMCs from young children with overweight/obesity exhibit adaptations to the metabolic stressors associated with insulin resistance, and PBMC metabolism correlates well with whole-body metabolism.

Stable isotope techniques serve as invaluable tools for kinetic measurements in metabolic research. In particular, deuterated water (D2O) administration is increasingly being applied in human health research. For use in protein kinetic studies, this includes measurements on gas chromatography-mass spectrometry (GC-MS) analysis of alanine (ALA) and deuterium-labeled alanines (d-ALAs) coming from D2O administration. However, the choice of the derivative of ALA and d-ALAs used in such analyses has not been evaluated thoroughly. Hence, we conducted a comprehensive head-to-head comparison to determine the most effective and reliable derivative. Two derivatization reagents, N,N-dimethylformamide dimethyl acetal (methyl-8 reagent) and N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MtBSTFA), were considered as candidates. Using chemical standards and available rodent muscle tissue, both reagents underwent testing, including the standard curve linear regression fit, sensitivity, reproducibility, and, importantly, column effectiveness. Our findings indicate that both reagents were suitable for ALA/d-ALAs analyses. However, the MtBSTFA derivative exhibited a better linear regression fit, higher sensitivity, and greater reproducibility than methyl-8. More importantly, the methyl-8 derivative resulted in severe column damage. In conclusion, our study highlights the MtBSTFA derivative as a preferred choice for ALA and d-ALAs GC-MS analysis, contributing to a reliable and sensitive analytical method for D2O administration studies for measurements of in vivo metabolic rates.

Circulating blood cells such as platelets represent a readily available sample type to determine mitochondrial function in humans. Here, we set out to determine the influence of sample preparation, assay buffer composition, and instrumental platform on the respiratory function of platelets isolated from human blood. Approximately 50 mL of whole blood was collected from healthy adults (n = 16) following an overnight (>12 h) fast. Platelets were immediately isolated from whole blood by centrifugation for respirometry. Respiratory function was assayed in intact and permeabilized platelets using an Oxygraph-2K (O2K) high-resolution respirometer in either RPMI or MIR05 (containing 5 mM glucose, 1 mM pyruvate, and 2 mM glutamine), or the participant's own plasma. In addition, respiratory function was determined in intact platelets using a Seahorse Extracellular Flux analyzer (XFe96) in RPMI buffer containing 1 mM pyruvate, 2 mM glutamine, and variable glucose concentrations (5, 10, and 10 mM). In assays performed in an O2K, routine and ATP-linked respiration were greater in cells assayed in RPMI compared to MIR05 (p < 0.001). However, compared to cells assayed in RPMI or MIR05, routine and ATP-linked respiration were higher in intact platelets assayed in their own plasma (p < 0.001). In digitonin-permeabilized platelets, state 3 respiration was greater when assayed in MIR05 compared to RPMI (p < 0.05). Across instrumental platforms, routine and leak respiration were lower in intact platelets assayed on an O2K versus an XFe96 (p < 0.05), whereas respiration available for ADP phosphorylation was greater in cells assayed on an O2K versus an XFe96 (p < 0.001), due to a diminished coupling response to oligomycin in cells assayed on the XFe96 (p < 0.001). Platelet respiratory function is influenced by assay buffer composition and instrumental platform. Consideration of these factors should be made by investigators planning to use platelet respiratory function as a readout of cellular energetics.

Purpose: To evaluate the association of platelet (PL) mitochondria respiration with markers of cardiovascular health in children ages 7–10 years. Methods: PL mitochondrial respiration (n = 91) was assessed by high resolution respirometry (HRR): Routine (R) respiration, complex (C) I linked respiration (CI), and maximal uncoupled electron transport capacity of CII (CIIE) were measured. The respiratory control ratio (RCR) was calculated as the ratio of maximal oxidative phosphorylation capacity of CI and CI leak respiration (PCI/LCI). Peak V ̇ O2 (incremental bike test) and body composition (dual-energy X-ray absorptiometry) were measured. Multiple generalized linear regression analysis was used to model the association of measures by HRR with variables of interest: adiposity, low-density lipoprotein (LDL-C) and triglyceride (TG) status (normal vs. elevated) HOMA2-IR, blood pressure status (normal vs. high), and demographics. Results: R and CI-linked respiration positively associated with adiposity, high blood pressure (HBP), and peak V ̇ O2. R and CI-linked respiration had inverse association with age and elevated LDL-C. CIIE was higher in children with elevated LDL-C (log-β = −0.54, p = 0.010). HBP and peak V ̇ O2 interacted in relation to RCR (log-β = −0.01, p = 0.028). Specifically, RCR was lowest among children with HBP and low aerobic capacity (i.e., mean peak V ̇ O2 -1SD). HOMA2-IR did not associate with measures of PL mitochondria respiration. Conclusion: In PL, R and CI-linked mitochondrial respiration directly associate with adiposity, peak V ̇ O2 and HBP. Elevated LDL-C associates with lower CI-linked respiration which is compensated by increasing CII respiration. PL bioenergetics phenotypes in children associate with whole-body metabolic health status.

In humans, circulating blood cells such as platelets are being increasingly used as a readily available and minimally invasive biospecimen to determine mitochondrial respiratory function. Here, we set out to determine the influence of sample preparation, assay buffer composition, and instrumental platform, on the respiratory function of platelets isolated from human blood.Approximately 50 mL of whole blood was collected from healthy adults (n=16) following an overnight (>12 hr.) fast. Platelets were immediately isolated from whole blood by centrifugation. Respiratory function was assayed in intact and permeabilized platelets using an Oxygraph-2K (O2K) high-resolution respirometer (Oroboros Instruments). Respiratory function was assayed in intact platelets suspended in either culture (RPMI) or respiration (MIR05) media (both supplemented with 5 mM glucose, 1 mM pyruvate and 2 mM glutamine), or the participants own plasma. Respiratory function was also assayed in digitonin-permeabilized platelets suspended in both RPMI and MIR05. In addition, respiratory function was determined in intact platelets using a Seahorse Extra-Cellular Flux analyzer (XFe) in RPMI buffer containing 5 mM glucose, 1 mM pyruvate and 2 mM glutamine.In intact platelets assayed in suspension using an O2K, routine and ATP-linked respiration were 35 and 34% greater in cells assayed in RPMI compared to MIR05, respectively (P<0.001). However, coupling control in response to oligomycin was comparable in cells assayed in RPMI and MIR05 (92±4 vs. 94±4%). Compared to cells assayed in RPMI (0.19±0.01 pmol/s/100 million cells) or MIR05 (0.14±0.02 pmol/s/100 million cells), ATP-linked respiration was greater in intact cells assayed in their own plasma (0.24±0.02 pmol/s/100 million cells, both P<0.001). In digitonin-permeabilized platelets, ATP-linked respiration was greater when assayed in MIR05 compared to RPMI (0.36±0.08 vs. 0.30±0.07 pmol/s/100 million cells, P<0.05). Across instrumental platforms, routine and leak respiration were lower in intact platelets assayed on an O2K vs. XFe (P<0.05), whereas ATP-linked respiration was greater in cells assayed on an O2K. (P<0.001), due to a diminished coupling response to oligomycin in cells assayed in the XFe (P<0.001). Platelet respiratory function is influenced by sample preparation, assay buffer and protocol, and the instrumental platform used. Consideration of these factors is necessary when using platelet respiratory function as a readout of cellular energetics. Supported by USDA-ARS Project 6026-51000-012-06S, NIH 5P20GM109096-07 and 5R35GM142744-02. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background Offspring of obese rodents develop a metabolic phenotype that favors fat deposition. Data regarding the impact of maternal obesity programing of offspring fuel usage in humans is scarce. Objective The objective of this study was to explore the association between maternal weight status and dietary palmitate oxidation (DPO) in 2-y-old offspring, taking into consideration potential confounders and modifiers. Methods Women (n = 56) were enrolled by the first trimester of gestation. Maternal physical activity (PA; measured with accelerometers) at enrollment and gestational weight gain (GWG) were measured. Offspring sex, race, and breastfeeding (BF) duration were self-reported. Human milk (HM) composition was determined at 6 mo postpartum. At age 2 y, dietary quality [healthy eating index (HEI)] and parental feeding practices [Child Feeding Questionnaire (CFQ)] were assessed. DPO in 2-y-olds (2-yo-DPO) was measured using deuterated palmitic acid. Generalized linear regression analysis was used to model the associations of 2-yo-DPO with maternal weight status [normal weight (NW), BMI <25 (in kg/m(2)) compared with excessive weight (EW), BMI >= 25]. Results DPO was higher in offspring of women with EW compared with NW (2.1 +/- 1.2%/h compared with 1.4 +/- 0.7%/h, P = 0.03). Maternal weight status interacted with BF duration in association with 2-yo-DPO (log ss: 0.05, P = 0.04). Specifically, 2-yo-DPO was higher in the EW compared with NW group if BF duration was >= 9 mo. HM insulin (log ss: 0.35, P = 0.002) and HM leptin (log ss: 0.81, P = 0.001) concentrations directly associated with 2-yo-DPO. PA (log ss: 0.06, P = 0.013), parental feeding restriction (log ss: 0.05, P < 0.0001), and male sex (log ss: 0.54, P < 0.001) were positively associated with 2-yo-DPO. HEI was negatively associated with 2-yo-DPO (log ss:-0.03, P < 0.0001). Conclusions Higher 2-yo-DPO in offspring of women with EW compared with NW were driven by BF duration. Higher HM insulin and leptin concentrations in women with EW may explain these finding. More studies are needed to confirm these results. This trial was registered at clinicaltrials.gov as NCT03281850.

Loss of muscle mass in response to injury or immobilization impairs functional capacity and metabolic health, thus hindering rehabilitation. Stable isotope techniques are powerful in determining skeletal muscle protein fluxes. Traditional tracer incorporation methods to measure muscle protein synthesis and breakdown are cumbersome and invasive to perform in vulnerable populations such as children. To circumvent these issues, a two-bolus stable isotope amino acid method has been developed; although, measured rates of protein synthesis and breakdown have not been validated simultaneously against an accepted technique such as the arterial-venous balance method. The purpose of the current analysis was to provide preliminary data from the simultaneous determination of the arteriovenous balance and two-bolus tracer incorporation methods on muscle fractional synthesis and breakdown rates in children with burns. Five were administered a primed-constant infusion of L-[15N]Threonine for 180 minutes (Prime: 8 µmol/kg; constant: 0.1 µmol·kg-1·minute-1). At 120 and 150 minutes, bolus injections of L-[ring-13C6]Phenylalanine and L-[15N]Phenylalanine (50 µmol/kg each) were administered, respectively. Blood and muscle tissue samples were collected to assess mixed muscle protein synthesis and breakdown rates. The preliminary results from this study indicate that there is no difference in either fractional synthesis rate (mean ± SD; arteriovenous balance: 0.19 ± 0.17 %/h; tracer incorporation: 0.14 ± 0.08 %/h; P = .42) or fractional breakdown rate (arteriovenous balance: 0.29 ± 0.22 %/h; tracer incorporation: 0.23 ± 0.14 %/h; P = .84) between methods. These data support the validity of both methods in quantifying muscle amino acid kinetics; however, the results are limited and adequately powered research is still required.

Platelets play a critical role in the pathogenesis of atherosclerotic disease (AD). In the absence of a nucleus, platelet function and viability are largely dependent on the health of their mitochondria. The purpose of this study was to evaluate the association between common markers of cardiometabolic health in 7 to 10-year-old children and measures of platelet mitochondria function. Sixty children participated in a single study visit. Fasting blood was collected, and cardiorespiratory fitness (CRF, cycle ergometer test), physical activity (PA, accelerometers), and blood pressure percentiles were determined. Routine respiration (R) was measured by high-resolution respirometry (Oxygraph-2k) before platelets were permeabilized by digitonin. Thereafter, Complex I (CI) supported respiration was assayed in the leak (L), coupled (P) and uncoupled (CIE) states. Complex IV (CIV) activity was assayed as a marker of respiratory capacity. Flux control ratios (FCR) were calculated by dividing respiratory fluxes by CIV. Multiple linear regression analyses were used to model FCRs (dependent variables) with age, sex, race, CRF, PA, Body Mass Index (BMI) percentile, HOMA2-IR, systolic (SBP-P)/diastolic blood pressure percentiles (DBP-P), triglyceride status, and Low Density Lipoprotein (LDL) status (status = high vs. normal) as dependent variables. Age, race, LDL status and the interaction between BMI percentiles with SBP-P were retained in the final regression models for R, L, P and CIE FCRs. Specifically, R and CI supported respiration in the L, P and CIE states decreased with age (P < .05), black race (P < .05), and high LDL-cholesterol status (P < .0001). To assess for the interaction between BMI and SBP-P, children were stratified as normal weight (NW) or overweight (OW = BMI ≥ 85th percentile) with high (≥90th percentile) or normal SBP. FCRs did not differ between NW groups. However, FCRs where ∼2 times higher in children with OW + high SBP compared to children with OW + normal SBP (P < 0.05). Age, race, LDL cholesterol status, and systolic blood pressure are determinants of platelet bioenergetics in children. SBP-P modifies the association between weight status and platelet mitochondria respiration. USDA-ARS 59-6250-4-001 and 6026-51,000-012-06S. NIH-NIGMS 5P20GM10909, NIH 8UG1OD024945, NIH-NCATS 1UL1TR003107-0.

Background Neonatal diet impacts many physiological systems and can modify risk for developing metabolic disease and obesity later in life. Less well studied is the effect of postnatal diet (e.g., comparing human milk (HM) or milk formula (MF) feeding) on mitochondrial bioenergetics. Such effects may be most profound in splanchnic tissues that would have early exposure to diet-associated or gut microbe-derived factors. Methods To address this question, we measured ileal and liver mitochondrial bioenergetics phenotypes in male piglets fed with HM or MF from day 2 to day 21 age. Ileal and liver tissue were processed for mitochondrial respiration (substrate only [pyruvate, malate, glutamate], substrate + ADP, and proton “leak” post-oligomycin; measured by Oroboros methods), mitochondrial DNA (mtDNA) and metabolically-relevant gene expression analyses. Results No differences between the diet groups were observed in mitochondrial bioenergetics indices in ileal tissue. In contrast, ADP-dependent liver Complex I-linked OXPHOS capacity and Complex I + II-linked OXPHOS capacity were significantly higher in MF animals relative to HM fed piglets. Interestingly, p53, Trap1, and Pparβ transcript abundances were higher in MF-fed relative to HM-fed piglets in the liver. Mitochondrial DNA copy numbers (normalized to nuclear DNA) were similar within-tissue regardless of postnatal diet, and were ~ 2–3 times higher in liver vs. ileal tissue. Conclusion While mechanisms remain to be identified, the data indicate that neonatal diet can significantly impact liver mitochondrial bioenergetics phenotypes, even in the absence of a change in mtDNA abundance. Since permeabilized liver mitochondrial respiration was increased in MF piglets only in the presence of ADP, it suggests that formula feeding led to a higher ATP turnover. Specific mechanisms and signals involved with neonatal diet-associated differences in liver bioenergetics remain to be elucidated.

CONTEXT Consuming calories later in the day is associated with obesity and metabolic syndrome. We hypothesized that eating a late dinner alters substrate metabolism during sleep in a manner that promotes obesity. OBJECTIVE To examine the impact of late dinner on nocturnal metabolism in healthy volunteers. DESIGN AND SETTING This is a randomized crossover trial of late dinner (LD, 22:00) versus routine dinner (RD, 18:00), with a fixed sleep period (23:00-07:00) in a laboratory setting. PARTICIPANTS 20 healthy volunteers (10 males, 10 females), aged 26.0 ± 0.6 years, BMI 23.2 ± 0.7 kg/m2, accustomed to a bedtime between 22:00-01:00. INTERVENTIONS An isocaloric macronutrient diet was administered on both visits. Dinner (35% daily kcal, 50% carbohydrate, 35% fat) with an oral lipid tracer ([2H31] palmitate, 15 mg/kg) was given at 18:00 on RD and 22:00 on LD. MAIN OUTCOME MEASURES Nocturnal and next-morning hourly plasma glucose, insulin, triglycerides, free fatty acids (FFAs), cortisol, dietary fatty acid oxidation, and overnight polysomnography. RESULTS LD caused a 4-hour shift in the postprandial period, overlapping with the sleep phase. Independent of this shift, the postprandial period following LD was characterized by higher glucose, a triglyceride peak delay, and lower FFA and dietary fatty acid oxidation. LD did not affect sleep architecture, but increased plasma cortisol. These metabolic changes were most pronounced in habitual earlier sleepers determined by actigraphy monitoring. CONCLUSION LD induces nocturnal glucose intolerance, and reduces fatty acid oxidation and mobilization, particularly in earlier sleepers. These effects might promote obesity if they recur chronically.

Introduction: Consuming calories later in the day is associated with obesity and metabolic syndrome. We hypothesized that eating a late dinner alters metabolic function during sleep in a manner that promotes obesity. Methods: We compared metabolic effects of routine dinnertime (RD, 18:00) versus late dinnertime (LD, 22:00) with a fixed sleep period (23:00-07:00) on the daily metabolic profile of healthy volunteers in a randomized crossover study. An isocaloric diet was administered at 8:00, 13:00, 18:00, or 22:00. For RD, dinner (35% daily kcal) was given at 18:00 and a snack (10% kcal) was given at 22:00; for LD, these meals were reversed. Peripheral venous blood samples were collected at 1-hour intervals from 17:00 to 12:00 the next day on both visits. We assessed plasma triglycerides (TG), free fatty acids (FFAs), glucose, insulin, cortisol, and sleep architecture. Participants ingested a lipid tracer, [ 2 H 31 ]palmitate with dinner to measure fatty acid oxidation. Time series data was analyzed using mixed effects regression models. Results: To date, eight participants (5 male and 3 females) aged 26.4 ± 0.7 years old, with a BMI of 24.1 ± 1.2 kg/m 2 completed the study. During sleep, LD increased plasma glucose ( β =16.8 mg/dl, P <0.001), insulin ( β =15.4 μ U/ml, P <0.001), cortisol ( β =1.5 μ g/dl, P =0.017), and decreased plasma FFA ( β =-0.1 mmol/l, P <0.001). Morning glucose, insulin, cortisol and FFA levels were not significantly different between two visits. Morning TG were increased by RD ( β =17.4 mg/dl, P <0.001). The evening postprandial period following LD was characterized by higher glucose and lower FFA as compared to RD ( P <0.05). Fatty acid oxidation will be measured by serial enrichment of plasma 2 H 2 0 (pending). Dinner time did not affect sleep architecture. Conclusion: LD increases nocturnal glucose, insulin, cortisol, and decreases FFA. The inhibition of nocturnal lipolysis with LD may lower morning TG. Overall, LD leads to an anabolic state during sleep that might promote the development of obesity. the plasma during sleep restriction. species richness and alpha diversity - the Chao1, ACE, Simpson, and Shannon indices - were regressed on self-reported sleep duration, extreme daytime sleepiness and the Epworth Sleepiness Scale (ESS), and actigraphy-measured sleep duration and wake after sleep onset (WASO). We estimated associations between each of the sleep and diversity measures separately, adjusting for age and sex and then additionally adjusting for BMI, moderate-vigorous physical activity, and dietary fat and fiber. Results: Adjusting for gender and age, greater WASO was stat-istically significantly associated with lower richness and alpha diversity. These associations remained significant (Chao1) or bor-derline significant (ACE and Shannon) after further adjustment for BMI, physical activity, and dietary fiber and fat; e.g., 60 minutes greater WASO was associated with an approximate 26% (95% CI=1%-50%) population standard deviation reduction in micro-bial richness measured by Chao1. In fully-adjusted models, greater sleepiness was associated with lower richness and diversity on all indices (p=0.01-0.06). The ESS and sleep duration were not associated with microbiota richness or diversity. Conclusion: Better sleep quality and less sleepiness are significantly associated with greater species richness and diversity of the gut microbiota in a general population sample of adults. Wisconsin (vita-min-D derivative), and PS(O-40:2) (a plasmalogen). Conclusion: Using discovery metabolomics we identified 13 metabolites associated with reduced S I during insufficient sleep. Increases in monoglycosylceramides are known to decrease S I , supporting our current findings. Decreases in overall phosphatidylcholine levels can disrupt cell-membrane integrity and are linked with metabolic disease. Links between the other identified metabolites and S I are less clear. Yet, these metabolites are all linked with oxidative stress suggesting oxidative stress during insufficient sleep may link these metabolites with S I . Collectively, our findings suggest altered lipid metabolism during insufficient sleep may contribute to reduced S I . Further lipidomics studies are required to validate our current discovery based metabolomics findings.

Elderly muscle seems less sensitive to the anabolic stimulus of a meal. Changes in blood concentrations of leucine are suggested as one important trigger of the anabolic response in muscle. The aim of this study was to investigate whether native whey protein, containing high amounts of leucine, may be a more potent stimulator of muscle protein synthesis (MPS) in elderly than regular whey protein (WPC-80) or milk.

Skeletal muscle mitochondrial respiration is thought to be altered in obesity, insulin resistance, and type 2 diabetes; however, the invasive nature of tissue biopsies is an important limiting factor for studying mitochondrial function. Recent findings suggest that bioenergetics profiling of circulating cells may inform on mitochondrial function in other tissues in lieu of biopsies. Thus, we sought to determine whether mitochondrial respiration in circulating cells [peripheral blood mononuclear cells (PBMCs) and platelets] reflects that of skeletal muscle fibers derived from the same subjects. PBMCs, platelets, and skeletal muscle (vastus lateralis) samples were obtained from 32 young (25–35 yr) women of varying body mass indexes. With the use of extracellular flux analysis and high-resolution respirometry, mitochondrial respiration was measured in intact blood cells as well as in permeabilized cells and permeabilized muscle fibers. Respiratory parameters were not correlated between permeabilized muscle fibers and intact PBMCs or platelets. In a subset of samples ( n = 12–13) with permeabilized blood cells available, raw measures of substrate (pyruvate, malate, glutamate, and succinate)-driven respiration did not correlate between permeabilized muscle (per mg tissue) and permeabilized PBMCs (per 106 cells); however, complex I leak and oxidative phosphorylation coupling efficiency correlated between permeabilized platelets and muscle (Spearman’s ρ = 0.64, P = 0.030; Spearman’s ρ = 0.72, P = 0.010, respectively). Our data indicate that bioenergetics phenotypes in circulating cells cannot recapitulate muscle mitochondrial function. Select circulating cell bioenergetics phenotypes may possibly inform on overall metabolic health, but this postulate awaits validation in cohorts spanning a larger range of insulin resistance and type 2 diabetes status.

With increasing rates of childhood obesity, diagnoses of insulin resistance and type 2 diabetes (DM) in children is on the rise. Mitochondrial dysfunction is associated with insulin resistance and DM, therefore bioenergetics profiling of circulating cells may serve as a marker for mitochondrial dysfunction in insulin resistant subjects. The advantage of this biomarker is that others currently in use may not exhibit alterations until symptoms are already present. Mitochondrial bioenergetics (Seahorse XF96) was measured in peripheral blood mononuclear cells (PBMCs) and platelets. Levels of oxidative stress, post-translational modifications of protein tyrosine residues, 3-Nitrotyrosine and 3-Chlorotyrosine, reduced (GSH) and oxidized (GSSG) glutathione, as well as whole-body D31-palmitate oxidation were also assessed in healthy lean (HL; n=18), and overweight/obese (OW/OB; n=29) (BMI ≥85th percentile) 5-9 years old children. While maximal respiration was not different, the reserve capacity (RC) was increased in OW/OB compared to HL subjects in PBMC (45,6±4,7 vs. 32,1±7.7; p=0.05) and platelet (65,3±3.7 vs. 31,2±6,7; p=0.001). Importantly, 20% of these children already presented HbA1c levels above 6.5%. Moreover, 3-Chlorotyrosine was elevated (52,0±3,1 vs. 40,7±1,8; p=0.05), while GSH/GSSG and cysteine/cysteine (intracellular, extracellular redox buffers, respectively) tended to be decreased. Palmitate oxidation is being quantified. RC is a critical component of the cell’s bioenergetics and can be used during increases in energy demand. Our results suggest significant metabolic adjustments to an excess in substrate availability and already compromised antioxidant defenses in 5-9 year old OW/OB children. Oxidative stress and damage occur when antioxidant defense mechanisms fail to counter-balance and control reactive oxygen species generated from endogenous oxidative metabolism or from pro-oxidant environmental exposures. Disclosure E. Carvalho: None. S. Rose: None. M. Cotter: None. S.C. Bennuri: None. L.M. Delhey: None. G.A. Goode: None. A. Beebe: None. E. Børsheim: None. Funding National Institute of General Medical Sciences (P20GM109096)

Background Protein intake is essential to maximally stimulate muscle protein synthesis, and the amino acid leucine seems to possess a superior effect on muscle protein synthesis compared to other amino acids. Native whey has higher leucine content and thus a potentially greater anabolic effect on muscle than regular whey (WPC-80). This study compared the acute anabolic effects of ingesting 2 × 20 g of native whey protein, WPC-80 or milk protein after a resistance exercise session.Methods A total of 24 young resistance trained men and women took part in this double blind, randomized, partial crossover, controlled study. Participants received either WPC-80 and native whey (n = 10), in a crossover design, or milk (n = 12). Supplements were ingested immediately (20 g) and two hours after (20 g) a bout of heavy-load lower body resistance exercise. Blood samples and muscle biopsies were collected to measure plasma concentrations of amino acids by gas-chromatography mass spectrometry, muscle phosphorylation of p70S6K, 4E–BP1 and eEF-2 by immunoblotting, and mixed muscle protein synthesis by use of [2H5]phenylalanine-infusion, gas-chromatography mass spectrometry and isotope-ratio mass spectrometry. Being the main comparison, differences between native whey and WPC-80 were analysed by a one-way ANOVA and comparisons between the whey supplements and milk were analysed by a two-way ANOVA.Results Native whey increased blood leucine concentrations more than WPC-80 and milk (P < 0.05). Native whey ingestion induced a greater phosphorylation of p70S6K than milk 180 min after exercise (P = 0.03). Muscle protein synthesis rates increased 1–3 h hours after exercise with WPC-80 (0.119%), and 1–5 h after exercise with native whey (0.112%). Muscle protein synthesis rates were higher 1–5 h after exercise with native whey than with milk (0.112% vs. 0.064, P = 0.023).Conclusions Despite higher-magnitude increases in blood leucine concentrations with native whey, it was not superior to WPC-80 concerning effect on muscle protein synthesis and phosphorylation of p70S6K during a 5-h post-exercise period. Native whey increased phosphorylation of p70S6K and muscle protein synthesis rates to a greater extent than milk during the 5-h post exercise period.Trial registration This study was retrospectively registered at clinicaltrials.gov as NCT02968888.

BackgroundSeveral studies have reported that skeletal muscle mitochondrial respiratory capacity is lower in older adults than in young adults, suggesting that mitochondrial dysfunction may occur as a consequence of the aging process. In addition, blood‐cell bioenergetics has been suggested as an indicator of systemic mitochondrial health. We have shown that supplementation with essential amino acids (EAAs) plus arginine decreases plasma and liver triacylglycerols in older adults (1). However, no studies to date have reported the impact of such supplementation on mitochondrial respiration within circulating cells. The purpose of this study was to assess the effect of eight weeks of EAA + arginine supplementation on circulating cell bioenergetics in older adults with hypertriacylglycerolaemia.MethodsSix (two men) older adults (age: 68.8±2.0 years) underwent testing before and after 8 weeks of supplementation with EAA + arginine (11 g of AAs ingested twice daily). Specifically, a skeletal muscle biopsy, peripheral blood mononuclear cells (PBMCs; n=4), and platelets (n=4) were collected pre‐ and post‐supplementation, in order to measure mitochondrial function by high‐resolution respirometry using the Oroboros and the Seahorse, respectively. Mitochondrial respiration was determined in permeabilized myofibers from the m. vastus lateralis after the addition of substrates, oligomycin, and FCCP. Thereafter, mitochondrial coupling control was calculated.ResultsMaximal coupled respiration (respiration linked to ATP production) in muscle was not significantly changed by 8 weeks of AA supplementation. On the other hand, ATP‐linked respiration (31.55±4.43 vs. 18.93±2.89 pmol/min, p=0.05), maximal respiration (127±25 vs. 60±9 pmol/min, p=0.05) and the reserve capacity (94.2±21.7 vs. 40.1±6.2, pmol/min, p=0.05) were decreased in isolated PBMCs following 8 weeks of supplementation. While no significant differences in mitochondrial respiration were observed for platelets, the glycolytic capacity of platelets was decreased (70.3±0.8 vs. 55.3±2.6 mpH/min, p=0.03) after AA supplementation. No significant alterations in glycolysis were observed in PBMCs.ConclusionsThese results indicate that EAA supplementation may lower respiration rates in PBMCs. The mechanisms underlying this may be that lowering lipids as previously observed decreases stress on cells and therefore lowers ATP demand, or that the substrate pool is altered and thus metabolism shifted. Future studies are needed in order to elucidate mechanisms and understand the implications of these novel findings.Support or Funding InformationFunding: NIH R01‐AG033761, NIH P30‐AG028718, Arkansas Biosciences Institute the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000, FEDER funds through the Operational Programme Competitiveness Factors ‐ COMPETE and UID/NEU/04539/2013