A ketone monoester drink reduces postprandial blood glucose concentrations in adults with type 2 diabetes: a randomised controlled trial.

AIMS/HYPOTHESIS: The aim of the present study was to conduct a randomised, placebo-controlled, double-blind, crossover trial to determine whether pre-meal ketone monoester ingestion reduces postprandial glucose concentrations in individuals with type 2 diabetes. METHODS: In this double-blind, placebo-controlled, crossover design study, ten participants with type 2 diabetes (age 59±1.7 years, 50% female, BMI 32±1 kg/m2, HbA1c 54±2 mmol/mol [7.1±0.2%]) were randomised using computer-generated random numbers. The study took place at the Nutritional Physiology Research Unit, University of Exeter, Exeter, UK. Using a dual-glucose tracer approach, we assessed glucose kinetics after the ingestion of a 0.5 g/kg body mass ketone monoester (KME) or a taste-matched non-caloric placebo before a mixed-meal tolerance test. The primary outcome measure was endogenous glucose production. Secondary outcome measures were total glucose appearance rate and exogenous glucose appearance rate, glucose disappearance rate, blood glucose, serum insulin, ß-OHB and NEFA levels, and energy expenditure. RESULTS: Data for all ten participants were analysed. KME ingestion increased mean ± SEM plasma beta-hydroxybutyrate from 0.3±0.03 mmol/l to a peak of 4.3±1.2 mmol/l while reducing 2 h postprandial glucose concentrations by ~18% and 4 h postprandial glucose concentrations by ~12%, predominately as a result of a 28% decrease in the 2 h rate of glucose appearance following meal ingestion (all p<0.05). The reduction in blood glucose concentrations was associated with suppressed plasma NEFA concentrations after KME ingestion, with no difference in plasma insulin concentrations between the control and KME conditions. Postprandial endogenous glucose production was unaffected by KME ingestion (mean ± SEM 0.76±0.15 and 0.88±0.10 mg kg-1 min-1 for the control and KME, respectively). No adverse effects of KME ingestion were observed. CONCLUSIONS/INTERPRETATION: KME ingestion appears to delay glucose absorption in adults with type 2 diabetes, thereby reducing postprandial glucose concentrations. Future work to explore the therapeutic potential of KME supplementation in type 2 diabetes is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT05518448. FUNDING: This project was supported by a Canadian Institutes of Health Research (CIHR) Project Grant (PJT-169116) and a Natural Sciences and Engineering Research Council (NSERC) Discovery Grant (RGPIN-2019-05204) awarded to JPL and an Exeter-UBCO Sports Health Science Fund Project Grant awarded to FBS and JPL.

The impact of forearm immobilization and acipimox administration on muscle amino acid metabolism and insulin sensitivity in healthy, young volunteers.

Although the mechanisms underpinning short-term muscle disuse atrophy and associated insulin resistance remain to be elucidated, perturbed lipid metabolism might be involved. Our aim was to determine the impact of acipimox administration [i.e., pharmacologically lowering circulating nonesterified fatty acid (NEFA) availability] on muscle amino acid metabolism and insulin sensitivity during short-term disuse. Eighteen healthy individuals (age: 22 ± 1 years; body mass index: 24.0 ± 0.6 kg·m-2) underwent 2 days forearm immobilization with placebo (PLA; n = 9) or acipimox (ACI; 250 mg Olbetam; n = 9) ingestion four times daily. Before and after immobilization, whole body glucose disposal rate (GDR), forearm glucose uptake (FGU; i.e., muscle insulin sensitivity), and amino acid kinetics were measured under fasting and hyperinsulinemic-hyperaminoacidemic-euglycemic clamp conditions using forearm balance and l-[ring-2H5]-phenylalanine infusions. Immobilization did not affect GDR but decreased insulin-stimulated FGU in both groups, more so in ACI (from 53 ± 8 to 12 ± 5 µmol·min-1) than PLA (from 52 ± 8 to 38 ± 13 µmol·min-1; P < 0.05). In ACI only, and in contrast to our hypothesis, fasting arterialized NEFA concentrations were elevated to 1.3 ± 0.1 mmol·L-1 postimmobilization (P < 0.05), and fasting forearm NEFA balance increased approximately fourfold (P = 0.10). Forearm phenylalanine net balance decreased following immobilization (P < 0.10), driven by an increased rate of appearance [from 32 ± 5 (fasting) and 21 ± 4 (clamp) preimmobilization to 53 ± 8 and 31 ± 4 postimmobilization; P < 0.05] while the rate of disappearance was unaffected by disuse or acipimox. Disuse-induced insulin resistance is accompanied by early signs of negative net muscle amino acid balance, which is driven by accelerated muscle amino acid efflux. Acutely elevated NEFA availability worsened muscle insulin resistance without affecting amino acid kinetics, suggesting increased muscle NEFA uptake may contribute to inactivity-induced insulin resistance but does not cause anabolic resistance.NEW & NOTEWORTHY We demonstrate that 2 days of forearm cast immobilization in healthy young volunteers leads to the rapid development of insulin resistance, which is accompanied by accelerated muscle amino acid efflux in the absence of impaired muscle amino acid uptake. Acutely elevated fasting nonesterified fatty acid (NEFA) availability as a result of acipimox supplementation worsened muscle insulin resistance without affecting amino acid kinetics, suggesting increased muscle NEFA uptake may contribute to inactivity-induced insulin resistance but does not cause anabolic resistance.

High-Moisture Extrusion of a Dietary Protein Blend Impairs In Vitro Digestion and Delays In Vivo Postprandial Plasma Amino Acid Availability in Humans.

BACKGROUND: Industrial processing can alter the structural complexity of dietary proteins and, potentially, their digestion and absorption upon ingestion. High-moisture extrusion (HME), a common processing method used to produce meat alternative products, affects in vitro digestion, but human data are lacking. We hypothesized that HME of a mycoprotein/pea protein blend would impair in vitro digestion and in vivo postprandial plasma amino acid availability. METHODS: In Study A, 9 healthy volunteers completed 2 experimental trials in a randomized, double-blind, crossover design. Participants consumed a beverage containing 25 g protein from a "dry" blend (CON) of mycoprotein/pea protein (39%/61%) or an HME content-matched blend (EXT). Arterialized venous blood samples were collected in the postabsorptive state and regularly over a 5-h postprandial period to assess plasma amino acid concentrations. In Study B, in vitro digestibility of the 2 beverages were assessed using bicinchoninic acid assay and optical fluorescence microscopy at baseline and during and following gastric and intestinal digestion using the INFOGEST model of digestion. RESULTS: Protein ingestion increased plasma total, essential (EAA), and branched-chain amino acid (BCAA) concentrations (time effect, P < 0.0001) but more rapidly and to a greater magnitude in the CON compared with the EXT condition (condition × time interaction, P < 0.0001). This resulted in greater plasma availability of EAA and BCAA concentrations during the early postprandial period (0-150 min). These data were corroborated by the in vitro approach, which showed greater protein availability in the CON (2150 ± 129 mg/mL) compared with the EXT (590 ± 41 mg/mL) condition during the gastric phase. Fluorescence microscopy revealed clear structural differences between the 2 conditions. CONCLUSIONS: These data demonstrate that HME delays in vivo plasma amino acid availability following ingestion of a mycoprotein/pea protein blend. This is likely due to impaired gastric phase digestion as a result of HME-induced aggregate formation in the pea protein. This trial was registered at clinicaltrials.gov as NCT05584358.

Nasogastric bolus administration of a protein-rich drink augments insulinaemia and aminoacidaemia but not whole-body protein turnover or muscle protein synthesis versus oral administration.

Nasogastric feeding of protein-rich liquids is a nutritional support therapy that attenuates muscle mass loss. However, whether administration via a nasogastric tube per se augments whole-body or muscle protein anabolism compared with oral administration is unknown. Healthy participants were administered a protein-rich drink (225 ml containing 21 g protein) orally (ORAL; n=13; age 21 ± 1 year; BMI 22.2 ± 0.6 kg·m-2) or via a nasogastric tube (NG; n=13; age 21 ± 1 yr; BMI 23.9 ± 0.9 kg·m-2) in a parallel group design, balanced for sex. L-[ring-2H5]-phenylalanine and L-[3,3-2H2]-tyrosine were infused to measure postabsorptive and postprandial whole-body protein turnover. Skeletal muscle biopsies were collected at -120, 0, 120 and 300 min relative to drink administration to quantify temporal myofibrillar fractional synthetic rates (myoFSR). Drink administration increased serum insulin and plasma amino acid concentrations, and to a greater extent and duration in NG versus ORAL (all interactions P<0.05). Drink administration increased whole-body protein synthesis (P<0.01), suppressed protein breakdown (P<0.001), and created positive net protein balance (P<0.001), but to a similar degree in ORAL and NG (interactions P>0.05). Drink administration increased myoFSR from the postabsorptive state (P<0.01), regardless of route of administration in ORAL and in NG (interaction P>0.05). Nasogastric bolus administration of a protein-rich drink induces insulinaemia and aminoacidaemia to a greater extent than oral administration, but the postprandial increase in whole-body protein turnover and muscle protein synthesis was equivalent between administration routes. Nasogastric administration is a potent intervention to increase postprandial amino acid availability. Future work should assess its utility in overcoming impaired sensitivity to protein feeding, such as that seen in ageing, disuse, and critical care.

Maximal sustainable energy intake during transatlantic ocean rowing is insufficient for total energy expenditure and skeletal muscle mass maintenance.

Studies of extreme endurance have suggested that there is an alimentary limit to energy intake (EI) of ∼2.5 × resting metabolic rate (RMR). To gain further insight, this study aimed to simultaneously measure EI, total energy expenditure (TEE) body mass and muscle mass in a large cohort of males and females of varying ages during a transatlantic rowing race. Forty-nine competitors (m = 32, f = 17; age 24-67 years; time at sea 46 ± 7 days) in the 2020 and 2021 Talisker Whisky Atlantic Challenge rowed 12-18 hday-1 for ∼3000 miles. TEE was assessed in the final week of the row using 2 H2 18 O doubly labelled water, and EI was analysed from daily ration packs over this period. Thickness of relatively active (vastus lateralis, intermedius, biceps brachaii and rectus abdominus) and inactive (gastrocnemius, soleus and triceps) muscles was measured pre (<7 days) and post (<24 h) row using ultrasound. Body mass was measured and used to calculate RMR from standard equations. There were no sex differences in males and females in EI (2.5 ± 0.5 and 2.3 ± 0.4 × RMR, respectively, P = 0.3050), TEE (2.5 ± 1.0 and 2.3 ± 0.4 × RMR, respectively, P = 0.5170), or body mass loss (10.2 ± 3.1% and 10.0 ± 3.0%, respectively, P = 0.8520), and no effect of age on EI (P = 0.5450) or TEE (P = 0.9344). Muscle loss occurred exclusively in the calf (15.7% ± 11.4% P < 0.0001), whilst other muscles remained unchanged. After 46 days of prolonged ultra-endurance ocean rowing incurring 10% body mass loss, maximal sustainable EI of ∼2.5 × RMR was unable to meet total TEE suggesting that there is indeed a physiological capacity to EI.

Ingestion of a variety of non-animal-derived dietary protein sources results in diverse postprandial plasma amino acid responses which differ between young and older adults.

Whole-body tissue protein turnover is regulated, in part, by the postprandial rise in plasma amino acid concentrations, although minimal data exist on the amino acid response following non-animal-derived protein consumption. We hypothesised that the ingestion of novel plant- and algae-derived dietary protein sources would elicit divergent plasma amino acid responses when compared with vegan- and animal-derived control proteins. Twelve healthy young (male (m)/female (f): 6/6; age: 22 ± 1 years) and 10 healthy older (m/f: 5/5; age: 69 ± 2 years) adults participated in a randomised, double-blind, cross-over trial. During each visit, volunteers consumed 30 g of protein from milk, mycoprotein, pea, lupin, spirulina or chlorella. Repeated arterialised venous blood samples were collected at baseline and over a 5-h postprandial period to assess circulating amino acid, glucose and insulin concentrations. Protein ingestion increased plasma total and essential amino acid concentrations (P < 0·001), to differing degrees between sources (P < 0·001), and the increase was further modulated by age (P < 0·001). Postprandial maximal plasma total and essential amino acid concentrations were highest for pea (2828 ± 106 and 1480 ± 51 µmol·l-1) and spirulina (2809 ± 99 and 1455 ± 49 µmol·l-1) and lowest for chlorella (2053 ± 83 and 983 ± 35 µmol·l-1) (P < 0·001), but were not affected by age (P > 0·05). Postprandial total and essential amino acid availabilities were highest for pea, spirulina and mycoprotein and lowest for chlorella (all P < 0·05), but no effect of age was observed (P > 0·05). The ingestion of a variety of novel non-animal-derived dietary protein sources elicits divergent plasma amino acid responses, which are further modulated by age.

Ingestion of mycoprotein, pea protein, and their blend support comparable postexercise myofibrillar protein synthesis rates in resistance-trained individuals.

Pea protein is an attractive nonanimal-derived protein source to support dietary protein requirements. However, although high in leucine, a low methionine content has been suggested to limit its anabolic potential. Mycoprotein has a complete amino acid profile which, at least in part, may explain its ability to robustly stimulate myofibrillar protein synthesis (MyoPS) rates. We hypothesized that an inferior postexercise MyoPS response would be seen following ingestion of pea protein compared with mycoprotein, which would be (partially) rescued by blending the two sources. Thirty-three healthy, young [age: 21 ± 1 yr, body mass index (BMI): 24 ± 1 kg·m-2] and resistance-trained participants received primed, continuous infusions of l-[ring-2H5]phenylalanine and completed a bout of whole body resistance exercise before ingesting 25 g of protein from mycoprotein (MYC, n = 11), pea protein (PEA, n = 11), or a blend (39% MYC, 61% PEA) of the two (BLEND, n = 11). Blood and muscle samples were taken pre-, 2 h, and 4 h postexercise/protein ingestion to assess postabsorptive and postprandial postexercise myofibrillar protein fractional synthetic rates (FSRs). Protein ingestion increased plasma essential amino acid and leucine concentrations (time effect; P < 0.0001), but more rapidly in BLEND and PEA compared with MYC (time × condition interaction; P < 0.0001). From similar postabsorptive values (MYC, 0.026 ± 0.008%·h-1; PEA, 0.028 ± 0.007%·h-1; BLEND, 0.026 ± 0.006%·h-1), resistance exercise and protein ingestion increased myofibrillar FSRs (time effect; P < 0.0001) over a 4-h postprandial period (MYC, 0.076 ± 0.004%·h-1; PEA, 0.087 ± 0.01%·h-1; BLEND, 0.085 ± 0.01%·h-1), with no differences between groups (all; P > 0.05). These data show that all three nonanimal-derived protein sources have utility in supporting postexercise muscle reconditioning.NEW & NOTEWORTHY This study provides evidence that pea protein (PEA), mycoprotein (MYC), and their blend (BLEND) can support postexercise myofibrillar protein synthesis rates following a bout of whole body resistance exercise. Furthermore, these data suggest that a methionine deficiency in pea may not limit its capacity to stimulate an acute increase in muscle protein synthesis (MPS).

Algae Ingestion Increases Resting and Exercised Myofibrillar Protein Synthesis Rates to a Similar Extent as Mycoprotein in Young Adults.

BACKGROUND: Spirulina [SPIR] (cyanobacterium) and chlorella [CHLO] (microalgae) are foods rich in protein and essential amino acids; however, their capacity to stimulate myofibrillar protein synthesis (MyoPS) in humans remains unknown. OBJECTIVES: We assessed the impact of ingesting SPIR and CHLO compared with an established high-quality nonanimal-derived dietary protein source (fungal-derived mycoprotein [MYCO]) on plasma amino acid concentrations, as well as resting and postexercise MyoPS rates in young adults. METHODS: Thirty-six healthy young adults (age: 22 ± 3 y; BMI: 23 ± 3 kg·m-2; male [m]/female [f], 18/18) participated in a randomized, double-blind, parallel-group trial. Participants received a primed, continuous infusion of L-[ring-2H5]-phenylalanine and completed a bout of unilateral-resistance leg exercise before ingesting a drink containing 25 g protein from MYCO (n = 12; m/f, 6/6), SPIR (n = 12; m/f, 6/6), or CHLO (n = 12; m/f, 6/6). Blood and bilateral muscle samples were collected at baseline and during a 4-h postprandial and postexercise period to assess the plasma amino acid concentrations and MyoPS rates in rested and exercised tissue. RESULTS: Protein ingestion increased the plasma total and essential amino acid concentrations (time effects; all P < 0.001), but most rapidly and with higher peak responses following the ingestion of SPIR compared with MYCO and CHLO (P < 0.05), and MYCO compared with CHLO (P < 0.05). Protein ingestion increased MyoPS rates (time effect; P < 0.001) in both rested (MYCO, from 0.041 ± 0.032 to 0.060 ± 0.015%·h-1; SPIR, from 0.042 ± 0.030 to 0.066 ± 0.022%·h-1; and CHLO, from 0.037 ± 0.007 to 0.055 ± 0.019%·h-1, respectively) and exercised tissue (MYCO, from 0.046 ± 0.014 to 0.092 ± 0.024%·h-1; SPIR, from 0.038 ± 0.011 to 0.086 ± 0.028%·h-1; and CHLO, from 0.048 ± 0.019 to 0.090 ± 0.024%·h-1, respectively), with no differences between groups (interaction effect; P > 0.05), but with higher rates in exercised compared with rested muscle (time × exercise effect; P < 0.001). CONCLUSIONS: The ingestion of a single bolus of algae-derived SPIR and CHLO increases resting and postexercise MyoPS rates to a comparable extent as MYCO, despite divergent postprandial plasma amino acid responses.

Vegan and Omnivorous High Protein Diets Support Comparable Daily Myofibrillar Protein Synthesis Rates and Skeletal Muscle Hypertrophy in Young Adults.

BACKGROUND: It remains unclear whether non-animal-derived dietary protein sources (and therefore vegan diets) can support resistance training-induced skeletal muscle remodeling to the same extent as animal-derived protein sources. METHODS: In Phase 1, 16 healthy young adults (m = 8, f = 8; age: 23 ± 1 y; BMI: 23 ± 1 kg/m2) completed a 3-d dietary intervention (high protein, 1.8 g·kg bm-1·d-1) where protein was derived from omnivorous (OMNI1; n = 8) or exclusively non-animal (VEG1; n = 8) sources, alongside daily unilateral leg resistance exercise. Resting and exercised daily myofibrillar protein synthesis (MyoPS) rates were assessed using deuterium oxide. In Phase 2, 22 healthy young adults (m = 11, f = 11; age: 24 ± 1 y; BMI: 23 ± 0 kg/m2) completed a 10 wk, high-volume (5 d/wk), progressive resistance exercise program while consuming an omnivorous (OMNI2; n = 12) or non-animal-derived (VEG2; n = 10) high-protein diet (∼2 g·kg bm-1·d-1). Muscle fiber cross-sectional area (CSA), whole-body lean mass (via DXA), thigh muscle volume (via MRI), muscle strength, and muscle function were determined pre, after 2 and 5 wk, and postintervention. OBJECTIVES: To investigate whether a high-protein, mycoprotein-rich, non-animal-derived diet can support resistance training-induced skeletal muscle remodeling to the same extent as an isonitrogenous omnivorous diet. RESULTS: Daily MyoPS rates were ∼12% higher in the exercised than in the rested leg (2.46 ± 0.27%·d-1 compared with 2.20 ± 0.33%·d-1 and 2.62 ± 0.56%·d-1 compared with 2.36 ± 0.53%·d-1 in OMNI1 and VEG1, respectively; P < 0.001) and not different between groups (P > 0.05). Resistance training increased lean mass in both groups by a similar magnitude (OMNI2 2.6 ± 1.1 kg, VEG2 3.1 ± 2.5 kg; P > 0.05). Likewise, training comparably increased thigh muscle volume (OMNI2 8.3 ± 3.6%, VEG2 8.3 ± 4.1%; P > 0.05), and muscle fiber CSA (OMNI2 33 ± 24%, VEG2 32 ± 48%; P > 0.05). Both groups increased strength (1 repetition maximum) of multiple muscle groups, to comparable degrees. CONCLUSIONS: Omnivorous and vegan diets can support comparable rested and exercised daily MyoPS rates in healthy young adults consuming a high-protein diet. This translates to similar skeletal muscle adaptive responses during prolonged high-volume resistance training, irrespective of dietary protein provenance. This trial was registered at clinicaltrials.gov as NCT03572127.

Skeletal Muscle Bioenergetics in Critical Limb Ischemia and Diabetes.

INTRODUCTION: Mitochondrial dysfunction is implicated in the metabolic myopathy accompanying peripheral artery disease (PAD) and critical limb ischemia (CLI). Type-2 diabetes mellitus (T2DM) is a major risk factor for PAD development and progression to CLI and may also independently be related to mitochondrial dysfunction. We set out to determine the effect of T2DM in the relationship between CLI and muscle mitochondrial respiratory capacity and coupling control. METHODS: We studied CLI patients undergoing revascularization procedures or amputation, and non-CLI patients with or without T2DM of similar age. Mitochondrial respiratory capacity and function were determined in lower limb permeabilized myofibers by high-resolution respirometry. RESULTS: Fourteen CLI patients (65 ± 10y) were stratified into CLI patients with (n = 8) or without (n = 6) T2DM and were compared to non-CLI patients with (n = 18; 69 ± 5y) or without (n = 19; 71 ± 6y) T2DM. Presence of CLI but not T2DM had a marked impact on all mitochondrial respiratory states in skeletal muscle, adjusted for the effects of sex. Leak respiration (State 2, P < 0.025 and State 4o, P < 0.01), phosphorylating respiration (P < 0.001), and maximal respiration in the uncoupled state (P < 0.001), were all suppressed in CLI patients, independent of T2DM. T2DM had no significant effect on mitochondrial respiratory capacity and function in adults without CLI. CONCLUSIONS: Skeletal muscle mitochondrial respiratory capacity was blunted by ∼35% in patients with CLI. T2DM was not associated with muscle oxidative capacity and did not moderate the relationship between muscle mitochondrial respiratory capacity and CLI.

Mycoprotein ingestion within or without its wholefood matrix results in equivalent stimulation of myofibrillar protein synthesis rates in resting and exercised muscle of young men.

Ingestion of mycoprotein stimulates skeletal muscle protein synthesis (MPS) rates to a greater extent than concentrated milk protein when matched for leucine content, potentially attributable to the wholefood nature of mycoprotein. We hypothesised that bolus ingestion of mycoprotein as part of its wholefood matrix would stimulate MPS rates to a greater extent compared with a leucine-matched bolus of protein concentrated from mycoprotein. Twenty-four healthy young (age, 21 ± 2 years; BMI, 24 ± 3 kg.m2) males received primed, continuous infusions of L-[ring-2H5]phenylalanine and completed a bout of unilateral resistance leg exercise before ingesting either 70 g mycoprotein (MYC; 31·4 g protein, 2·5 g leucine; n 12) or 38·2 g of a protein concentrate obtained from mycoprotein (PCM; 28·0 g protein, 2·5 g leucine; n 12). Blood and muscle samples (vastus lateralis) were taken pre- and (4 h) post-exercise/protein ingestion to assess postabsorptive and postprandial myofibrillar protein fractional synthetic rates (FSR) in resting and exercised muscle. Protein ingestion increased plasma essential amino acid and leucine concentrations (P < 0·0001), but more rapidly (both 60 v. 90 min; P < 0·0001) and to greater magnitudes (1367 v. 1346 µmol·l-1 and 298 v. 283 µmol·l-1, respectively; P < 0·0001) in PCM compared with MYC. Protein ingestion increased myofibrillar FSR (P < 0·0001) in both rested (MYC, Δ0·031 ± 0·007 %·h-1 and PCM, Δ0·020 ± 0·008 %·h-1) and exercised (MYC, Δ0·057 ± 0·011 %·h-1 and PCM, Δ0·058 ± 0·012 %·h-1) muscle, with no differences between conditions (P > 0·05). Mycoprotein ingestion results in equivalent postprandial stimulation of resting and post-exercise myofibrillar protein synthesis rates irrespective of whether it is consumed within or without its wholefood matrix.

Daily protein-polyphenol ingestion increases daily myofibrillar protein synthesis rates and promotes early muscle functional gains during resistance training.

Factors underpinning the time-course of resistance-type exercise training (RET) adaptations are not fully understood. This study hypothesized that consuming a twice-daily protein-polyphenol beverage (PPB; n = 15; age, 24 ± 1 yr; BMI, 22.3 ± 0.7 kg·m-2) previously shown to accelerate recovery from muscle damage and increase daily myofibrillar protein synthesis (MyoPS) rates would accelerate early (10 sessions) improvements in muscle function and potentiate quadriceps volume and muscle fiber cross-sectional area (fCSA) following 30 unilateral RET sessions in healthy, recreationally active, adults. Versus isocaloric placebo (PLA; n = 14; age, 25 ± 2 yr; BMI, 23.9 ± 1.0 kg·m-2), PPB increased 48 h MyoPS rates after the first RET session measured using deuterated water (2.01 ± 0.15 vs. 1.51 ± 0.16%·day-1, respectively; P < 0.05). In addition, PPB increased isokinetic muscle function over 10 sessions of training relative to the untrained control leg (%U) from 99.9 ± 1.8 pretraining to 107.2 ± 2.4%U at session 10 (vs. 102.6 ± 3.9 to 100.8 ± 2.4%U at session 10 in PLA; interaction P < 0.05). Pre to posttraining, PPB increased type II fCSA (PLA: 120.8 ± 8.2 to 109.5 ± 8.6%U; PPB: 92.8 ± 6.2 to 108.4 ± 9.7%U; interaction P < 0.05), but the gain in quadriceps muscle volume was similar between groups. Similarly, PPB did not further increase peak isometric torque, muscle function, or MyoPS measured posttraining. This suggests that although PPB increases MyoPS and early adaptation, it may not influence longer term adaptations to unilateral RET.NEW & NOTEWORTHY Using a unilateral model of resistance training, we show for the first time that a protein-polyphenol beverage increases initial rates of myofibrillar protein synthesis and promotes early functional improvements. Following a prolonged period of training, this strategy also increases type II fiber hypertrophy and causes large individual variation in gains in quadricep muscle cross-sectional area.

Muscle damaging eccentric exercise attenuates disuse-induced declines in daily myofibrillar protein synthesis and transiently prevents muscle atrophy in healthy men.

Short-term disuse leads to muscle loss driven by lowered daily myofibrillar protein synthesis (MyoPS). However, disuse commonly results from muscle damage, and its influence on muscle deconditioning during disuse is unknown. Twenty-one males [20 ± 1 yr, BMI = 24 ± 1 kg·m-2 (± SE)] underwent 7 days of unilateral leg immobilization immediately preceded by 300 bilateral, maximal, muscle-damaging eccentric quadriceps contractions (DAM; subjects n = 10) or no exercise (CON; subjects n = 11). Participants ingested deuterated water and underwent temporal bilateral thigh MRI scans and vastus lateralis muscle biopsies of immobilized (IMM) and nonimmobilized (N-IMM) legs. N-IMM quadriceps muscle volume remained unchanged throughout both groups. IMM quadriceps muscle volume declined after 2 days by 1.7 ± 0.5% in CON (P = 0.031; and by 1.3 ± 0.6% when corrected to N-IMM; P = 0.06) but did not change in DAM, and declined equivalently in CON [by 6.4 ± 1.1% (5.0 ± 1.6% when corrected to N-IMM)] and DAM [by 2.6 ± 1.8% (4.0 ± 1.9% when corrected to N-IMM)] after 7 days. Immobilization began to decrease MyoPS compared with N-IMM in both groups after 2 days (P = 0.109), albeit with higher MyoPS rates in DAM compared with CON (P = 0.035). Frank suppression of MyoPS was observed between days 2 and 7 in CON (IMM = 1.04 ± 0.12, N-IMM = 1.86 ± 0.10%·day-1; P = 0.002) but not DAM (IMM = 1.49 ± 0.29, N-IMM = 1.90 ± 0.30%·day-1; P > 0.05). Declines in MyoPS and quadriceps volume after 7 days correlated positively in CON (r2 = 0.403; P = 0.035) but negatively in DAM (r2 = 0.483; P = 0.037). Quadriceps strength declined following immobilization in both groups, but to a greater extent in DAM. Prior muscle-damaging eccentric exercise increases MyoPS and prevents loss of quadriceps muscle volume after 2 (but not 7) days of disuse.NEW & NOTEWORTHY We investigated the impact of prior muscle-damaging eccentric exercise on disuse-induced muscle deconditioning. Two and 7 days of muscle disuse per se lowered quadriceps muscle volume in association with lowered daily myofibrillar protein synthesis (MyoPS). Prior eccentric exercise prevented the decline in muscle volume after 2 days and attenuated the decline in MyoPS after 2 and 7 days. These data indicate eccentric exercise increases MyoPS and transiently prevents quadriceps muscle atrophy during muscle disuse.

Improved recovery from skeletal muscle damage is largely unexplained by myofibrillar protein synthesis or inflammatory and regenerative gene expression pathways.

The contribution of myofibrillar protein synthesis (MyoPS) to recovery from skeletal muscle damage in humans is unknown. Recreationally active men and women consumed a daily protein-polyphenol beverage targeted at increasing amino acid availability and reducing inflammation (PPB; n = 9), both known to affect MyoPS, or an isocaloric placebo (PLA; n = 9) during 168 h of recovery from 300 maximal unilateral eccentric contractions (EE). Muscle function was assessed daily. Muscle biopsies were collected for 24, 27, 36, 72, and 168 h for MyoPS measurements using 2H2O and expression of 224 genes using RT-qPCR and pathway analysis. PPB improved recovery of muscle function, which was impaired for 5 days after EE in PLA (interaction P < 0.05). Acute postprandial MyoPS rates were unaffected by nutritional intervention (24-27 h). EE increased overnight (27-36 h) MyoPS versus the control leg (PLA: 33 ± 19%; PPB: 79 ± 25%; leg P < 0.01), and PPB tended to increase this further (interaction P = 0.06). Daily MyoPS rates were greater with PPB between 72 and 168 h after EE, albeit after function had recovered. Inflammatory and regenerative signaling pathways were dramatically upregulated and clustered after EE but were unaffected by nutritional intervention. These results suggest that accelerated recovery from EE is not explained by elevated MyoPS or suppression of inflammation.NEW & NOTEWORTHY The present study investigated the contribution of myofibrillar protein synthesis (MyoPS) and associated gene signaling to recovery from 300 muscle-damaging, eccentric contractions. Measured with 2H2O, MyoPS rates were elevated during recovery and observed alongside expression of inflammatory and regenerative signaling pathways. A nutritional intervention accelerated recovery; however, MyoPS and gene signaling were unchanged compared with placebo. These data indicate that MyoPS and associated signaling do not explain accelerated recovery from muscle damage.

Liver-Specific Nonviral Gene Delivery of Fibroblast Growth Factor 21 Protein Expression in Mice Regulates Body Mass and White/Brown Fat Respiration.

Viral-mediated in vivo gene delivery methods currently dominate among therapeutic strategies within the clinical and experimental settings, albeit with well documented limitations arising from immunologic constraints. In this study, we demonstrate the utility of nonviral hepatotropic in vivo gene delivery of unpackaged expression constructs, including one encoding fibroblast growth factor 21 (FGF21). FGF21 is an important hepatokine whose expression positively correlates with therapeutic outcomes across various animal models of obesity. Our data demonstrate that FGF21 expression can be restored into the livers of immunocompetent FGF21 knockout mice for at least 2 weeks after a single injection with an FGF21 expression plasmid. In wild-type C57BL6/J mice, in vivo transfection with an FGF21-expressing plasmid induced weight loss, decreased adiposity, and activated thermogenesis in white fat within 2 weeks. Furthermore, in vivo FGF21 gene delivery protected C57BL6/J mice against diet-induced obesity by decreasing adiposity and increasing uncoupling protein 1-dependent thermogenesis in brown fat and by boosting respiratory capacity in subcutaneous and perigonadal white fat. Together, the data illustrate a facile and effective methodology for delivering prolonged protein expression specifically to the liver. We contend that this method will find utility in basic science research as a practical means to enhance in vivo studies characterizing liver protein function. We further believe our data provide a rationale for further exploring the potential clinical utility of nonviral gene therapy in mouse models of disease. SIGNIFICANCE STATEMENT: This study presents a valuable method for nonviral gene delivery in mice that improves upon existing techniques. The data provide a rationale for further exploring the potential clinical utility of nonviral gene therapy in mouse models of disease and will likely enhance in vivo studies characterizing liver protein function.

A mycoprotein-based high-protein vegan diet supports equivalent daily myofibrillar protein synthesis rates compared with an isonitrogenous omnivorous diet in older adults: a randomised controlled trial.

Animal-derived dietary protein ingestion and physical activity stimulate myofibrillar protein synthesis rates in older adults. We determined whether a non-animal-derived diet can support daily myofibrillar protein synthesis rates to the same extent as an omnivorous diet. Nineteen healthy older adults (aged 66 (sem 1) years; BMI 24 (sem 1) kg/m2; twelve males, seven females) participated in a randomised, parallel-group, controlled trial during which they consumed a 3-d isoenergetic high-protein (1·8 g/kg body mass per d) diet, where the protein was provided from predominantly (71 %) animal (OMNI; n 9; six males, three females) or exclusively vegan (VEG; n 10; six males, four females; mycoprotein providing 57 % of daily protein intake) sources. During the dietary control period, participants conducted a daily bout of unilateral resistance-type leg extension exercise. Before the dietary control period, participants ingested 400 ml of deuterated water, with 50-ml doses consumed daily thereafter. Saliva samples were collected throughout to determine body water 2H enrichments, and muscle samples were collected from rested and exercised muscle to determine daily myofibrillar protein synthesis rates. Deuterated water dosing resulted in body water 2H enrichments of approximately 0·78 (sem 0·03) %. Daily myofibrillar protein synthesis rates were 13 (sem 8) (P = 0·169) and 12 (sem 4) % (P = 0·016) greater in the exercised compared with rested leg (1·59 (sem 0·12) v. 1·77 (sem 0·12) and 1·76 (sem 0·14) v. 1·93 (sem 0·12) %/d) in OMNI and VEG groups, respectively. Daily myofibrillar protein synthesis rates did not differ between OMNI and VEG in either rested or exercised muscle (P > 0·05). Over the course of a 3-d intervention, omnivorous- or vegan-derived dietary protein sources can support equivalent rested and exercised daily myofibrillar protein synthesis rates in healthy older adults consuming a high-protein diet.

Leucine and mTORc1 act independently to regulate 2-deoxyglucose uptake in L6 myotubes.

Chronic mTORc1 hyperactivation via obesity-induced hyperleucinaemia has been implicated in the development of insulin resistance, yet the direct impact of leucine on insulin-stimulated glucose uptake in muscle cells remains unclear. To address this, differentiated L6 myotubes were subjected to various compounds designed to either inhibit mTORc1 activity (rapamycin), blunt leucine intracellular import (BCH), or activate mTORc1 signalling (3BDO), prior to the determination of the uptake of the glucose analogue, 2-deoxyglucose (2-DG), in response to 1 mM insulin. In separate experiments, L6 myotubes were subject to various media concentrations of leucine (0-0.8 mM) for 24 h before 2-DG uptake in response to insulin was assessed. Both rapamycin and BCH blunted 2-DG uptake, irrespective of insulin administration, and this occurred in parallel with a decline in mTOR, 4E-BP1, and p70S6K phosphorylation status, but little effect on AKT phosphorylation. In contrast, reducing leucine media concentrations suppressed 2-DG uptake, both under insulin- and non-insulin-stimulated conditions, but did not alter the phosphorylation state of AKT-mTORc1 components examined. Unexpectedly, 3BDO failed to stimulate mTORc1 signalling, but, nonetheless, caused a significant increase in 2-DG uptake under non-insulin-stimulated conditions. Both leucine and mTORc1 influence glucose uptake in muscle cells independent of insulin administration, and this likely occurs via distinct but overlapping mechanisms.

The commercial pig as a model of spontaneously-occurring osteoarthritis.

BACKGROUND: Preclinical osteoarthritis models where damage occurs spontaneously may better reflect the initiation and development of human osteoarthritis. The aim was to assess the commercial pig as a model of spontaneous osteoarthritis development by examining pain-associated behaviour, joint cartilage integrity, as well as the use of porcine cartilage explants and isolated chondrocytes and osteoblasts for ex vivo and in vitro studies. METHODS: Female pigs (Large white x Landrace x Duroc) were examined at different ages from 6 weeks to 3-4 years old. Lameness was assessed as a marker of pain-associated behaviour. Femorotibial joint cartilage integrity was determined by chondropathy scoring and histological staining of proteoglycan. IL-6 production and proteoglycan degradation was assessed in cartilage explants and primary porcine chondrocytes by ELISA and DMMB assay. Primary porcine osteoblasts from damaged and non-damaged joints, as determined by chondropathy scoring, were assessed for mineralisation, proliferative and mitochondrial function as a marker of metabolic capacity. RESULTS: Pigs aged 80 weeks and older exhibited lameness. Osteoarthritic lesions in femoral condyle and tibial plateau cartilage were apparent from 40 weeks and increased in severity with age up to 3-4 years old. Cartilage from damaged joints exhibited proteoglycan loss, which positively correlated with chondropathy score. Stimulation of porcine cartilage explants and primary chondrocytes with either IL-1ß or visfatin induced IL-6 production and proteoglycan degradation. Primary porcine osteoblasts from damaged joints exhibited reduced proliferative, mineralisation, and metabolic capacity. CONCLUSION: In conclusion, the commercial pig represents an alternative model of spontaneous osteoarthritis and an excellent source of tissue for in vitro and ex vivo studies.

The addition of mycoprotein to a mixed-meal impacts postprandial glucose kinetics without altering blood glucose concentrations: a randomised controlled trial.

BACKGROUND: Mycoprotein is a high-fibre food previously shown to reduce postprandial glucose concentrations when ingested within a mixed-meal. We applied a dual stable isotope tracer approach to determine whether this is due to a reduced rate of appearance of glucose, in participants of ranging BMI. METHODS: Twenty-four adults (F = 8, BMI 30 ± 6 kg·m-2) attended 2 trials in a double-blind, randomised, cross-over design. Participants ingested two energy and macronutrient matched milk-based drinks (enriched with 1000 mg [U-13C6] glucose in a subset of 12 participants), containing 50 g glucose and either 0 (CON) or 20 g (MYC) mycoprotein. A primed continuous intravenous infusion of D-[6,6-2H2] glucose determined plasma glucose kinetics over 6 h. Postprandial time-course, and AUC, of glucose and insulin concentration, rate of disappearance (RdT) and appearance of exogenous (RaEx), endogenous (EGP), and total (RaT) plasma glucose were assessed using two- and one-way ANOVA. RESULTS: Drink ingestion increased blood glucose and serum insulin concentrations (P < 0.05) and were comparable between conditions (P > 0.05). Both RaT and RdT were higher with MYC compared with CON over 6 h (mean 6 h glucose appearance and disappearance increased by 5 and 9%, respectively, P < 0.05). RaEx was not affected by MYC ingestion over 6 h (P > 0.05). The mean contribution of EGP to total glucose appearance was 15% greater with MYC, with a trend towards significance (P = 0.05). There was no relationship between BMI and the response to MYC ingestion for any of the variables (P < 0.05). CONCLUSION: The ingestion of mycoprotein within a mixed-meal impacted postprandial glucose kinetics, but not blood glucose or serum insulin concentrations, in individuals of ranging BMI. CLINICAL TRIAL REGISTRY NUMBER AND WEBSITE: This trial was registered at clinicaltrials.gov as NCT04084639 and can be accessed at https://clinicaltrials.gov/ct2/show/NCT04084639 .

Plant Protein Blend Ingestion Stimulates Postexercise Myofibrillar Protein Synthesis Rates Equivalently to Whey in Resistance-Trained Adults.

PURPOSE: Whey protein ingestion is typically considered an optimal dietary strategy to maximize myofibrillar protein synthesis (MyoPS) after resistance exercise. Although single-source plant protein ingestion is typically less effective, at least partly, due to less favorable amino acid profiles, this could theoretically be overcome by blending plant-based proteins with complementary amino acid profiles. We compared the postexercise MyoPS response after the ingestion of a novel plant-derived protein blend with an isonitrogenous bolus of whey protein. METHODS: Ten healthy, resistance-trained, young adults (male/female: 8/2; age: 26 ± 6 yr; BMI: 24 ± 3 kg·m -2 ) received a primed continuous infusion of L-[ ring - 2 H 5 ]-phenylalanine and completed a bout of bilateral leg resistance exercise before ingesting 32 g protein from whey (WHEY) or a plant protein blend (BLEND; 39.5% pea, 39.5% brown rice, 21.0% canola) in a randomized, double-blind crossover fashion. Blood and muscle samples were collected at rest, and 2 and 4 h after exercise and protein ingestion, to assess plasma amino acid concentrations, and postabsorptive and postexercise MyoPS rates. RESULTS: Plasma essential amino acid availability over the 4 h postprandial postexercise period was ~44% higher in WHEY compared with BLEND ( P = 0.04). From equivalent postabsorptive values (WHEY, 0.042 ± 0.020%·h -1 ; BLEND, 0.043 ± 0.015%·h -1 ) MyoPS rates increased after exercise and protein ingestion (time effect; P < 0.001) over a 0- to 2-h period (WHEY, 0.085 ± 0.037%·h -1 ; BLEND, 0.080 ± 0.037%·h -1 ) and 2- to 4-h period (WHEY, 0.085 ± 0.036%·h -1 ; BLEND, 0.086 ± 0.034%·h -1 ), with no differences between conditions during either period or throughout the entire (0-4 h) postprandial period (time-condition interactions; all P > 0.05). CONCLUSIONS: Ingestion of a novel plant-based protein blend stimulates postexercise MyoPS to an equivalent extent as whey protein, demonstrating the utility of plant protein blends to optimize postexercise skeletal muscle reconditioning.