Rate Of Mitochondrial Protein Synthesis Research Articles

Divergence in aerobic capacity and energy expenditure influence metabolic tissue mitochondrial protein synthesis rates in aged rats.

Age-associated declines in aerobic capacity promote the development of various metabolic diseases. In rats selectively bred for high/low intrinsic aerobic capacity, greater aerobic capacity reduces susceptibility to metabolic disease while increasing longevity. However, little remains known how intrinsic aerobic capacity protects against metabolic disease, particularly with aging. Here, we tested the effects of aging and intrinsic aerobic capacity on systemic energy expenditure, metabolic flexibility and mitochondrial protein synthesis rates using 24-month-old low-capacity (LCR) or high-capacity runner (HCR) rats. Rats were fed low-fat diet (LFD) or high-fat diet (HFD) for eight weeks, with energy expenditure (EE) and metabolic flexibility assessed utilizing indirect calorimetry during a 48h fast/re-feeding metabolic challenge. Deuterium oxide(D2O) labeling was used to assess mitochondrial protein fraction synthesis rates (FSR) over a 7-day period. HCR rats possessed greater EE during the metabolic challenge. Interestingly, HFD induced changes in respiratory exchange ratio (RER) in male and female rats, while HCR female rat RER was largely unaffected by diet. In addition, analysis of protein FSR in skeletal muscle, brain, and liver mitochondria showed tissue-specific adaptations between HCR and LCR rats. While brain and liver protein FSR were altered by aerobic capacity and diet, these effects were less apparent in skeletal muscle. Overall, we provide evidence that greater aerobic capacity promotes elevated EE in an aged state, while also regulating metabolic flexibility in a sex-dependent manner. Modulation of mitochondrial protein FSR by aerobic capacity is tissue-specific with aging, likely due to differential energetic requirements by each tissue.

Dietary nitrate preserves mitochondrial bioenergetics and mitochondrial protein synthesis rates during short-term immobilization in mice.

Skeletal muscle disuse reduces muscle protein synthesis rates and induces atrophy, events associated with decreased mitochondrial respiration and increased reactive oxygen species. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation attenuates disuse-induced impairments in mitochondrial function and muscle protein synthesis rates. Female C57Bl/6N mice were subjected to single-limb casting (3 or 7days) and consumed drinking water with or without 1mM sodium nitrate. Compared with the contralateral control limb, 3days of immobilization lowered myofibrillar fractional synthesis rates (FSR, P< 0.0001), resulting in muscle atrophy. Although FSR and mitophagy-related proteins were higher in subsarcolemmal (SS) compared with intermyofibrillar (IMF) mitochondria, immobilization for 3days decreased FSR in both SS (P=0.009) and IMF (P=0.031) mitochondria. Additionally, 3days of immobilization reduced maximal mitochondrial respiration, decreased mitochondrial protein content, and increased maximal mitochondrial reactive oxygen species emission, without altering mitophagy-related proteins in muscle homogenate or isolated mitochondria (SS and IMF). Although nitrate consumption did not attenuate the decline in muscle mass or myofibrillar FSR, intriguingly, nitrate completely prevented immobilization-induced reductions in SS and IMF mitochondrial FSR. In addition, nitrate prevented alterations in mitochondrial content and bioenergetics after both 3 and 7days of immobilization. However, in contrast to 3days of immobilization, nitrate did not prevent the decline in SS and IMF mitochondrial FSR after 7days of immobilization. Therefore, although nitrate supplementation was not sufficient to prevent muscle atrophy, nitrate may represent a promising therapeutic strategy to maintain mitochondrial bioenergetics and transiently preserve mitochondrial protein synthesis rates during short-term muscle disuse. KEY POINTS: Alterations in mitochondrial bioenergetics (decreased respiration and increased reactive oxygen species) are thought to contribute to muscle atrophy and reduced protein synthesis rates during muscle disuse. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation could attenuate immobilization-induced skeletal muscle impairments in female mice. Dietary nitrate prevented short-term (3day) immobilization-induced declines in mitochondrial protein synthesis rates, reductions in markers of mitochondrial content, and alterations in mitochondrial bioenergetics. Despite these benefits and the preservation of mitochondrial content and bioenergetics during more prolonged (7 day) immobilization, nitrate consumption did not preserve skeletal muscle mass or myofibrillar protein synthesis rates. Overall, although dietary nitrate did not prevent atrophy, nitrate supplementation represents a promising nutritional approach to preserve mitochondrial function during muscle disuse.

Restoration of mitochondrial function through activation of hypomodified tRNAs with pathogenic mutations associated with mitochondrial diseases.

Mutations in mitochondrial (mt-)tRNAs frequently cause mitochondrial dysfunction. Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and myoclonus epilepsy associated with ragged red fibers (MERRF) are major clinical subgroups of mitochondrial diseases caused by pathogenic point mutations in tRNA genes encoded in mtDNA. We previously reported a severe reduction in the frequency of 5-taurinomethyluridine (τm5U) and its 2-thiouridine derivative (τm5s2U) in the anticodons of mutant mt-tRNAs isolated from the cells of patients with MELAS and MERRF, respectively. The hypomodified tRNAs fail to decode cognate codons efficiently, resulting in defective translation of respiratory chain proteins in mitochondria. To restore the mitochondrial activity of MELAS patient cells, we overexpressed MTO1, a τm5U-modifying enzyme, in patient-derived myoblasts. We used a newly developed primer extension method and showed that MTO1 overexpression almost completely restored the τm5U modification of the MELAS mutant mt-tRNALeu(UUR). An increase in mitochondrial protein synthesis and oxygen consumption rate suggested that the mitochondrial function of MELAS patient cells can be activated by restoring the τm5U of the mutant tRNA. In addition, we confirmed that MTO1 expression restored the τm5s2U of the mutant mt-tRNALys in MERRF patient cells. These findings pave the way for epitranscriptomic therapies for mitochondrial diseases.

Pre-sleep Protein Ingestion Increases Mitochondrial Protein Synthesis Rates During Overnight Recovery from Endurance Exercise: A Randomized Controlled Trial

BackgroundCasein protein ingestion prior to sleep has been shown to increase myofibrillar protein synthesis rates during overnight sleep. It remains to be assessed whether pre-sleep protein ingestion can also increase mitochondrial protein synthesis rates. Though it has been suggested that casein protein may be preferred as a pre-sleep protein source, no study has compared the impact of pre-sleep whey versus casein ingestion on overnight muscle protein synthesis rates.ObjectiveWe aimed to assess the impact of casein and whey protein ingestion prior to sleep on mitochondrial and myofibrillar protein synthesis rates during overnight recovery from a bout of endurance-type exercise.MethodsThirty-six healthy young men performed a single bout of endurance-type exercise in the evening (19:45 h). Thirty minutes prior to sleep (23:30 h), participants ingested 45 g of casein protein, 45 g of whey protein, or a non-caloric placebo. Continuous intravenous l-[ring-13C6]-phenylalanine infusions were applied, with blood and muscle tissue samples being collected to assess overnight mitochondrial and myofibrillar protein synthesis rates.ResultsPooled protein ingestion resulted in greater mitochondrial (0.087 ± 0.020 vs 0.067 ± 0.016%·h−1, p = 0.005) and myofibrillar (0.060 ± 0.014 vs 0.047 ± 0.011%·h−1, p = 0.012) protein synthesis rates when compared with placebo. Casein and whey protein ingestion did not differ in their capacity to stimulate mitochondrial (0.082 ± 0.019 vs 0.092 ± 0.020%·h−1, p = 0.690) and myofibrillar (0.056 ± 0.009 vs 0.064 ± 0.018%·h−1, p = 0.440) protein synthesis rates.ConclusionsProtein ingestion prior to sleep increases both mitochondrial and myofibrillar protein synthesis rates during overnight recovery from exercise. The overnight muscle protein synthetic response to whey and casein protein does not differ.Clinical Trial RegistrationNTR7251.

Variability in Skeletal Muscle Protein Synthesis Rates in Critically Ill Patients.

(1) Background: Muscle protein synthesis in critically ill patients is, on average, normal despite dramatic muscle loss, but the variation is much larger than in controls. Here, we evaluate if this variation is due to 1) heterogeneity in synthesis rates, 2) morphological variation or infiltrating cells, or 3) heterogeneity in the synthesis of different protein fractions. (2) Methods: Muscle biopsies were taken from both legs of critically ill patients (n = 17). Mixed and mitochondrial protein synthesis rates and morphologies were evaluated in both legs. Synthesis rates of myosin and actin were determined in combined biopsies and compared with controls. (3) Results: Muscle protein synthesis rates had a large variability in the patients (1.4–10.8%/day). No differences in mixed and mitochondrial protein synthesis rates between both legs were observed. A microscopic examination revealed no morphological differences between the two legs or any infiltrating inflammatory cells. The synthesis rates for myosin were lower and for actin they were higher in the muscles of critically ill patients, compared with the controls. (4) Conclusions: The large variation in muscle protein synthesis rates in critically ill patients is not the result of heterogeneity in synthesis rates, nor due to infiltrating cells. There are differences in the synthesis rates of different proteins, but these do not explain the larger variations.

AMP deamination is sufficient to replicate an atrophy-like metabolic phenotype in skeletal muscle

BackgroundSkeletal muscle atrophy, whether caused by chronic disease, acute critical illness, disuse or aging, is characterized by tissue-specific decrease in oxidative capacity and broad alterations in metabolism that contribute to functional decline. However, the underlying mechanisms responsible for these metabolic changes are largely unknown. One of the most highly upregulated genes in atrophic muscle is AMP deaminase 3 (AMPD3: AMP → IMP + NH3), which controls the content of intracellular adenine nucleotides (AdN; ATP + ADP + AMP). Given the central role of AdN in signaling mitochondrial gene expression and directly regulating metabolism, we hypothesized that overexpressing AMPD3 in muscle cells would be sufficient to alter their metabolic phenotype similar to that of atrophic muscle. MethodsAMPD3 and GFP (control) were overexpressed in mouse tibialis anterior (TA) muscles via plasmid electroporation and in C2C12 myotubes using adenovirus vectors. TA muscles were excised one week later, and AdN were quantified by UPLC. In myotubes, targeted measures of AdN, AMPK/PGC-1α/mitochondrial protein synthesis rates, unbiased metabolomics, and transcriptomics by RNA sequencing were measured after 24 h of AMPD3 overexpression. Media metabolites were measured as an indicator of net metabolic flux. At 48 h, the AMPK/PGC-1α/mitochondrial protein synthesis rates, and myotube respiratory function/capacity were measured. ResultsTA muscles overexpressing AMPD3 had significantly less ATP than contralateral controls (−25%). In myotubes, increasing AMPD3 expression for 24 h was sufficient to significantly decrease ATP concentrations (−16%), increase IMP, and increase efflux of IMP catabolites into the culture media, without decreasing the ATP/ADP or ATP/AMP ratios. When myotubes were treated with dinitrophenol (mitochondrial uncoupler), AMPD3 overexpression blunted decreases in ATP/ADP and ATP/AMP ratios but exacerbated AdN degradation. As such, pAMPK/AMPK, pACC/ACC, and phosphorylation of AMPK substrates, were unchanged by AMPD3 at this timepoint. AMPD3 significantly altered 191 out of 639 detected intracellular metabolites, but only 30 transcripts, none of which encoded metabolic enzymes. The most altered metabolites were those within purine nucleotide, BCAA, glycolysis, and ceramide metabolic pathways. After 48 h, AMPD3 overexpression significantly reduced pAMPK/AMPK (−24%), phosphorylation of AMPK substrates (−14%), and PGC-1α protein (−22%). Moreover, AMPD3 significantly reduced myotube mitochondrial protein synthesis rates (−55%), basal ATP synthase-dependent (−13%), and maximal uncoupled oxygen consumption (−15%). ConclusionsIncreased expression of AMPD3 significantly decreased mitochondrial protein synthesis rates and broadly altered cellular metabolites in a manner similar to that of atrophic muscle. Importantly, the changes in metabolites occurred prior to reductions in AMPK signaling, gene expression, and mitochondrial protein synthesis, suggesting metabolism is not dependent on reductions in oxidative capacity, but may be consequence of increased AMP deamination. Therefore, AMP deamination in skeletal muscle may be a mechanism that alters the metabolic phenotype of skeletal muscle during atrophy and could be a target to improve muscle function during muscle wasting.

Early resistance training-mediated stimulation of daily muscle protein synthetic responses to higher habitual protein intake in middle-aged adults.

The ingestion of protein potentiates the stimulation of myofibrillar protein synthesis rates after an acute bout of resistance exercise. Protein supplementation (eating above the protein Recommended Dietary Allowance) during resistance training has been shown to maximize lean mass and strength gains in healthy young and older adults. Here, contractile, oxidative, and structural protein synthesis were assessed in skeletal muscle in response to a moderate or higher protein diet during the early adaptive phase of resistance training in middle-aged adults. The stimulation of myofibrillar, mitochondrial or collagen protein synthesis rates during 0-3weeks of resistance training is not further enhanced by a higher protein diet. These results show that moderate protein diets are sufficient to support the skeletal muscle adaptive response during the early phase of a resistance training programme. Protein ingestion augments muscle protein synthesis (MPS) rates acutely after resistance exercise and can offset age-related loss in muscle mass. Skeletal muscle contains a variety of protein pools, such as myofibrillar (contractile), mitochondrial (substrate oxidation), and collagen (structural support) proteins, and the sensitivity to nutrition and exercise seems to be dependent on the major protein fraction studied. However, it is unknown how free-living conditions with high dietary protein density and habitual resistance exercise mediates muscle protein subfraction synthesis. Therefore, we investigated the effect of moderate (MOD: 1.06±0.22gkg-1 day-1 ) or high (HIGH: 1.55±0.25gkg-1 day-1 ) protein intake on daily MPS rates within the myofibrillar (MyoPS), mitochondrial (MitoPS) and collagen (CPS) protein fractions in middle-aged men and women (n=20, 47±1years, BMI 28±1kgm-2 ) during the early phase (0-3weeks) of a dietary counselling-controlled resistance training programme. Participants were loaded with deuterated water, followed by daily maintenance doses throughout the intervention. Muscle biopsies were collected at baseline and after weeks 1, 2 and 3. MyoPS in the HIGH condition remained constant (P=1.000), but MOD decreased over time (P=0.023). MitoPS decreased after 0-3weeks when compared to 0-1week (P=0.010) with no effects of protein intake (P=0.827). A similar decline with no difference between groups (P=0.323) was also observed for CPS (P=0.007). Our results demonstrated that additional protein intake above moderate amounts does not potentiate the stimulation of longer-term MPS responses during the early stage of resistance training adaptations in middle-aged adults.

Mitochondrial mistranslation modulated by metabolic stress causes cardiovascular disease and reduced lifespan.

Changes in the rate and fidelity of mitochondrial protein synthesis impact the metabolic and physiological roles of mitochondria. Here we explored how environmental stress in the form of a high‐fat diet modulates mitochondrial translation and affects lifespan in mutant mice with error‐prone (Mrps12ep / ep ) or hyper‐accurate (Mrps12ha / ha ) mitochondrial ribosomes. Intriguingly, although both mutations are metabolically beneficial in reducing body weight, decreasing circulating insulin and increasing glucose tolerance during a high‐fat diet, they manifest divergent (either deleterious or beneficial) outcomes in a tissue‐specific manner. In two distinct organs that are commonly affected by the metabolic disease, the heart and the liver, Mrps12ep / ep mice were protected against heart defects but sensitive towards lipid accumulation in the liver, activating genes involved in steroid and amino acid metabolism. In contrast, enhanced translational accuracy in Mrps12ha / ha mice protected the liver from a high‐fat diet through activation of liver proliferation programs, but enhanced the development of severe hypertrophic cardiomyopathy and led to reduced lifespan. These findings reflect the complex transcriptional and cell signalling responses that differ between post‐mitotic (heart) and highly proliferative (liver) tissues. We show trade‐offs between the rate and fidelity of mitochondrial protein synthesis dictate tissue‐specific outcomes due to commonly encountered stressful environmental conditions or aging.

Skeletal Muscle Mitochondrial Bioenergetics in a Model of Primary Osteoarthritis after Treatment with Rapamycin and/or Metformin

Impaired mitochondrial function is associated with the loss of skeletal muscle function during aging and age-related osteoarthritis (OA). Emerging evidence suggests Rapamycin (Rap), metformin (Met) or the combination of rapamycin plus metformin (Rap+Met) can extend lifespan and preserve skeletal muscle mass and function during aging. However, the mechanisms of action are not completely understood and have not been studied in specific models of age-related disease. The goal of this study was to test to test the hypothesis that Rap, Met, or Rap+Met would improve skeletal muscle mitochondrial function in an animal model of naturally occurring, age-related OA. We chose the Dunkin Hartley guinea pig because they are an outbred strain that develops naturally occurring OA by 5 months of age with a progression and pathology that highly resembles aging humans. After 5 months of age, Dunkin Hartley guinea pigs also have an age-related decline in skeletal muscle mitochondrial protein synthesis rates. Therefore, 5-month old Dunkin Hartley guinea pigs were fed a standard diet (Con; n=8), or a diet enriched with Rap (14ppm; n=8), Met (1000ppm, n=8) or Rap+Met (14+1000ppm; n=7) for 12-weeks. Mitochondrial respiration and hydrogen peroxide (H2O2) emissions were evaluated in permeabilized muscle fibers from the soleus via high-resolution respirometry and fluorometry using either a saturating bolus or titrating doses of ADP. Michaelis-Menten kinetics was used to evaluate Vmax and ADP sensitivity. All comparisons were made to control group. In the current study, Met had no effect while Rap and Rap+Met modified skeletal muscle mitochondrial bioenergetics only when ADP was titrated but not with a saturating bolus. Specifically, Rap (P<0.05) and Rap+Met (P=0.07) increased ADP sensitivity but decreased (P<0.05) maximal complex-I (CI) linked respiration. Rap and Rap+Met also tended to decrease mitochondrial H2O2 emissions but this trend was no longer apparent when mitochondrial H2O2 emissions were expressed relative to respiration. The decrease in CI-linked respiration and H2O2 emissions was attributed to both a lower CI protein abundance and a decreased index of intrinsic mitochondrial function. This is the first inquiry into how lifespan extending treatments can influence skeletal muscle mitochondria in a model of age-related OA. Further research is needed to understand if increased mitochondrial ADP sensitivity at the cost of CI-linked maximal capacity by Rap and Rap+Met is a beneficial strategy to maintain cellular energy homeostasis during age-related OA.

Ammonium chloride administration prior to exercise has muscle-specific effects on mitochondrial and myofibrillar protein synthesis in rats.

AimExercise is able to increase both muscle protein synthesis and mitochondrial biogenesis. However, acidosis, which can occur in pathological states as well as during high‐intensity exercise, can decrease mitochondrial function, whilst its impact on muscle protein synthesis is disputed. Thus, the aim of this study was to determine the effect of a mild physiological decrease in pH, by administration of ammonium chloride, on myofibrillar and mitochondrial protein synthesis, as well as associated molecular signaling events.MethodsMale Wistar rats were given either a placebo or ammonium chloride prior to a short interval training session. Rats were killed before exercise, immediately after exercise, or 3 h after exercise.ResultsMyofibrillar (p = 0.036) fractional protein synthesis rates was increased immediately after exercise in the soleus muscle of the placebo group, but this effect was absent in the ammonium chloride group. However, in the gastrocnemius muscle NH4Cl increased myofibrillar (p = 0.044) and mitochondrial protein synthesis (0 h after exercise p = 0.01; 3 h after exercise p = 0.003). This was accompanied by some small differences in protein phosphorylation and mRNA expression.ConclusionThis study found ammonium chloride administration immediately prior to a single session of exercise in rats had differing effects on mitochondrial and myofibrillar protein synthesis rates in soleus (type I) and gastrocnemius (type II) muscle in rats.

Endurance-Type Exercise Increases Bulk and Individual Mitochondrial Protein Synthesis Rates in Rats.

Physical activity increases muscle protein synthesis rates. However, the impact of exercise on the coordinated up- and/or downregulation of individual protein synthesis rates in skeletal muscle tissue remains unclear. The authors assessed the impact of exercise on mixed muscle, myofibrillar, and mitochondrial protein synthesis rates as well as individual protein synthesis rates in vivo in rats. Adult Lewis rats either remained sedentary (n = 3) or had access to a running wheel (n = 3) for the last 2 weeks of a 3-week experimental period. Deuterated water was injected and subsequently administered in drinking water over the experimental period. Blood and soleus muscle were collected and used to assess bulk mixed muscle, myofibrillar, and mitochondrial protein synthesis rates using gas chromatography-mass spectrometry and individual muscle protein synthesis rates using liquid chromatography-mass spectrometry (i.e.,dynamic proteomic profiling). Wheel running resulted in greater myofibrillar (3.94 ± 0.26 vs. 3.03 ± 0.15%/day; p < .01) and mitochondrial (4.64 ± 0.24 vs. 3.97 ± 0.26%/day; p < .05), but not mixed muscle (2.64 ± 0.96 vs. 2.38 ± 0.62%/day; p = .71) protein synthesis rates, when compared with the sedentary condition. Exercise impacted the synthesis rates of 80 proteins, with the difference from the sedentary condition ranging between -64% and +420%. Significantly greater synthesis rates were detected for F1-ATP synthase, ATP synthase subunit alpha, hemoglobin, myosin light chain-6, and synaptopodin-2 (p < .05). The skeletal muscle protein adaptive response to endurance-type exercise involves upregulation of mitochondrial protein synthesis rates, but it is highly coordinated as reflected by the up- and downregulation of various individual proteins across different bulk subcellular protein fractions.

The mitoribosome-specific protein mS38 is preferentially required for synthesis of cytochrome c oxidase subunits.

Message-specific translational regulation mechanisms shape the biogenesis of multimeric oxidative phosphorylation (OXPHOS) enzyme in mitochondria from the yeast Saccharomyces cerevisiae. These mechanisms, driven mainly by the action of mRNA-specific translational activators, help to coordinate synthesis of OXPHOS catalytic subunits by the mitoribosomes with both the import of their nucleus-encoded partners and their assembly to form the holocomplexes. However, little is known regarding the role that the mitoribosome itself may play in mRNA-specific translational regulation. Here, we show that the mitoribosome small subunit protein Cox24/mS38, known to be necessary for mitoribosome-specific intersubunit bridge formation and 15S rRNA H44 stabilization, is required for efficient mitoribogenesis. Consequently, mS38 is necessary to sustain the overall mitochondrial protein synthesis rate, despite an adaptive ∼2-fold increase in mitoribosome abundance in mS38-deleted cells. Additionally, the absence of mS38 preferentially disturbs translation initiation of COX1, COX2, and COX3 mRNAs, without affecting the levels of mRNA-specific translational activators. We propose that mS38 confers the mitochondrial ribosome an intrinsic capacity of translational regulation, probably acquired during evolution from bacterial ribosomes to facilitate the translation of mitochondrial mRNAs, which lack typical anti-Shine-Dalgarno sequences.

Brain Protein Synthesis Rates in the UM-HET3 Mouse Following Treatment With Rapamycin or Rapamycin With Metformin.

Treatment with the mechanistic target of rapamycin (mTOR) inhibitor, rapamycin (RAP), alone and in combination with the antidiabetic drug, metformin (RAP+MET), extends lifespan in mice. The mechanisms underlying lifespan extension are unclear. One possibility is improved capacity for proteostatic maintenance. We have previously characterized peripheral protein synthesis rates following treatment with RAP. However, it is unknown if RAP+MET elicits similar changes, or if either treatment affects protein synthesis in the brain. We hypothesized that 8 weeks of treatment with RAP and RAP+MET would alter brain protein synthesis rates to reflect proteostatic processes. Using the stable isotopic tracer, deuterium oxide (D2O), we demonstrate in UM-HET3 mice that protein synthesis rates measured in whole brain were unaffected by treatment in young male mice, whereas RAP+MET decreased mitochondrial protein synthesis in young females. Conversely, RAP increased mitochondrial protein synthesis rates in older females. Activity through the AMPK/mTOR pathway was affected in a sex-specific manner in young mice, and minimal changes were observed in the older cohort. Thus, we establish D2O for measurements of biogenesis in the brain. These results provide initial insights into the effects of RAP and RAP+MET on brain protein synthesis. Additionally, these data emphasize that responses to slowed aging treatments vary with sex and age.

Myofibrillar and Mitochondrial Protein Synthesis Rates Do Not Differ in Young Men Following the Ingestion of Carbohydrate with Milk Protein, Whey, or Micellar Casein after Concurrent Resistance- and Endurance-Type Exercise

ABSTRACTBackgroundWhey and micellar casein are high-quality dairy proteins that can stimulate postprandial muscle protein synthesis rates. How whey and casein compare with milk protein in their capacity to stimulate postprandial myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis rates during postexercise recovery is currently unknown.ObjectiveThe objective of this study was to compare postprandial MyoPS and MitoPS rates after protein-carbohydrate co-ingestion with milk protein, whey, or micellar casein during recovery from a single bout of concurrent resistance- and endurance-type exercise in young healthy men.MethodsIn a randomized, double-blind, parallel-group design, 48 healthy, young, recreationally active men (mean ± SEM age: 23 ± 0.3 y) received a primed continuous infusion of L-[ring-13C6]-phenylalanine and L-[ring-3,5-2H2]-tyrosine and ingested 45 g carbohydrate with 0 g protein (CHO), 20 g milk protein (MILK), 20 g whey protein (WHEY), or 20 g micellar casein protein (CASEIN) after a sequential bout of resistance- and endurance-type exercise (i.e., concurrent exercise). Blood and muscle biopsies were collected over 360 min during recovery from exercise to assess MyoPS and MitoPS rates and signaling through mammalian target of rapamycin complex 1 (mTORC1).ResultsDespite temporal differences in postprandial plasma leucine concentrations between treatments (P < 0.001), MyoPS rates over 360 min of recovery did not differ between treatments (CHO: 0.049% ± 0.003%/h; MILK: 0.059% ± 0.003%/h; WHEY: 0.054% ± 0.002%/h; CASEIN: 0.059% ± 0.005%/h; P = 0.11). When MILK, WHEY, and CASEIN were pooled into a single group (PROTEIN), protein co-ingestion resulted in greater MyoPS rates compared with CHO (PROTEIN: 0.057% ± 0.002%/h; CHO: 0.049% ± 0.003%/h; P = 0.04). MitoPS rates and signaling through the mTORC1 pathway were similar between treatments.ConclusionMyoPS and MitoPS rates do not differ after co-ingestion of either milk protein, whey protein, or micellar casein protein with carbohydrate during recovery from a single bout of concurrent resistance- and endurance-type exercise in recreationally active young men. Co-ingestion of protein with carbohydrate results in greater MyoPS, but not MitoPS rates, when compared with the ingestion of carbohydrate only during recovery from concurrent exercise. This trial was registered at Nederlands Trial Register: NTR5098.

Myofibrillar and Mitochondrial Protein Synthesis Rates Do Not Differ in Young Men Following the Ingestion of Carbohydrate with Whey, Soy, or Leucine-Enriched Soy Protein after Concurrent Resistance- and Endurance-Type Exercise

ABSTRACTBackgroundProtein ingestion during recovery from resistance-type exercise increases postexercise muscle protein synthesis rates. Whey protein has been reported to have greater anabolic properties than soy protein, an effect which may be attributed to the higher leucine content of whey.ObjectiveThe objective of this study was to compare postprandial myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis rates after ingestion of carbohydrate with whey, soy, or soy protein enriched with free leucine (to match the leucine content of whey) during recovery from a single bout of concurrent resistance- and endurance-type exercise in young healthy men.MethodsIn a randomized, double-blind, parallel-group design, 36 healthy young recreationally active men (mean ± SEM age: 23 ± 0.4 y) received a primed continuous infusion of l-[ring-13C6]-phenylalanine and l-[ring-3,5-2H2]-tyrosine and ingested 45 g carbohydrate with 20 g protein from whey (WHEY), soy (SOY), or leucine-enriched soy (SOY + LEU) after concurrent resistance- and endurance-type exercise. Blood and muscle biopsies were collected over a 360 min postexercise recovery period to assess MyoPS and MitoPS rates, and associated signaling through the mammalian target of rapamycin complex 1 (mTORC1).ResultsPostprandial peak plasma leucine concentrations were significantly higher in WHEY (mean ± SEM: 322 ± 10 μmol/L) and SOY + LEU (328 ± 14 μmol/L) compared with SOY (216 ± 6 μmol/L) (P < 0.05). Despite the apparent differences in plasma leucinemia, MyoPS (WHEY: 0.054 ± 0.002; SOY: 0.053 ± 0.004; SOY + LEU: 0.056 ± 0.004%·h−1; P = 0.83), and MitoPS (WHEY: 0.061 ± 0.004; SOY: 0.061 ± 0.006; SOY + LEU: 0.063 ± 0.004%·h−1; P = 0.96) rates over the entire 360 min recovery period did not differ between treatments. Similarly, signaling through mTORC1Ser2448, p70S6kThr389, 4E-BP1Thr37/46, and rpS6Ser235/236 was similar between treatments.ConclusionPostexercise MyoPS and MitoPS rates do not differ after co-ingestion of carbohydrate with 20 g protein from whey, soy, or leucine-enriched soy protein during 360 min of recovery from concurrent resistance- and endurance-type exercise in young, recreationally active men. This trial was registered at Nederlands Trial Register as NTR5098.

Skeletal Muscle Mitochondrial Protein Synthesis and Respiration Increase With Low-Load Blood Flow Restricted as Well as High-Load Resistance Training.

Purpose: It is well established that high-load resistance exercise (HLRE) can stimulate myofibrillar accretion. Additionally, recent studies suggest that HLRE can also stimulate mitochondrial biogenesis and respiratory function. However, in several clinical situations, the use of resistance exercise with high loading may not constitute a viable approach. Low-load blood flow restricted resistance exercise (BFRRE) has emerged as a time-effective low-load alternative to stimulate myofibrillar accretion. It is unknown if BFRRE can also stimulate mitochondrial biogenesis and respiratory function. If so, BFRRE could provide a feasible strategy to stimulate muscle metabolic health.Methods: To study this, 34 healthy previously untrained individuals (24 ± 3 years) participated in BFRRE, HLRE, or non-exercise control intervention (CON) 3 times per week for 6 weeks. Skeletal muscle biopsies were collected; (1) before and after the 6-week intervention period to assess mitochondrial biogenesis and respiratory function and; (2) during recovery from single-bout exercise to assess myocellular signaling events involved in transcriptional regulation of mitochondrial biogenesis. During the 6-week intervention period, deuterium oxide (D2O) was continuously administered to the participants to label newly synthesized skeletal muscle mitochondrial proteins. Mitochondrial respiratory function was assessed in permeabilized muscle fibers with high-resolution respirometry. Mitochondrial content was assessed with a citrate synthase activity assay. Myocellular signaling was assessed with immunoblotting.Results: Mitochondrial protein synthesis rate was higher with BFRRE (1.19%/day) and HLRE (1.15%/day) compared to CON (0.92%/day) (P < 0.05) but similar between exercise groups. Mitochondrial respiratory function increased to similar degree with both exercise regimens and did not change with CON. For instance, coupled respiration supported by convergent electron flow from complex I and II increased 38% with BFRRE and 24% with HLRE (P < 0.01). Training did not alter citrate synthase activity compared to CON. BFRRE and HLRE elicited similar myocellular signaling responses.Conclusion: These results support recent findings that resistance exercise can stimulate mitochondrial biogenesis and respiratory function to support healthy skeletal muscle and whole-body metabolism. Intriquingly, BFRRE produces similar mitochondrial adaptations at a markedly lower load, which entail great clinical perspective for populations in whom exercise with high loading is untenable.

Metformin inhibits mitochondrial adaptations to aerobic exercise training in older adults.

Metformin and exercise independently improve insulin sensitivity and decrease the risk of diabetes. Metformin was also recently proposed as a potential therapy to slow aging. However, recent evidence indicates that adding metformin to exercise antagonizes the exercise‐induced improvement in insulin sensitivity and cardiorespiratory fitness. The purpose of this study was to test the hypothesis that metformin diminishes the improvement in insulin sensitivity and cardiorespiratory fitness after aerobic exercise training (AET) by inhibiting skeletal muscle mitochondrial respiration and protein synthesis in older adults (62 ± 1 years). In a double‐blinded fashion, participants were randomized to placebo (n = 26) or metformin (n = 27) treatment during 12 weeks of AET. Independent of treatment, AET decreased fat mass, HbA1c, fasting plasma insulin, 24‐hr ambulant mean glucose, and glycemic variability. However, metformin attenuated the increase in whole‐body insulin sensitivity and VO2max after AET. In the metformin group, there was no overall change in whole‐body insulin sensitivity after AET due to positive and negative responders. Metformin also abrogated the exercise‐mediated increase in skeletal muscle mitochondrial respiration. The change in whole‐body insulin sensitivity was correlated to the change in mitochondrial respiration. Mitochondrial protein synthesis rates assessed during AET were not different between treatments. The influence of metformin on AET‐induced improvements in physiological function was highly variable and associated with the effect of metformin on the mitochondria. These data suggest that prior to prescribing metformin to slow aging, additional studies are needed to understand the mechanisms that elicit positive and negative responses to metformin with and without exercise.

Metformin Blunts Exercise‐Induced Improvements in Skeletal Muscle Mitochondrial Respiration Independent of Changes in Mitochondrial Biogenesis

The anti‐hyperglycemic agent Metformin and exercise independently prevent the progression from prediabetes to overt type two diabetes (T2D) and are both implicated in healthspan extension. However, emerging evidence indicates the addition of Metformin to exercise may inhibit the health benefits of exercise. The purpose of the current study was to test the hypothesis that Metformin blunts the positive adaptations to exercise by inhibiting skeletal muscle mitochondrial fractional protein synthesis rates (FSR) and respiration. Older adults (n=53, 62±5 yrs) with at least one risk factor for T2D were randomized in a double‐blinded fashion to consume Metformin or placebo (1500–2000 mg/day) during 12‐weeks of aerobic exercise training (AET). VO2max and skeletal muscle mitochondrial respiration were assessed before (PRE) and after (POST) the 12‐week intervention. Skeletal muscle biopsy samples were obtained 48 hrs after the last exercise bout and 36 hrs after the last metformin dose. High‐resolution respirometry was performed on permeabilized skeletal muscle fibers to assess submaximal and maximal oxidative phosphorylation (OXPHOS; P) primarily linked to complex‐I (CIP), fatty acid oxidation (CIP&amp;FAO), and complex II (CI+IIP&amp;FAO). Subsequent addition of FCCP determined maximal uncoupled electron transport system (ETS) capacity. During the last four weeks of the intervention, participants consumed the stable isotope deuterium oxide (2H2O) to measure cumulative FSR (%/day) in a mitochondrial enriched skeletal muscle fraction. Compared to placebo, Metformin tended (P=0.10) to attenuate the increase in VO2max after 12 weeks of AET (Metformin: 0.06±0.02 vs. Placebo: 0.13±0.03 L/min). Congruent with these findings, Metformin abrogated (P&lt;0.05) the exercise‐mediated increase in submaximal mitochondrial respiration, CIp, CI+IIP, and CI+IIP&amp;FAO after AET (Metformin: −0.7 to 2.6 vs Placebo: 5.3 to 22.8 pmol/s/mg tissue). Unlike the effect of Metformin on mitochondrial respiration, mitochondrial protein synthesis rates assessed during AET were not different between Metformin and placebo (1.06±0.05 vs. 1.00±0.04 %/day; P=0.38). Here we demonstrate that metformin tended to diminish the improvement in VO2max and inhibited the increase in skeletal muscle mitochondrial respiration after AET in older adults at risk for T2D. However, the effects on mitochondrial respiration do not appear to be related to changes in mitochondrial biogenesis. Future research is warranted to determine mechanisms by which metformin influences skeletal muscle mitochondrial respiration and impacts the health benefits of exercise.Support or Funding InformationThis study was supported by the National Dairy CouncilThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

AMP Deaminase 3 Overexpression Reduces Mitochondrial Content in C2C12 Myotubes by Decreasing PGC‐1α Promotor Activation

Skeletal muscles undergoing atrophy have decreased intracellular [ATP], PGC‐1α expression, and mitochondrial content. This combination of findings is unexpected considering that decreased [ATP] is typically associated with increases in [ADP] and [AMP], activation of AMPK and PGC‐1α, and subsequent mitochondrial biogenesis in order to increase energy production. Therefore, it is unclear why atrophying muscles display deficits in PGC‐1α and mitochondrial content despite having decreased [ATP]. A possible explanation is increased activity of the enzyme AMP Deaminase 3 (AMP→IMP+NH3), which is highly upregulated during muscle atrophy. We tested the hypothesis that increased expression of AMPD3 can reduce mitochondrial content in skeletal muscle cells by decreasing the AMPK→ PGC‐1α→ mitochondrial biogenesis intracellular signaling cascade by decreasing [AMP].MethodsC2C12 myotubes were transduced with adenovirus encoding AMPD3 or GFP (control) and allowed to differentiate further for 5 days. Myotubes were then collected and analyzed for nucleotide concentrations (UPLC), protein expression of AMPK, phosphorylated AMPK(Thr172), AMPKα substrates, mitochondrial OXPHOS proteins (Western Blot), 2 Kb PGC‐1α promotor activity (PGC‐1α‐Luciferase assay), and citrate synthase enzyme activity (marker of mitochondrial content).ResultsIncreased AMPD activity was confirmed by the presence of IMP (0.20 μmol/g) in AMPD3 groups compared to undetectable in GFP controls. Overexpression of AMPD3 decreased [ATP] by 32%, [ADP] by 19%, [AMP] by 28%, and the total adenine nucleotide pool + IMP by 31% (p&lt;0.001 for all). Notably, despite the pronounced decrease in [ATP], the [AMP]/[ATP] did not differ between groups. This was reflected by no differences in protein expression of AMPK, pAMPK(Thr172), and AMPKα substrate. However, PGC‐1α‐luciferase activity was significantly lower after 1 and 5 days of AMPD3 overexpression compared to GFP (p&lt;0.0001, p&lt;0.05), suggesting decreased PGC‐1α promotor region activation despite no measurable difference in pAMPK(Thr172). Furthermore, citrate synthase activity was 17% lower in myotubes overexpressing AMPD3 compared to GFP (p=0.01), yet no differences in mitochondrial OXPHOS proteins were detected.Summary/ConclusionsOur results show that increased expression of AMPD3 can decrease adenine nucleotide concentrations without disrupting the relative ATP:ADP:AMP ratios. Consequently, we do not detect measurable differences in AMPK activation. However, we do find significantly lower PGC‐1α promotor activity and citrate synthase activity. These findings indicate that increased expression of AMPD3, as occurs during muscle atrophy, can reduce PGC‐1α activation and mitochondrial biogenesis, ultimately resulting in reduced mitochondrial content. Interestingly, our data suggests that the reduction in PGC‐1α activity occurs independent of AMPK activation. Future studies should be conducted to determine the specific mechanism of PGC‐1α activation along with direct measures of mitochondrial biogenesis, such as, mitochondrial protein synthesis rate.Support or Funding InformationFunded by NIH R01 AR070200This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Translocation and protein complex co-localization of mTOR is associated with postprandial myofibrillar protein synthesis at rest and after endurance exercise.

Translocation and colocalization of mechanistic target of rapamycin complex 1 (mTORC1) with regulatory proteins represents a critical step in translation initiation of protein synthesis in vitro. However, mechanistic insight into the control of postprandial skeletal muscle protein synthesis rates at rest and after an acute bout of endurance exercise in humans is lacking. In crossover trials, eight endurance‐trained men received primed‐continuous infusions of L‐[ring‐2H5]phenylalanine and consumed a mixed‐macronutrient meal (18 g protein, 60 g carbohydrates, 17 g fat) at rest (REST) and after 60 min of treadmill running at 70% VO2peak (EX). Skeletal muscle biopsies were collected to measure changes in phosphorylation and colocalization in the mTORC1‐pathway, in addition to rates of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis. MyoPS increased (P < 0.05) above fasted in REST (~2.1‐fold) and EX (~twofold) during the 300 min postprandial period, with no corresponding changes in MitoPS (P > 0.05). TSC2/Rheb colocalization decreased below fasted at 60 and 300 min after feeding in REST and EX (P < 0.01). mTOR colocalization with Rheb increased above fasted at 60 and 300 min after feeding in REST and EX (P < 0.01), which was consistent with an increased phosphorylation 4E‐BP1Thr37/46 and rpS6ser240/244 at 60 min. Our data suggest that MyoPS, but not MitoPS, is primarily nutrient responsive in trained young men at rest and after endurance exercise. The postprandial increase in MyoPS is associated with an increase in mTOR/Rheb colocalization and a reciprocal decrease in TSC2/Rheb colocalization and thus likely represent important regulatory events for in vivo skeletal muscle myofibrillar mRNA translation in humans.