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 7 days) and consumed drinking water with or without 1 mM sodium nitrate. Compared with the contralateral control limb, 3 days 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 3 days decreased FSR in both SS (P = 0.009) and IMF (P = 0.031) mitochondria. Additionally, 3 days 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 7 days of immobilization. However, in contrast to 3 days of immobilization, nitrate did not prevent the decline in SS and IMF mitochondrial FSR after 7 days 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 (3 day) 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.

Higher Muscle Protein Synthesis Rates Following Ingestion of an Omnivorous Meal Compared with an Isocaloric and Isonitrogenous Vegan Meal in Healthy, Older Adults.

BACKGROUND: Plant-derived proteins are considered to have fewer anabolic properties when compared with animal-derived proteins. The anabolic properties of isolated proteins do not necessarily reflect the anabolic response to the ingestion of whole foods. The presence or absence of the various components that constitute the whole-food matrix can strongly impact protein digestion and amino acid absorption and, as such, modulate postprandial muscle protein synthesis rates. So far, no study has compared the anabolic response following ingestion of an omnivorous compared with a vegan meal. OBJECTIVES: This study aimed to compare postprandial muscle protein synthesis rates following ingestion of a whole-food omnivorous meal providing 100 g lean ground beef with an isonitrogenous, isocaloric whole-food vegan meal in healthy, older adults. METHODS: In a randomized, counter-balanced, cross-over design, 16 older (65-85 y) adults (8 males, 8 females) underwent 2 test days. On one day, participants consumed a whole-food omnivorous meal containing beef as the primary source of protein (0.45 g protein/kg body mass; MEAT). On the other day, participants consumed an isonitrogenous and isocaloric whole-food vegan meal (PLANT). Primed continuous L-[ring-13C6]-phenylalanine infusions were applied with blood and muscle biopsies being collected frequently for 6 h to assess postprandial plasma amino acid profiles and muscle protein synthesis rates. Data are presented as means ± standard deviations and were analyzed by 2 way-repeated measures analysis of variance and paired-samples t tests. RESULTS: MEAT increased plasma essential amino acid concentrations more than PLANT over the 6-h postprandial period (incremental area under curve 87 ± 37 compared with 38 ± 54 mmol·6 h/L, respectively; P-interaction < 0.01). Ingestion of MEAT resulted in ∼47% higher postprandial muscle protein synthesis rates when compared with the ingestion of PLANT (0.052 ± 0.023 and 0.035 ± 0.021 %/h, respectively; paired-samples t test: P = 0.037). CONCLUSIONS: Ingestion of a whole-food omnivorous meal containing beef results in greater postprandial muscle protein synthesis rates when compared with the ingestion of an isonitrogenous whole-food vegan meal in healthy, older adults. This study was registered at clinicaltrials.gov as NCT05151887.

Co-overexpression of CD36 and FABPpm increases fatty acid transport additively, not synergistically, within muscle.

We aimed to determine the combined effects of overexpressing plasma membrane fatty acid binding protein (FABPpm) and fatty acid translocase (CD36) on skeletal muscle fatty acid transport to establish if these transport proteins function collaboratively. Electrotransfection with either FABPpm or CD36 increased their protein content at the plasma membrane (+75% and +64%), increased fatty acid transport rates by +24% for FABPpm and +62% for CD36, resulting in a calculated transport efficiency of ∼0.019 and ∼0.053 per unit protein change for FABPpm and CD36, respectively. We subsequently used these data to determine if increasing both proteins additively or synergistically increased fatty acid transport. Cotransfection of FABPpm and CD36 simultaneously increased protein content in whole muscle (FABPpm, +46%; CD36, +45%) and at the sarcolemma (FABPpm, +41%; CD36, +42%), as well as fatty acid transport rates (+50%). Since the relative effects of changing FABPpm and CD36 content had been independently determined, we were able to a predict a change in fatty acid transport based on the overexpression of plasmalemmal transporters in the cotransfection experiments. This prediction yielded an increase in fatty acid transport of +0.984 and +1.722 pmol/mg prot/15 s for FABPpm and CD36, respectively, for a total increase of +2.96 pmol/mg prot/15 s. This calculated determination was remarkably consistent with the measured change in transport, namely +2.89 pmol/mg prot/15 s. Altogether, these data indicate that increasing CD36 and FABPpm alters fatty acid transport rates additively, but not synergistically, suggesting an independent mechanism of action within muscle for each transporter. This conclusion was further supported by the observation that plasmalemmal CD36 and FABPpm did not coimmunoprecipitate.

Dietary nitrate increases submaximal SERCA activity and ADP transfer to mitochondria in slow-twitch muscle of female mice.

Rapid oscillations in cytosolic calcium (Ca2+) coordinate muscle contraction, relaxation, and physical movement. Intriguingly, dietary nitrate decreases ATP cost of contraction, increases force production, and increases cytosolic Ca2+, which would seemingly necessitate a greater demand for sarcoplasmic reticulum Ca2+ ATPase (SERCA) to sequester Ca2+ within the sarcoplasmic reticulum (SR) during relaxation. As SERCA is highly regulated, we aimed to determine the effect of 7-day nitrate supplementation (1 mM via drinking water) on SERCA enzymatic properties and the functional interaction between SERCA and mitochondrial oxidative phosphorylation. In soleus, we report that dietary nitrate increased force production across all stimulation frequencies tested, and throughout a 25 min fatigue protocol. Mice supplemented with nitrate also displayed an ∼25% increase in submaximal SERCA activity and SERCA efficiency (P = 0.053) in the soleus. To examine a possible link between ATP consumption and production, we established a methodology coupling SERCA and mitochondria in permeabilized muscle fibers. The premise of this experiment is that the addition of Ca2+ in the presence of ATP generates ADP from SERCA to support mitochondrial respiration. Similar to submaximal SERCA activity, mitochondrial respiration supported by SERCA-derived ADP was increased by ∼20% following nitrate in red gastrocnemius. This effect was fully attenuated by the SERCA inhibitor cyclopiazonic acid and was not attributed to differences in mitochondrial oxidative capacity, ADP sensitivity, protein content, or reactive oxygen species emission. Overall, these findings suggest that improvements in submaximal SERCA kinetics may contribute to the effects of nitrate on force production during fatigue.NEW & NOTEWORTHY We show that nitrate supplementation increased force production during fatigue and increased submaximal SERCA activity. This was also evident regarding the high-energy phosphate transfer from SERCA to mitochondria, as nitrate increased mitochondrial respiration supported by SERCA-derived ADP. Surprisingly, these observations were only apparent in muscle primarily expressing type I (soleus) but not type II fibers (EDL). These findings suggest that alterations in SERCA properties are a possible mechanism in which nitrate increases force during fatiguing contractions.

Insulin rapidly increases skeletal muscle mitochondrial ADP sensitivity in the absence of a high lipid environment.

Reductions in mitochondrial function have been proposed to cause insulin resistance, however the possibility that impairments in insulin signaling negatively affects mitochondrial bioenergetics has received little attention. Therefore, we tested the hypothesis that insulin could rapidly improve mitochondrial ADP sensitivity, a key process linked to oxidative phosphorylation and redox balance, and if this phenomenon would be lost following high-fat diet (HFD)-induced insulin resistance. Insulin acutely (60 min post I.P.) increased submaximal (100-1000 µM ADP) mitochondrial respiration ∼2-fold without altering maximal (>1000 µM ADP) respiration, suggesting insulin rapidly improves mitochondrial bioenergetics. The consumption of HFD impaired submaximal ADP-supported respiration ∼50%, however, despite the induction of insulin resistance, the ability of acute insulin to stimulate ADP sensitivity and increase submaximal respiration persisted. While these data suggest that insulin mitigates HFD-induced impairments in mitochondrial bioenergetics, the presence of a high intracellular lipid environment reflective of an HFD (i.e. presence of palmitoyl-CoA) completely prevented the beneficial effects of insulin. Altogether, these data show that while insulin rapidly stimulates mitochondrial bioenergetics through an improvement in ADP sensitivity, this phenomenon is possibly lost following HFD due to the presence of intracellular lipids.

Independent of mitochondrial respiratory function, dietary nitrate attenuates HFD-induced lipid accumulation and mitochondrial ROS emission within the liver.

The liver is particularly susceptible to the detrimental effects of a high-fat diet (HFD), rapidly developing lipid accumulation and impaired cellular homeostasis. Recently, dietary nitrate has been shown to attenuate HFD-induced whole body glucose intolerance and liver steatosis, however, the underlying mechanism(s) remain poorly defined. In the current study, we investigated the ability of dietary nitrate to minimize possible impairments in liver mitochondrial bioenergetics following 8 wk of HFD (60% fat) in male C57BL/6J mice. Consumption of a HFD caused whole body glucose intolerance (P < 0.0001), and within the liver, increased lipid accumulation (P < 0.0001), mitochondrial-specific reactive oxygen species emission (P = 0.007), and markers of oxidative stress. Remarkably, dietary nitrate attenuated almost all of these pathological responses. Despite the reduction in lipid accumulation and redox stress (reduced TBARS and nitrotyrosine), nitrate did not improve insulin signaling within the liver or whole body pyruvate tolerance (P = 0.313 HFD vs. HFD + nitrate). Moreover, the beneficial effects of nitrate were independent of changes in weight gain, 5' AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) signaling, mitochondrial content, mitochondrial respiratory capacity and ADP sensitivity or antioxidant protein content. Combined, these data suggest nitrate supplementation represents a potential therapeutic strategy to attenuate hepatic lipid accumulation and decrease mitochondrial ROS emission following HFD, processes linked to improvements in whole body glucose tolerance. However, the beneficial effects of nitrate within the liver do not appear to be a result of increased oxidative capacity or mitochondrial substrate sensitivity.NEW & NOTEWORTHY The mechanism(s) for how dietary nitrate prevents high-fat diet (HFD)-induced glucose intolerance remain poorly defined. We show that dietary nitrate attenuates HFD-induced increases in lipid accumulation, mitochondrial-specific reactive oxygen species (ROS) emission, and markers of oxidative stress within the liver. The beneficial effects of nitrate were independent of changes 5' AMP-activated protein kinase signaling, mitochondrial content/respiratory capacity, or lipid-supported respiratory sensitivity. Combined, these data provide potential mechanisms underlying the therapeutic potential of dietary nitrate.

Exercise alters cardiac function independent of acute systemic inflammation in healthy men.

Acute elevations in inflammatory cytokines have been demonstrated to increase aortic and left ventricular stiffness and reduce endothelial function in healthy subjects. As vascular and cardiac functions are often transiently reduced following prolonged exercise, it is possible that cytokines released during exercise may contribute to these alterations. The a priori aims of this study were to determine whether vaccine-induced increases in inflammatory cytokines would reduce vascular and left ventricular function, whether vascular alterations would drive cardiac impairments, and whether this would be potentiated by moderate exercise. In a randomized crossover fashion, 16 male participants were tested under control (CON) and inflammatory (INF) conditions, wherein INF testing occurred 8 h following administration of an influenza vaccine. On both days, participants underwent measures of echocardiography performed during light cycling (stress-echocardiography), carotid-femoral pulse wave velocity (cf-PWV), and superficial femoral flow-mediated dilation (FMD) before and after cycling for 90 min at ∼85% of their first ventilatory threshold. IL-6 increased significantly (Δ1.9 ± 1.3 pg/mL, P < 0.001), whereas TNFα was nonsignificantly augmented (Δ0.05 ± 0.11 pg/mL, P = 0.09), 8 h following vaccination. Vascular function was unaltered following cycling or inflammation (all P > 0.05). The use of echocardiography during light cycling revealed cardiac alterations traditionally expected to occur only with greater exercise loads, with reduced systolic (e.g., longitudinal strain CON: Δ3.3 ± 4.4%, INF: Δ1.7 ± 2.7%, P = 0.002) and diastolic function (e.g., E/A ratio CON: Δ-0.32 ± 0.34 a.u., INF:Δ-0.25 ± 0.27 a.u., P = 0.002) following cycling, independent of inflammation. The vaccine reduced stroke volume (SV) (main effect of condition P = 0.009) before-and-after cycling. These findings indicate that reduced cardiac function following exercise occurs largely independent of additional inflammatory load.NEW & NOTEWORHTHY This experimental investigation sought to determine the role of inflammation on the occurrence of cardiovascular alterations following exercise. Despite successfully stimulating systemic inflammation via vaccination, vascular and cardiac functions were largely unaltered. Prolonged exercise itself reduced cardiac function assessed via echocardiography performed during light exercise stress. This demonstrates a potential advantage to using stress-echocardiography for measuring exercise-induced cardiac fatigue, as typical resting measures following similar exercise exposures commonly suggest no effect.

In vitro ketone-supported mitochondrial respiration is minimal when other substrates are readily available in cardiac and skeletal muscle.

KEY POINTS: Ketone bodies are proposed to represent an alternative fuel source driving energy production, particularly during exercise. Biologically, the extent to which mitochondria utilize ketone bodies compared to other substrates remains unknown. We demonstrate in vitro that maximal mitochondrial respiration supported by ketone bodies is low when compared to carbohydrate-derived substrates in the left ventricle and red gastrocnemius muscle from rodents, and in human skeletal muscle. When considering intramuscular concentrations of ketone bodies and the presence of other carbohydrate and lipid substrates, biological rates of mitochondrial respiration supported by ketone bodies are predicted to be minimal. At the mitochondrial level, it is therefore unlikely that ketone bodies are an important source for energy production in cardiac and skeletal muscle, particularly when other substrates are readily available. ABSTRACT: Ketone bodies (KB) have recently gained popularity as an alternative fuel source to support mitochondrial oxidative phosphorylation and enhance exercise performance. However, given the low activity of ketolytic enzymes and potential inhibition from carbohydrate oxidation, it remains unknown if KBs can contribute to energy production. We therefore determined the ability of KBs (sodium dl-ß-hydroxybutyrate, ß-HB; lithium acetoacetate, AcAc) to stimulate in vitro mitochondrial respiration in the left ventricle (LV) and red gastrocnemius (RG) of rats, and in human vastus lateralis. Compared to pyruvate, the ability of KBs to maximally drive respiration was low in isolated mitochondria and permeabilized fibres (PmFb) from the LV (∼30-35% of pyruvate), RG (∼10-30%), and human vastus lateralis (∼2-10%). In PmFb, the concentration of KBs required to half-maximally drive respiration (LV: 889 µm ß-HB, 801 µm AcAc; RG: 782 µm ß-HB, 267 µm AcAc) were greater than KB content representative of the muscle microenvironment (∼100 µm). This would predict low rates (∼1-4% of pyruvate) of biological KB-supported respiration in the LV (8-14 pmol s-1 mg-1 ) and RG (3-6 pmol s-1 mg-1 ) at rest and following exercise. Moreover, KBs did not increase respiration in the presence of saturating pyruvate, submaximal pyruvate (100 µm) reduced the ability of physiological ß-HB to drive respiration, and addition of other intracellular substrates (succinate + palmitoylcarnitine) decreased maximal KB-supported respiration. As a result, product inhibition is likely to limit KB oxidation. Altogether, the ability of KBs to drive mitochondrial respiration is minimal and they are likely to be outcompeted by other substrates, compromising their use as an important energy source.

Nitrate attenuates high fat diet-induced glucose intolerance in association with reduced epididymal adipose tissue inflammation and mitochondrial reactive oxygen species emission.

KEY POINTS: Dietary nitrate is a prominent therapeutic strategy to mitigate some metabolic deleterious effects related to obesity. Mitochondrial dysfunction is causally linked to adipose tissue inflammation and insulin resistance. Whole-body glucose tolerance is prevented by nitrate independent of body weight and energy expenditure. Dietary nitrate reduces epididymal adipose tissue inflammation and mitochondrial reactive oxygen species emission while preserving insulin signalling. Metabolic beneficial effects of nitrate consumption are associated with improvements in mitochondrial redox balance in hypertrophic adipose tissue. ABSTRACT: Evidence has accumulated to indicate that dietary nitrate alters energy expenditure and the metabolic derangements associated with a high fat diet (HFD), but the mechanism(s) of action remain incompletely elucidated. Therefore, we aimed to determine if dietary nitrate (4 mm sodium nitrate via drinking water) could prevent HFD-mediated glucose intolerance in association with improved mitochondrial bioenergetics within both white (WAT) and brown (BAT) adipose tissue in mice. HFD feeding caused glucose intolerance (P < 0.05) and increased body weight. As a result of higher body weight, energy expenditure increased proportionally. HFD-fed mice displayed greater mitochondrial uncoupling and a twofold increase in uncoupling protein 1 content within BAT. Within epididymal white adipose tissue (eWAT), HFD increased cell size (i.e. hypertrophy), mitochondrial H2 O2 emission, oxidative stress, c-Jun N-terminal kinase phosphorylation and leucocyte infiltration, and induced insulin resistance. Remarkably, dietary nitrate consumption attenuated and/or mitigated all these responses, including rendering mitochondria more coupled within BAT, and normalizing mitochondrial H2 O2 emission and insulin-mediated Akt-Thr308 phosphorylation within eWAT. Intriguingly, the positive effects of dietary nitrate appear to be independent of eWAT mitochondrial respiratory capacity and content. Altogether, these data suggest that dietary nitrate attenuates the development of HFD-induced insulin resistance in association with attenuating WAT inflammation and redox balance, independent of changes in either WAT or BAT mitochondrial respiratory capacity/content.

Short-term bed rest-induced insulin resistance cannot be explained by increased mitochondrial H2 O2 emission.

KEY POINTS: We determined if bed rest increased mitochondrially derived reactive oxygen species and cellular redox stress, contributing to the induction of insulin resistance. Bed rest decreased maximal and submaximal ADP-stimulated mitochondrial respiration. Bed rest did not alter mitochondrial H2 O2 emission in the presence of ADP concentrations indicative of resting muscle, the ratio of H2 O2 emission to mitochondrial O2 consumption or markers of oxidative stress The present data suggest strongly that mitochondrial H2 O2 does not contribute to bed rest-induced insulin resistance ABSTRACT: Mitochondrial H2 O2 has been causally linked to diet-induced insulin resistance, although it remains unclear if muscle disuse similarly increases mitochondrial H2 O2 . Therefore, we investigated the potential that an increase in skeletal muscle mitochondrial H2 O2 emission, potentially as a result of decreased ADP sensitivity, contributes to cellular redox stress and the induction of insulin resistance during short-term bed rest in 20 healthy males. Bed rest led to a decline in glucose infusion rate during a hyperinsulinaemic-euglycaemic clamp (-42 ± 2%; P < 0.001), and in permeabilized skeletal muscle fibres it decreased OXPHOS protein content (-16 ± 8%) and mitochondrial respiration across a range of ADP concentrations (-13 ± 5%). While bed rest tended to increase maximal mitochondrial H2 O2 emission rates (P = 0.053), H2 O2 emission in the presence of ADP concentrations indicative of resting muscle, the ratio of H2 O2 emission to mitochondrial O2 consumption, and markers of oxidative stress were not altered following bed rest. Altogether, while bed rest impairs mitochondrial ADP-stimulated respiration, an increase in mitochondrial H2 O2 emission does not contribute to the induction of insulin resistance following short-term bed rest.

Acute insulin deprivation results in altered mitochondrial substrate sensitivity conducive to greater fatty acid transport.

Type 1 and type 2 diabetes are both tightly associated with impaired glucose control. Although both pathologies stem from different mechanisms, a reduction in insulin action coincides with drastic metabolic dysfunction in skeletal muscle and metabolic inflexibility. However, the underlying explanation for this response remains poorly understood, particularly since it is difficult to distinguish the role of attenuated insulin action from the detrimental effects of reactive lipid accumulation, which impairs mitochondrial function and promotes reactive oxygen species (ROS) emission. We therefore utilized streptozotocin to examine the effects of acute insulin deprivation, in the absence of a high-lipid/nutrient excess environment, on the regulation of mitochondrial substrate sensitivity and ROS emission. The ablation of insulin resulted in reductions in absolute mitochondrial oxidative capacity and ADP-supported respiration and reduced the ability for malonyl-CoA to inhibit carnitine palmitoyltransferase I (CPT-I) and suppress fatty acid-supported respiration. These bioenergetic responses coincided with increased mitochondrial-derived H2O2 emission and lipid transporter content, independent of major mitochondrial substrate transporter proteins and enzymes involved in fatty acid oxidation. Together, these data suggest that attenuated/ablated insulin signaling does not affect mitochondrial ADP sensitivity, whereas the increased reliance on fatty acid oxidation in situations where insulin action is reduced may occur as a result of altered regulation of mitochondrial fatty acid transport through CPT-I.

Long-term, high-fat feeding exacerbates short-term increases in adipose mitochondrial reactive oxygen species, without impairing mitochondrial respiration.

White adipose tissue (WAT) dysfunction in obesity is implicated in the onset of whole body insulin resistance. Alterations in mitochondrial bioenergetics, namely impaired mitochondrial respiration and increased mitochondrial reactive oxygen species (mtROS) production, have been suggested to contribute to this metabolic dysregulation. However, techniques investigating mitochondrial function are classically normalized to tissue weight, which may be confounding when considering obesity-related adipocyte hypertrophy. Furthermore, the effect of long-term high-fat diet (HFD) on mtROS in WAT has yet to be elucidated. Therefore, we sought to determine the HFD-mediated temporal changes in mitochondrial respiration and mtROS emission in WAT. C57BL/6N mice received low-fat diet or HFD for 1 or 8 wk and changes in inguinal WAT (iWAT) and epididymal WAT (eWAT) were assessed. While tissue weight-normalized mitochondrial respiration was reduced in iWAT following 8-wk HFD-feeding, this effect was mitigated when adipocyte cell size and/or number were considered. These data suggest HFD does not impair mitochondrial respiratory capacity per adipocyte within WAT. In support of this assertion, within eWAT compensatory increases in lipid-supported and maximal succinate-supported respiration occurred at 8 wk despite cell hypertrophy and increases in WAT inflammation. Although these data suggest impairments in mitochondrial respiration do not contribute to HFD-mediated WAT phenotype, lipid-supported mtROS emission increased following 1-wk HFD in eWAT, while both lipid and carbohydrate-supported mtROS were increased at 8 wk in both depots. Combined, these data establish that while HFD does not impair adipocyte mitochondrial respiratory capacity, increased mtROS is an enduring physiological occurrence within WAT in HFD-induced obesity.

Low-load resistance training to task failure with and without blood flow restriction: muscular functional and structural adaptations.

The application of blood flow restriction (BFR) during resistance exercise is increasingly recognized for its ability to improve rehabilitation and for its effectiveness in increasing muscle hypertrophy and strength among healthy populations. However, direct comparison of the skeletal muscle adaptations to low-load resistance exercise (LL-RE) and low-load BFR resistance exercise (LL-BFR) performed to task failure is lacking. Using a within-subject design, we examined whole muscle group and skeletal muscle adaptations to 6 wk of LL-RE and LL-BFR training to repetition failure. Muscle strength and size outcomes were similar for both types of training, despite ~33% lower total exercise volume (load × repetition) with LL-BFR than LL-RE (28,544 ± 1,771 vs. 18,949 ± 1,541 kg, P = 0.004). After training, only LL-BFR improved the average power output throughout the midportion of a voluntary muscle endurance task. Specifically, LL-BFR training sustained an 18% greater power output from baseline and resulted in a greater change from baseline than LL-RE (19 ± 3 vs. 3 ± 4 W, P = 0.008). This improvement occurred despite histological analysis revealing similar increases in capillary content of type I muscle fibers following LL-RE and LL-BFR training, which was primarily driven by increased capillary contacts (4.53 ± 0.23 before training vs. 5.33 ± 0.27 and 5.17 ± 0.25 after LL-RE and LL-BFR, respectively, both P < 0.05). Moreover, maximally supported mitochondrial respiratory capacity increased only in the LL-RE leg by 30% from baseline (P = 0.006). Overall, low-load resistance training increased indexes of muscle oxidative capacity and strength, which were not further augmented with the application of BFR. However, performance on a muscle endurance test was improved following BFR training.

High intensity exercise inhibits carnitine palmitoyltransferase-I sensitivity to l-carnitine.

The decline in fat oxidation at higher power outputs of exercise is a complex interaction between several mechanisms; however, the influence of mitochondrial bioenergetics in this process remains elusive. Therefore, using permeabilized muscle fibers from mouse skeletal muscle, we aimed to determine if acute exercise altered mitochondrial sensitivity to (1) adenosine diphosphate (ADP) and inorganic phosphate (Pi), or (2) carnitine palmitoyltransferase-I (CPT-I) independent (palmitoylcarnitine, PC) and dependent [palmitoyl-CoA (P-CoA), malonyl-CoA (M-CoA), and l-carnitine] substrates, in an intensity-dependent manner. As the apparent ADP Km increased to a similar extent following low (LI) and high (HI) intensity exercise compared with sedentary (SED) animals, and Pi sensitivity was unaltered by exercise, regulation of phosphate provision likely does not contribute to the well-established intensity-dependent shift in substrate utilization. Mitochondrial sensitivity to PC and P-CoA was not influenced by exercise, while M-CoA sensitivity was attenuated similarly following LI and HI. In contrast, CPT-I sensitivity to l-carnitine was only altered following HI, as HI exercise attenuated l-carnitine sensitivity by ∼40%. Moreover, modeling the in vivo concentrations of l-carnitine and P-CoA during exercise suggests that CPT-I flux is ∼25% lower following HI, attributed equally to reductions in l-carnitine content and l-carnitine sensitivity. Altogether, these data further implicate CPT-I flux as a key event influencing metabolic interactions during exercise, as a decline in l-carnitine sensitivity in addition to availability at higher power outputs could impair mitochondrial fatty acid oxidation.

Blood flow restricted resistance exercise and reductions in oxygen tension attenuate mitochondrial H2 O2 emission rates in human skeletal muscle.

KEY POINTS: Blood flow restricted resistance exercise (BFR-RE) is capable of inducing comparable adaptations to traditional resistance exercise (RE), despite a lower total exercise volume. It has been suggested that an increase in reactive oxygen species (ROS) production may be involved in this response; however, oxygen partial pressure ( PO2 ) is reduced during BFR-RE, and the influence of PO2 on mitochondrial redox balance remains poorly understood. In human skeletal muscle tissue, we demonstrate that both maximal and submaximal mitochondrial ROS emission rates are acutely decreased 2 h following BFR-RE, but not RE, occurring along with a reduction in tissue oxygenation during BFR-RE. We further suggest that PO2 is involved in this response because an in vitro analysis revealed that reducing PO2 dramatically decreased mitochondrial ROS emissions and electron leak to ROS. Altogether, these data indicate that mitochondrial ROS emission rates are attenuated following BFR-RE, and such a response is likely influenced by reductions in PO2 . ABSTRACT: Low-load blood flow restricted resistance exercise (BFR-RE) training has been proposed to induce comparable adaptations to traditional resistance exercise (RE) training, however, the acute signalling events remain unknown. Although a suggested mechanism of BFR-RE is an increase in reactive oxygen species (ROS) production, oxygen partial pressure ( PO2 ) is reduced during BFR-RE, and the influence of O2 tension on mitochondrial redox balance remains ambiguous. We therefore aimed to determine whether skeletal muscle mitochondrial bioenergetics were altered following an acute bout of BFR-RE or RE, and to further examine the role of PO2 in this response. Accordingly, muscle biopsies were obtained from 10 males at rest and 2 h after performing three sets of single-leg squats (RE or BFR-RE) to failure at 30% one-repetition maximum. We determined that mitochondrial respiratory capacity and ADP sensitivity were not altered in response to RE or BFR-RE. Although maximal (succinate) and submaximal (non-saturating ADP) mitochondrial ROS emission rates were unchanged following RE, BFR-RE attenuated these responses by ∼30% compared to pre-exercise, occurring along with a reduction in skeletal muscle tissue oxygenation during BFR-RE (P < 0.01 vs. RE). In a separate cohort of participants, evaluation of mitochondrial bioenergetics in vitro revealed that mild O2 restriction (50 µm) dramatically attenuated maximal (∼4-fold) and submaximal (∼50-fold) mitochondrial ROS emission rates and the fraction of electron leak to ROS compared to room air (200 µm). Combined, these data demonstrate that mitochondrial ROS emissions are attenuated following BFR-RE, a response which may be mediated by a reduction in skeletal muscle PO2 .

Cytosolic reverse CrAT activity in cardiac tissue: potential importance for fuel selection.

The movement of lipids across mitochondrial membranes represents a rate-limiting step in fatty acid oxidation within the heart. A key regulatory point in this process is flux through carnitine palmitoyltransferase-I (CPT-I), an enzyme located on the outer mitochondrial membrane. Malonyl-CoA (M-CoA) is a naturally occurring inhibitor of CPT-I; therefore, the abundance of M-CoA has long been considered a major regulator of fatty acid oxidation. A recent paper published in the Biochemical Journal by Altamimi et al. (Biochem. J. (2018) 475, 959-976) provides evidence for a novel mechanism to produce M-CoA. Specifically, these authors identified carnitine acetyltransferase within the cytosol and further show that flux in the reverse direction forms acetyl-CoA, which is the necessary substrate for the subsequent synthesis of M-CoA. The elegant study design and intriguing data presented by Altamimi et al. provide further insights into the reciprocal regulation of substrate selection within the heart, with implications for fuel utilization and the development of cardiac diseases.

Acute oral antioxidant consumption does not alter brachial artery flow mediated dilation in young adults independent of exercise training status.

Endothelium-dependent vasodilation can be tested using a variety of shear stress paradigms, some of which may involve the production of reactive oxygen species. The purpose of this study was to compare different methods for assessing endothelial function and their specific involvement of reactive oxygen species and influence of aerobic training status. Twenty-nine (10 F) young and healthy participants (VO2max: 34-74 mL·kg-1·min-1) consumed either an antioxidant cocktail (AOC; vitamin C, vitamin E, α-lipoic acid) or placebo (PLA) on each of two randomized visits. Endothelial function was measured via three different brachial artery flow-mediated dilation (FMD) tests: reactive hyperemia (RH-FMD: 5 min cuff occlusion and release), sustained shear (SS-FMD: 6 min rhythmic handgrip), and progressive sustained shear (P-SS-FMD: three intensities of 3 min of rhythmic handgrip). Baseline artery diameter decreased (all tests: 3.8 ± 0.5 to 3.7 ± 0.6 mm, p = 0.004), and shear rate stimulus increased (during RH-FMD test, p = 0.021; during SS-FMD test, p = 0.36; during P-SS-FMD test, p = 0.046) following antioxidant consumption. However, there was no difference in FMD following AOC consumption (RH-FMD, PLA: 8.1 ± 2.6%, AOC: 8.2 ± 3.5%, p = 0.92; SS-FMD, PLA: 6.9 ± 3.9%, AOC: 7.8 ± 5.2%, p = 0.15) or FMD per shear rate slope (P-SS-FMD: PLA: 0.0039 ± 0.0035 mm·s-1, AOC: 0.0032 ± 0.0017 mm·s-1, p = 0.28) and this was not influenced by training status/fitness (all p > 0.60). Allometric scaling did not alter these outcomes (all p > 0.40). Reactive oxygen species may not be integral to endothelium-dependent vasodilation tested using reactive, sustained, or progressive shear protocols in young males and females, regardless of fitness level.