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.

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.

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.