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.

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.

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.

Dietary Nitrate and Corresponding Gut Microbiota Prevent Cardiac Dysfunction in Obese Mice.

Impaired heart function can develop in individuals with diabetes in the absence of coronary artery disease or hypertension, suggesting mechanisms beyond hypertension/increased afterload contribute to diabetic cardiomyopathy. Identifying therapeutic approaches that improve glycemia and prevent cardiovascular disease are clearly required for clinical management of diabetes-related comorbidities. Since intestinal bacteria are important for metabolism of nitrate, we examined whether dietary nitrate and fecal microbial transplantation (FMT) from nitrate-fed mice could prevent high-fat diet (HFD)-induced cardiac abnormalities. Male C57Bl/6N mice were fed a low-fat diet (LFD), HFD, or HFD+Nitrate (4 mmol/L sodium nitrate) for 8 weeks. HFD-fed mice presented with pathological left ventricle (LV) hypertrophy, reduced stroke volume, and increased end-diastolic pressure, in association with increased myocardial fibrosis, glucose intolerance, adipose inflammation, serum lipids, LV mitochondrial reactive oxygen species (ROS), and gut dysbiosis. In contrast, dietary nitrate attenuated these detriments. In HFD-fed mice, FMT from HFD+Nitrate donors did not influence serum nitrate, blood pressure, adipose inflammation, or myocardial fibrosis. However, microbiota from HFD+Nitrate mice decreased serum lipids, LV ROS, and similar to FMT from LFD donors, prevented glucose intolerance and cardiac morphology changes. Therefore, the cardioprotective effects of nitrate are not dependent on reducing blood pressure, but rather mitigating gut dysbiosis, highlighting a nitrate-gut-heart axis. ARTICLE HIGHLIGHTS: Identifying therapeutic approaches that prevent cardiometabolic diseases are clearly important, and nitrate represents one such potential compound given its multifactorial metabolic effects. We aimed to determine whether nitrate could prevent high-fat diet (HFD)-induced cardiac abnormalities and whether this was dependent on the gut microbiome. Dietary nitrate attenuated HFD-induced pathological changes in cardiac remodelling, left ventricle reactive oxygen species, adipose inflammation, lipid homeostasis, glucose intolerance, and gut dysbiosis. Fecal microbial transplantation from nitrate-fed mice also prevented serum dyslipidemia, left ventricle reactive oxygen species, glucose intolerance, and cardiac dysfunction. Therefore, the cardioprotective effects of nitrate are related to mitigating gut dysbiosis, highlighting a nitrate-gut-heart axis.

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.

Nitrate consumption preserves HFD-induced skeletal muscle mitochondrial ADP sensitivity and lysine acetylation: A potential role for SIRT1.

Dietary nitrate supplementation, and the subsequent serial reduction to nitric oxide, has been shown to improve glucose homeostasis in several pre-clinical models of obesity and insulin resistance. While the mechanisms remain poorly defined, the beneficial effects of nitrate appear to be partially dependent on AMPK-mediated signaling events, a central regulator of metabolism and mitochondrial bioenergetics. Since AMPK can activate SIRT1, we aimed to determine if nitrate supplementation (4 mM sodium nitrate via drinking water) improved skeletal muscle mitochondrial bioenergetics and acetylation status in mice fed a high-fat diet (HFD: 60% fat). Consumption of HFD induced whole-body glucose intolerance, and within muscle attenuated insulin-induced Akt phosphorylation, mitochondrial ADP sensitivity (higher apparent Km), submaximal ADP-supported respiration, mitochondrial hydrogen peroxide (mtH2O2) production in the presence of ADP and increased cellular protein carbonylation alongside mitochondrial-specific acetylation. Consumption of nitrate partially preserved glucose tolerance and, within skeletal muscle, normalized insulin-induced Akt phosphorylation, mitochondrial ADP sensitivity, mtH2O2, protein carbonylation and global mitochondrial acetylation status. Nitrate also prevented the HFD-mediated reduction in SIRT1 protein, and interestingly, the positive effects of nitrate ingestion on glucose homeostasis and mitochondrial acetylation levels were abolished in SIRT1 inducible knock-out mice, suggesting SIRT1 is required for the beneficial effects of dietary nitrate. Altogether, dietary nitrate preserves mitochondrial ADP sensitivity and global lysine acetylation in HFD-fed mice, while in the absence of SIRT1, the effects of nitrate on glucose tolerance and mitochondrial acetylation were abrogated.

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.

Ckmt1 is Dispensable for Mitochondrial Bioenergetics Within White/Beige Adipose Tissue.

Within brown adipose tissue (BAT), the brain isoform of creatine kinase (CKB) has been proposed to regulate the regeneration of ADP and phosphocreatine in a futile creatine cycle (FCC) that stimulates energy expenditure. However, the presence of FCC, and the specific creatine kinase isoforms regulating this theoretical model within white adipose tissue (WAT), remains to be fully elucidated. In the present study, creatine did not stimulate respiration in cultured adipocytes, isolated mitochondria or mouse permeabilized WAT. Additionally, while creatine kinase ubiquitous-type, mitochondrial (CKMT1) mRNA and protein were detected in human WAT, shRNA-mediated reductions in Ckmt1 did not decrease submaximal respiration in cultured adipocytes, and ablation of CKMT1 in mice did not alter energy expenditure, mitochondrial responses to pharmacological ß3-adrenergic activation (CL 316, 243) or exacerbate the detrimental metabolic effects of consuming a high-fat diet. Taken together, these findings solidify CKMT1 as dispensable in the regulation of energy expenditure, and unlike in BAT, they do not support the presence of FCC within WAT.

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.

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.

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.

Endurance and Sprint Training Improve Glycemia and V˙O2peak but only Frequent Endurance Benefits Blood Pressure and Lipidemia.

PURPOSE: Sprint interval training (SIT) has gained popularity as a time-effective alternative to moderate-intensity endurance training (END). However, whether SIT is equally effective for decreasing cardiometabolic risk factors remains debatable, as many beneficial effects of exercise are thought to be transient, and unlike END, SIT is not recommended daily. Therefore, in line with current exercise recommendations, we examined the ability of SIT and END to improve cardiometabolic health in overweight/obese males. METHODS: Twenty-three participants were randomized to perform 6 wk of constant workload SIT (3 d·wk-1, 4-6 × 30 s ~170% Wpeak, 2 min recovery, n = 12) or END (5 d·wk-1, 30-40 min, ~60% Wpeak, n = 11) on cycle ergometers. Aerobic capacity (V˙O2peak), body composition, blood pressure (BP), arterial stiffness, endothelial function, glucose and lipid tolerance, and free-living glycemic regulation were assessed pre- and posttraining. RESULTS: Both END and SIT increased V˙O2peak (END ~15%, SIT ~5%) and glucose tolerance (~20%). However, only END decreased diastolic BP, abdominal fat, and improved postprandial lipid tolerance, representing improvements in cardiovascular risk factors that did not occur after SIT. Although SIT, but not END, increased endothelial function, arterial stiffness was not altered in either group. Indices of free-living glycemic regulation were improved after END and trended toward an improvement after SIT (P = 0.06-0.09). However, glycemic control was better on exercise compared with rest days, highlighting the importance of exercise frequency. Furthermore, in an exploratory nature, favorable individual responses (V˙O2peak, BP, glucose tolerance, lipidemia, and body fat) were more prevalent after END than low-frequency SIT. CONCLUSION: As only high-frequency END improved BP and lipid tolerance, free-living glycemic regulation was better on days that participants exercised, and favorable individual responses were consistent after END, high-frequency END may favorably improve cardiometabolic health.

Vascular Function Is Differentially Altered by Distance after Prolonged Running.

PURPOSE: Ultraendurance exercise is steadily growing in popularity; however, the effect of increasingly prolonged durations of exercise on the vascular endothelium is unknown. The aim of this study was to characterize the effect of various ultramarathon running distances on vascular form and function. METHODS: We evaluated vascular endothelial function via flow-mediated dilation (FMD) in the superficial femoral artery, as well as microvascular function, inflammatory factors, and central artery stiffness, before and after participants completed 25-km (7M:2F), 50-km (11M:10F), 80-km (9M:4F), or 160-km (9M:2F) trail races all run on the same day and course. RESULTS: Completion required 149 ± 20, 386 ± 111, 704 ± 130, and 1470 ± 235 min, with corresponding average paces of 6.0 ± 0.8, 7.7 ± 2.2, 8.6 ± 1.3, and 9.6 ± 1.3 min·km-1, respectively. At baseline, there were no differences in participant characteristics across race distance groups. Shear rate stimulus trended toward an increase after the race (P = 0.07), but resting postrace artery diameter (P < 0.001) was elevated to a similar extent in all conditions. There was a reduction in FMD after the 50-km race (Δ -1.9% ± 2.2%, P < 0.01), but not the 25-km (Δ +0.3% ± 2.9%, P = 0.8), the 80-km (Δ -1.5% ± 3.2%, P = 0.1), or the 160-km (Δ +0.5% ± 2.5%, P = 0.5) race. Inflammatory markers increased most after 160 km, but arterial stiffness and microvascular function were not differently affected by race distance. CONCLUSIONS: Although the superficial femoral artery baseline diameter was larger postexercise regardless of race distance, only the 50-km race reduced FMD, whereas a short-duration higher-intensity race (25 km) and longer-duration lower-intensity races (160 km) did not. Therefore, a 50-km ultramarathon may represent the intersection between higher-intensity exercise over a prolonged duration, causing reduced endothelial function not seen in shorter or longer distances.

Alterations in Cardiac Function Following Endurance Exercise Are Not Duration Dependent.

Cardiac function has been shown to transiently decrease following prolonged exercise, with greater durations related to increased impairment. However, the prospective assessment of exercise duration on cardiac performance is rare, and the influence of relative exercise intensity is typically not assessed in relation to these changes. The aim of this study was to determine whether progressively longer running distances over the same course would elicit greater cardiac impairment. The present investigation examined cardiac alterations in 49 athletes, following trail-running races of 25, 50, 80, and 160 km, performed on the same course on the same day. Echocardiography, including conventional and speckle tracking imaging, was performed with legs-raised to 60° to mitigate alterations in preload both pre- and post-race. Race-intensities were monitored via heart rate (HR). Following the races, mean arterial pressure (Δ-11 ± 7 mmHg, P < 0.0001), and HR (Δ19 ± 14 bpm, P < 0.0001) were altered independent of race distance. Both left and right ventricular (LV and RV) diastolic function were reduced (ΔLV E/A -0.54 ± 0.49, P < 0.0001; ΔRV A' + 0.02 ± 0.04 m/s, P = 0.01) and RV systolic function decreased (ΔTAPSE -0.25 ± 0.9 cm, P = 0.01), independent of race distance. Cardiac impairment was not apparent using speckle tracking analysis with cubic spline interpolation. While race duration was unrelated to cardiac alterations, increased racing HR was related to greater RV base dilation (r = -0.37, P = 0.03). Increased time spent at higher exercise intensities was related to reduced LV ejection fraction following 25 km (r = -0.81, P = 0.03), LV systolic strain rate following 50 km (r = 0.59, P = 0.04), and TAPSE (r = -0.81, P = 0.03) following 80 km races. Increased running duration did not affect the extent of exercise-induced cardiac fatigue, however, intensity may be a greater driver of cardiac alterations.

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.

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.