Exposure to mechanical stimuli can modulate skeletal muscle structure and metabolism, yet the extent to which repeated, isolated mechanical stress promotes adaptive remodeling in humans has not been defined. We investigated whether repeated percussive massage (PM)—a widely used but poorly validated therapeutic modality—induces beneficial skeletal muscle adaptations under ambulatory conditions and whether such adaptations confer resilience during limb disuse in humans. In a 6-wk randomized trial, PM did not alter myofiber cross-sectional area, satellite cell abundance, or capillary density, but RNA-Seq pathway analysis revealed enrichment of extracellular matrix (ECM) remodeling networks, which was supported by increases in the expression of basement membrane and focal adhesion components. PM also reduced subcutaneous fat thickness and increased fatty acid-supported mitochondrial respiration while lowering mitochondrial H2O2 emission. In a separate 10-day immobilization study, PM failed to attenuate unloading-induced reductions in muscle size or strength. However, PM partially preserved fatty acid-supported respiratory capacity relative to a control group, indicating a selective metabolic resilience. Finally, in an acute mechanistic experiment, unilateral PM did not increase subcutaneous adipose tissue lipolysis, as interstitial glycerol concentrations rose similarly in treated and untreated limbs, suggesting that chronic reductions in subcutaneous fat thickness were not driven by lipolytic activation. Collectively, these findings demonstrate that repeated PM promotes targeted skeletal muscle metabolic adaptations, yet is insufficient to induce overt structural remodeling or prevent disuse-induced functional decline.

In this study, we investigated the impact of aspartame and sucrose on short-term cognitive performance. We employed a quasi-randomized-controlled, double-anonymous design, and had 130 participants undergo two study visits in a randomized order. The shorter visit included a dual-energy x-ray absorptiometry (DXA) scan, a fasting blood glucose (BG) test, and cognitive assessments from the NIH-toolbox. During the longer visit, participants underwent fasting BG testing before consuming a 500 ml beverage of either a 0.05% aspartame solution, an 11% sucrose solution, or water (with these beverage groups randomized). BG tests and cognitive assessments were completed twice more, at 20-min post-beverage consumption (20m PC) and at 60-min post-beverage consumption (60m PC). BG responses were as predicted, with participants in the water and AS groups responding similarly and remaining at fasting levels, and the sucrose group showing a spike in BG at 20m PC and returning to baseline by 60m PC (p < .0001). We found no group differences in cognitive performance at any time point (p = .6172). Cognitive test performance improved from the first test to subsequent tests regardless of beverage group (p < .0001). Although we found no group differences in short-term cognitive performance as a function of sweetener condition, more research is needed to determine the short and long-term cognitive effects of artificial sweeteners.

The menstrual cycle influences function in various tissues in the body. We sought to determine if menstrual cycle phase could influence mitochondrial function in skeletal muscle in females. Twenty-nine females with regular menstrual cycles were randomized to have a vastus lateralis muscle biopsy during either the early follicular or luteal phase. High-resolution respirometry was used to determine mitochondrial respiration on permeabilized muscle fibers. Glutamate/malate LEAK respiration was significantly higher during the luteal phase compared to the early follicular phase. Glutamate/malate/succinate LEAK respiration was the same during both menstrual cycle phases, as was maximal coupled and uncoupled respiration. There were no differences in fatty acid-supported respiration. The fatty acid-supported coupling efficiency ratios of 1-OcM (octanoylcarnitine/malate) LEAK over maximal coupled respiration and 1-OcM LEAK over maximal uncoupled respiration were both significantly higher in mitochondria from the early follicular phase than in the luteal phase. Mitochondrial H2O2 emission (glutamate/malate/succinate supported) was significantly increased in muscle from the early follicular phase. We detected no differences in mitochondrial content using citrate synthase activity between phases of the menstrual cycle. Collectively, our observations demonstrate a limited influence of the menstrual cycle on certain measures of submaximal respiration, coupling efficiencies, and H2O2 emission.

The purpose of this study was to examine the effects of 6 weeks of localized, muscle-focused (quadriceps femoris) passive heat therapy (PHT) on resistance artery function, exercise haemodynamics and exercise performance relative to knee extension (KE) exercise training (EX). We randomized 34 healthy adults (ages 18-36; n = 17 female, 17 male) to receive either PHT or sham heating sessions (120 min, 3 days/week), or EX (40 min, 3 days/week) over 6 weeks. Blood flow was assessed with Doppler ultrasound of the femoral artery during both passive leg movement (PLM) and a KE graded exercise test. Muscle biopsies were taken from the vastus lateralis at baseline and after 6 weeks. Peak blood flow during PLM increased to the same extent in both the EX (∼10.5% increase, P = 0.009) and PHT groups (∼8.5% increase, P = 0.044). Peak flow during knee extension exercise increased in EX (∼19%, P = 0.005), but did not change in PHT (P = 0.523) and decreased in SHAM (∼7%, P = 0.020). Peak vascular conductance during KE increased by ∼25% in EX (P = 0.030) and PHT (P = 0.012). KE peak power increased in EX by ∼27% (P = 0.001) but did not significantly change in PHT and SHAM groups. Expression of endothelial nitric oxide synthase increased significantly in both EX (P = 0.028) and PHT (P = 0.0095), but only EX resulted in increased angiogenesis. In conclusion, 6 weeks of localized PHT improved resistance artery function at rest and during exercise to the same extent as exercise training but did not yield significant improvements in performance. KEY POINTS: Many for whom exercise would be most beneficial are either unable to exercise or have a very low exercise tolerance. In these cases, an alternative treatment to combat declines in resistance artery function is needed. We tested the hypothesis that passive heat therapy (PHT) would increase resistance artery function, improve exercise haemodynamics and enhance exercise performance compared to a sham treatment, but less than aerobic exercise training. This report shows that 6 weeks of localized PHT improved resistance artery function at rest and during exercise to the same extent as exercise training but did not improve exercise performance. Additionally, muscle biopsy analyses revealed that endothelial nitric oxide synthase expression increased in both PHT and exercise training groups, but only exercise resulted in increased angiogenesis. Our data demonstrate the efficacy of applying passive heat as an alternative treatment to improve resistance artery function for those unable to receive the benefits of regular exercise.

AbstractThe Tour Divide (TD) is a 4385 km ultra‐endurance bicycle race that follows the continental divide from Canada to Mexico. In this case study, we performed a comprehensive molecular and physiological profile before and after the completion of the TD. Assessments were performed 35 days before the start (Pre‐TD) and ∼36 h after the finish (Post‐TD). Total energy expenditure was assessed during the first 9 days by doubly labelled water (2H218O), abdominal and leg tissue volumes via MRI, and graded exercise tests to quantify fitness and substrate preference. Vastus lateralis muscle biopsies were taken to measure mitochondrial function via respirometry, and vascular function was assessed using Doppler ultrasound. The 47‐year‐old male subject took 16 days 7 h 45 min to complete the route. He rode an average of 16.8 h/day. Neither maximal O2 uptake nor maximal power output changed pre‐ to post‐TD. Measurement of total energy expenditure and dietary recall records suggested maintenance of energy balance, which was supported by the lack of change in body weight. The subject lost both appendicular and trunk fat mass and gained leg lean mass pre‐ to post‐TD. Skeletal muscle mitochondrial and vascular endothelial function decreased pre‐ to post‐TD. Overall, exercise performance was maintained despite reductions in muscle mitochondrial and vascular endothelial function post‐TD, suggesting a metabolic reserve in our highly trained athlete.

The purpose of this study was to examine the effects of menstrual cycle phase on myofiber injury, regenerative events, and inflammation after electrical stimulation (ES)-induced myofiber damage. Twenty-eight premenopausal women (20.8 ± 2 yr) were randomized into early follicular (EF; n = 14) and late follicular (LF; n = 14) groups. After menstrual cycle tracking and phase confirmation, subjects underwent 200 electrically stimulated eccentric muscle contractions 1 wk after providing a muscle biopsy. Seven days post-ES, subjects provided a final biopsy. Primary outcomes included serum estradiol, indirect markers of muscle damage, direct indicators of myofiber necrosis and regeneration, satellite cell number, and macrophage infiltration. Women in the LF group had higher serum estradiol (122.1 ± 23.4 vs. 81.7 ± 30.8 pg/mL; P < 0.001) than in the EF group on the day of ES. Although the EF group recovered baseline maximal isometric strength by 4 days post-ES, the LF group did not. Only women in the LF group showed significant and consistent evidence of myofiber necrosis and regeneration pre- to post-ES. Despite showing more evidence of myofiber damage, women in the LF group also experienced reduced total and CD206+ macrophage infiltration relative to the EF group. Satellite cell quantity increased significantly post-ES in both groups, with no differences between groups. Collectively, the data suggest that the high-estrogen LF phase may be associated with increased susceptibility to myofiber injury while also limiting the subsequent intramuscular inflammatory response.NEW & NOTEWORTHY The menstrual cycle has widespread physiological effects across many systems, including skeletal muscle. In this study, we show that women in the late follicular phase of the menstrual cycle may be more susceptible to myofiber necrosis following electrical stimulation. We also show reduced evidence of inflammation in the late follicular phase. This is the first study to demonstrate a difference in the response of human skeletal muscle to a necrotic stimulus across a menstrual cycle.

Epidemiological data indicate that repeated heat stress improves cardiovascular health, making passive heat therapy (PHT) a potential alternative for those unable to exercise. Few studies to date have examined the potential exercise mimetic effects in humans, and it is unclear how adaptations compare in magnitude to exercise training. OBJECTIVE: To examine the effects of 6 weeks of localized, muscle-focused PHT on resistance artery vascular function, exercise hemodynamics, and exercise performance relative to the adaptations observed following high-intensity aerobic exercise training focused on the same muscles. HYPOTHESIS: 6 weeks of PHT, applied through pulsed shortwave diathermy (2 hr, 3 days/week), would increase resistance artery function, improve exercise hemodynamics, and enhance exercise performance more than a sham treatment, but less than single-leg knee extension (KE) exercise training (EX; 40 min, 3 days/week). We also hypothesized that these functional adaptations would be accompanied by increased skeletal muscle capillarity. METHODS: We randomized 34 sedentary but otherwise healthy, young adults (ages 18–36; n = 17 female, 17 male) to receive PHT, EX, or sham heating sessions (SHAM; 2 hr, 3 days/week) over 6 weeks. Vascular function was determined through the blood flow response during both a passive leg movement (PLM) assessment and a knee extension graded exercise test (GXTmax). Muscle biopsies were taken from the vastus lateralis at baseline and after 6 weeks of intervention. RESULTS: Peak muscle treatment temperature was significantly different between all groups with PHT exhibiting a higher peak temperature (~40.80°C) than those in the EX (~37.75°C, P<0.001) and SHAM groups (~36.10°C, P<0.001). Peak blood flow during PLM increased to the same extent (P=0.625) in both the EX (~10.5% increase, P=0.009) and PHT groups (~8.5% increase, P=0.044); but tended to decrease in the SHAM group (P=0.087). KE peak flow increased in EX (~19%, P=0.005), but did not change in PHT (P=0.523) and decreased in SHAM (~7%, P=0.020). Peak vascular conductance during KE significantly increased by ~25% in EX (P=0.030) and PHT (P=0.012). KE peak power increased in EX by ~27% (P=0.001) but did not significantly change in PHT(P=0.175) and SHAM groups (P=0.111). EX, but not PHT or SHAM increased muscle capillary-to-fiber ratio (P = 0.0003), capillary density (P = 0.0428), and the Capillary to Fiber Perimeter Exchange Index (P = 0.0089). CONCLUSIONS: 6 weeks of localized PHT, when applied to young healthy individuals, improved resistance artery function at rest and during exercise to the same extent as exercise training. However, PHT did not lead to increased KE peak flow, microvascular remodeling, or improved exercise performance. Therefore, PHT mimics many, but not all the vascular benefits of exercise training. Further research is necessary to determine the mechanism by which 6 weeks of PHT led to improved vascular function at rest and during exercise. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The mitochondria are central to skeletal muscle metabolic health. Impaired mitochondrial function is associated with various muscle pathologies, including insulin resistance and muscle atrophy. As a result, continuous efforts are made to find ways to improve mitochondrial health in the context of disuse and disease. While exercise is known to cause robust improvements in mitochondrial health, not all individuals are able to exercise. This creates a need for alternate interventions which elicit some of the same benefits as exercise. Passive heating (i.e., application of heat in the absence of muscle contractions) is one potential intervention which has been shown to increase mitochondrial enzyme content and activity, and to improve mitochondrial respiration. Associated with increases in mitochondrial content and/or function, passive heating can also improve insulin sensitivity in the context of type II diabetes and preserve muscle mass in the face of limb disuse. This area of research remains in its infancy, with many questions yet to be answered about how to maximize the benefits of passive heating and elucidate the mechanisms by which heat stress affects muscle mitochondria.

To examine body shape perception in 218 adults without obesity or history of eating disorders during caloric restriction (CR). Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) is a 2-year, randomized clinical trial using a 2:1 assignment (CR, 25 CR is associated with reduced concern for body shape in men and women without obesity and with no history of eating disorders. Body shape perception among this sample was complex and influenced by multiple factors. Level I, randomized controlled trial.

The beneficial effects of exercise on skeletal muscle health and function are well-established. The intracellular signaling that mediates exercise's effects on muscle are undoubtedly complex and are not entirely understood. Nonetheless, increasing adenosine monophosphate (AMP) levels during muscle contraction signals a declining energy charge in the cell and this certainly drives many of the beneficial adaptations of exercise. AMP-activated protein kinase (AMPK) is one of AMP's best-defined targets and has been described as the cell's master energy regulator. Because it is activated by exercise, AMPK has gained recent attention as an attractive pharmacological target in harnessing at least some of the benefits of exercise for those who cannot exercise due to old age or other disorders. The prodrug AICAR effectively activates AMPK as it is taken up into the cell and phosphorylated to form ZMP, an AMP mimetic. However, AICAR's therapeutic potential is severely limited by its poor pharmacokinetics. Here, we describe a novel approach to ZMP delivery in a novel prodrug (Prodrug-39; P39). We found that similar to AICAR, P39 concentrations above ~400 μM effectively achieved significant AMPK activation and glycogen depletion. Surprisingly, given AMPK's well-documented inhibition of the mechanistic target of rapamycin (mTORC1), P39 was also effective at stimulating p70S6k and rpS6 phosphorylation concentrations as low as 10 uM and early in the timecourse after administration of P39 at higher concentrations. Given this evidence, we conclude that Prodrug-39 is indeed an effective AMPK activator and may also have some utility in mTOR activation at lower concentrations.

AIM: Mild heat stress can improve mitochondrial respiratory capacity in skeletal muscle. However, long-term heat interventions are scarce, and the effects of heat therapy need to be understood in the context of the adaptations which follow the more complex combination of stimuli from exercise training. The purpose of this work was to compare the effects of 6 weeks of localized heat therapy on human skeletal muscle mitochondria to single-leg interval training. METHODS: Thirty-five subjects were assigned to receive sham therapy, short-wave diathermy heat therapy, or single-leg interval exercise training, localized to the quadriceps muscles of the right leg. All interventions took place 3 times per week. Muscle biopsies were performed at baseline, and after 3 and 6 weeks of intervention. Mitochondrial respiratory capacity was assessed on permeabilized muscle fibers via high-resolution respirometry. RESULTS: The primary finding of this work was that heat therapy and exercise training significantly improved mitochondrial respiratory capacity by 24.8 ± 6.2% and 27.9 ± 8.7%, respectively (p < 0.05). Fatty acid oxidation and citrate synthase activity were also increased following exercise training by 29.5 ± 6.8% and 19.0 ± 7.4%, respectively (p < 0.05). However, contrary to our hypothesis, heat therapy did not increase fatty acid oxidation or citrate synthase activity. CONCLUSION: Six weeks of muscle-localized heat therapy significantly improves mitochondrial respiratory capacity, comparable to exercise training. However, unlike exercise, heat does not improve fatty acid oxidation capacity.

Doxorubicin (DOX) is a common, effective chemotherapy drug. However, DOX treatment causes the negative side‐effects of skeletal muscle atrophy and weakness. One of the key mechanisms by which DOX can lead to increased atrophy is through increased mitochondrial damage and inhibition of protein synthesis via down‐regulation of mTOR. Leucine (LEU), an essential branched‐chain amino acid, activates mTOR, increases mitochondrial biogenesis, and muscle protein synthesis. It is not clear if enhanced activation of mTOR by LEU might moderate the damaging effects of DOX in skeletal muscle. The purpose of this study is to determine if LEU supplementation attenuates the loss of mitochondrial function caused by DOX. C2C12 myoblast cells were grown to confluence and then treated with 10 mM LEU, 0.5 μM DOX, or both for 24 hours. High‐resolution respirometry was performed on cells using the Oroboros O2K respirometer to assess mitochondrial function in leak, coupled, and uncoupled states. Total cells used for respiration were assessed by aliquoting a portion of cells before respirometry and performing a BCA protein assay on this representative aliquot. Citrate synthase activity was also evaluated on cells used for respirometry as a marker of mitochondrial content. We report that DOX caused reduced leak, maximum coupled respiration, and uncoupled respiration by 33%, 42%, and 38%, respectively (p<0.05). DOX also lowered citrate synthase activity by 44% (p<0.05). Total protein, measured for total cell homogenates was reduced by 63% with DOX treatment (p<0.05). Contrary to our hypothesis that LEU may mitigate the increase in cellular degradation seen with DOX treatment, there was no significant difference between DOX treatment and DOX + LEU treatment. These findings suggest DOX interferes with LEU’s ability to upregulate mTOR activity. Interestingly, the total protein content was reduced significantly more by DOX (63%) than either reductions seen in mitochondrial respiration (~40%) and citrate synthase activity (44%). These findings suggest that the effects of DOX on general cellular protein content is far greater than the negative effects observed on mitochondrial content or function.

Exercise training is known to elicit many beneficial adaptations. However, the mechanisms by which this happens are not entirely understood. During muscle contraction, increased intracellular AMP levels mediate many exercise-induced adaptations by activating AMP-activated protein kinase (AMPK), among other targets. AMP mimicry may, therefore, be an attractive pharmacological approach to stimulate some of exercise's benefits. The prodrug AICAR is a well-established AMPK activator when phosphorylated in the cell to form ZMP, an AMP mimetic. However, it has limited pharmacological potential due to poor pharmacokinetics. Prodrug 39 (P39) uses an alternative approach to deliver ZMP to the cell. The purpose of this study was to compare AMPK and P39 effects on blood glucose levels, AMPK activation, and anabolic signaling in the fasted and refed states. C57BL/6 mice were subjected to a 12 hour fast, followed by 1 hour of refeeding. Following the 12-hour fast, blood glucose was measured, and mice were injected with saline, AICAR or P39 (400 mg/kg BW). Mice were immediately transferred to cages with food, except for a fasted saline group. After 1 hour of refeeding, food was removed from cages. Blood glucose was measured 25 minutes later, and mice were sacrificed. Gastrocnemius-plantaris-soleus complex (GPS) muscles were quick-frozen for protein analysis. Fasting blood glucose levels were 80 ± 4 mg/dL prior to refeeding and were elevated to 144 ± 8 mg/dL (p<0.0001) after food reintroduction. Both AICAR and P39 caused a significant reduction in blood glucose compared to refed controls (p<0.0001, 54 ± 9, and 56 ± 10 mg/dL, respectively). In GPS muscle, AMPK phosphorylation status was unaffected by any treatment. However, ACC phosphorylation, a downstream target of AMPK, was ~3-fold higher in AICAR-treated animals compared to refed controls (p<0.01), with no significant effect of P39. Refeeding induced an increase in anabolic signaling, as indicated by an increase in S6 phosphorylation (~4-fold, p<0.01) and a trend for increased eIF4E-binding protein (4E-BP1) phosphorylation (p = 0.1). This increase was entirely prevented in AICAR and P39-treated animals. Overall, these results suggest that AICAR and P39 are similarly effective in lowering blood glucose and dampening anabolic signaling following refeeding. However, AICAR was more effective in activating AMPK signaling in skeletal muscle at this dose.

High intensity and prolonged exercise have been known to cause at least a temporary reduction in mitochondrial function. However, it is unclear how long mitochondrial function may remain impaired. Electrically stimulated eccentric muscle contractions (EEMC) may offer insights into the mechanisms by which mitochondrial function is restored following damaging muscle contractions like what is seen following high intensity exercise. The effects of EEMC on mitochondrial function have not been investigated. The aim of this study was to determine the impact that EEMC has on mitochondrial function one week following a single bout of the electrical stimulation. Muscle biopsies were taken from the vastus lateralis of female subjects one week prior and one week after EEMC. Muscle fibers were then prepared for High Resolution Respirometry and Fluorometry analysis on an Oroboros O2K Fluo-Respirometer. This approach allowed for real-time evaluation of mitochondrial function and production of reactive oxygen species (ROS). We measured mitochondrial function using two different substrate-uncoupler-inhibitor-titrations (SUITs) designed to evaluate carbohydrate-supported respiration (SUIT-1) and fatty-acid supported respiration (SUIT-2). Under SUIT-1 conditions, maximum coupled respiration (OXPHOS) was not significantly different between the pre and post EEMC biopsies. Under SUIT-2 fatty-acid supported and phosphorylating conditions (with ADP), there was a significant increase in respiratory function in the post EEMC biopsy (p=0.045). Mitochondrial ROS production was not significantly different following recovery than the baseline measures under SUIT-1 conditions. These data represent the first evaluation of mitochondrial function one week following EEMC. We also show that the previously demonstrated negative effect of high-intensity exercise on mitochondrial function may be fully recovered one week after damaging contractions and that fatty-acid supported respiration is improved at the same time point.

Heat, the major byproduct of exercise, may mediate many of the vascular adaptations that come from exercise, making heat therapy a potential alternative to exercise in situations where exercise is not possible. PURPOSE: Compare the effects of 6 weeks of exercise or heat training on resistance artery function at rest and during exercise. METHODS: Thirty-five (18 female) untrained subjects completed either 6-weeks of high-intensity, single-leg, knee-extension exercise (40 mins, 3x per week, n = 11), localized heat training (pulsed short-wave diathermy for 120 minutes, 3x per week n = 13), or a sham heat training protocol (120 minutes, 3x per week, n = 11). Vascular function was measured before and after the training. Specifically, resistance artery function was assessed at rest with the passive leg movement induced hyperemia (PLM). Leg blood flow and vascular conductance were also measured with Doppler Ultrasound and finometry during submaximal, steady-state knee extension (KE) exercise at 10 W and during a maximal graded KE exercise test. RESULTS: Resistance artery function, assessed by peak blood flow during PLM, increased in the exercise (~10.5% increase, P = 0.009) and heating groups (~8.5% increase, P = 0.044) to the same extent (P = 0.625); but tended to decrease in the sham group (P = 0.087). While blood flow during submaximal KE (10 W) did not change in any group, vascular conductance at 10 W tended to increase in the exercise group (19%, P = 0.056) and the heat group (16%, P = 0.036), but did not change in the sham group (P = 0.915). Likewise, maximum vascular conductance during KE significantly increased by ~25% in the exercise (P = 0.030) and heat (P = 0.012) groups but did not change in the sham group (P = 0.699). Importantly, the magnitude of change in resistance artery function, assessed by PLM, was related to the change in maximal vascular conductance (r = 0.50, P = 0.006). Maximum power achieved during KE only increased in the exercise group (~27%, P = 0.001). CONCLUSIONS: Localized heat therapy improved resistance artery function and exercise vascular conductance to the same extent as exercise training but did not yield significant improvements in exercise performance. Thus, heat therapy may be used to achieve many of the vascular benefits of exercise.

While changes maximum exercise blood flow and maximum coupled mitochondrial respiration were related to training-induced changes in P , the change in resistance artery function best predicted the training-induced increase in P . While not tested in this study, resistance artery function may potentially relate to P by improving the precision of oxygen delivery and reducing oxygen deficit at the start of exercise. Interventions seeking to improve P and exercise tolerance, should consider focusing on strategies to enhance resistance artery function in the exercising muscles.

Exercise training (EX) imposes stress on skeletal muscle, resulting in improved mitochondrial respiratory capacity and fat oxidation. Heat, which is one of the stresses of exercise, has been previously shown to increase mitochondrial respiration in human skeletal muscle. Still, it is unknown how the effects of localized heat therapy (HEAT) compare to the effects of EX, or if HEAT improves fat oxidation capacity. Therefore, the objective of this study was to determine the extent to which 6 weeks of HEAT improve mitochondrial function compared to EX. We hypothesized that HEAT would increase mitochondrial respiratory capacity, fatty acid oxidation and citrate synthase (CS) activity, but to a lesser extent than EX. Subjects were randomized into three groups: HEAT (n=11), EX (n=8), and a sham control group (CON, n=12). Subjects in the HEAT group were exposed to 2 hrs/day, 3 days/wk of short‐wave diathermy, which raised internal muscle temperature by 3.2 ± 0.33° C, while CON subjects experienced no change in muscle temperature during sham diathermy sessions. EX consisted of single‐leg extension exercise on a modified cycle ergometer for 45 min/day, 3 days/week, and resulted in a 1.8 ± 0.42° C increase in muscle temperature. Muscle biopsies were performed at baseline, and after 3 and 6 weeks of intervention. Small muscle fiber bundles (2‐4 mg wet wt.) were permeabilized, and mitochondrial respiration was assessed using high‐resolution respirometry (Oroboros O2k, Innsbruck, Austria). CS and 3‐hydroxyacyl‐CoA dehydrogenase (HAD) activity assays were performed in duplicate on supernatant from homogenized muscle samples. At 6 weeks, EX resulted in 27.9 ± 8.7% and 33.2 ± 11.7% increases in maximal coupled and uncoupled respiration, respectively, which were significantly different from baseline (p<0.05). At 6 weeks, HEAT resulted in 24.8 ± 6.2% and 24.5 ± 6.2% increases in maximal coupled and uncoupled respiration, respectively, which were significantly different from baseline (p<0.05), but not different from EX. Fatty acid oxidation was improved only in EX subjects after 6 weeks (29.5 ± 6.8%), compared to baseline (p<0.05), with no change in HEAT or CON subjects. CS activity increased in EX subjects (25.9 ± 10.5 µmol·min‐1·mg protein‐1 or 19.0 ± 7.4%) compared to all others (p<0.05), but no changes were observed in HEAT or CON subjects. Furthermore, no change in HAD activity occurred in any group. These results suggest that HEAT improves mitochondrial respiratory capacity to a similar extent as EX but does not result in other EX‐induced adaptations like increased fat oxidation and CS activity. Still, HEAT‐induced improvements in respiratory capacity may have therapeutic potential in the context of skeletal muscle disease or disuse, and further investigation is warranted.

Chronic inflammation is associated with low-dose oxidative stress. This exposure has previously been shown to disrupt iron regulation by downregulating iron storage and increasing iron import. As a result, the increased unbound intracellular iron is able to generate damaging free radicals via Fenton reaction chemistry. Using Glucose Oxidase and Catalase, we produced a steady-state, low-dose hydrogen peroxide exposure for human hepatocytes in the HepG2 cell line to simulate this dysregulation caused by chronic inflammation. We have previously shown that curcumin is able to attenuate the effects of this oxidative stress in mouse C2C12 myoblasts. Because of the liver's key role in human iron homeostasis, we investigated curcumin's ability to produce the same effect in HepG2 cells. Purpose To determine if curcumin can adequately prevent oxidative-stress induced iron dysregulation in human hepatocytes. Methods Cell viability was measured using MTT assays. Protein content was qualitatively compared between treatment groups using standard western blotting protocol. HepG2 cells were grown to 80% confluency and then subsequently treated with combinations of FeCl3, H2O2, and Curcumin. FeCl3 was added directly into culture media. Except during H2O2 treatments (Dulbecco's high-glucose medium, Sigma D6429), Eagle's Minimal Essential Minimum from ATCC(30-2003) was used for culture. Curcumin was also added as a bolus into the culture medium prior to treatment. Combined, 36-hour treatments were carried out as described here: Iron (0-12 hours); Iron + H2O2 (12-24 hours); Curcumin alone (24-36 hours); followed by a 2x rinse with PBS and harvesting in RIPA buffer (Millipore 20-188). Results MTT assays showed no significant cell death. Transferrin receptor (TfR) protein content is significantly decreased following 24-hour iron exposure (Control=1±0.1 SEM vs. 0.71±0.1; p<0.01; n=12). Interestingly, The addition of H2O2 to that treatment (Fe + H2O2) resulted in TfR content that showed no significant difference compared to control–likely indicating that the oxidative stress had impaired the normal cellular response to iron exposure (Control=1±0.09 SEM vs. 0.88±0.04 SEM; p=0.8; n=4). Our most recent findings showed that when these two treatments were followed by a 12-hour curcumin exposure, doses of 20 and 30 µM were able to significantly reduce TfR content when compared to control (Control=1±0.12 SEM vs. 30 µM 0.49±0.09 SEM; p=0.01; n=6). When all three treatments were blotted simultaneously for comparison, Ferritin light chain, a protein responsible for storing intracellular iron in hepatocytes, increased 13-fold (Control=1±0.21 SEM vs. 13.1±2.4 SEM; p=0.01; n=4). compared to control in the Fe + H2O2 + Curc group. This was a slight decrease from the Fe + H2O2 group (data not shown). Conclusion Our results indicate that curcumin may effectively mitigate oxidative stress on hepatocytes and subsequent iron dysregulation through altering the expression of crucial iron regulatory proteins such as TfR and FLC. We are continuing this work to determine curcumin's effects on modulating the intracellular iron concentrations as a result of this protein regulation. Importantly, our results suggest that curcumin could reduce the effects of chronic inflammation on human hepatocytes.

Purpose: Very little research has investigated the effects of ultraendurance exercise on the bioenergetic status of muscle. The primary objective of this case study was to characterize the changes that occur in skeletal muscle mitochondria in response to a 100-km ultramarathon in monozygotic twins. A second objective was to determine whether mitochondrial function is altered by consuming a periodized lowcarbohydrate, high-fat diet during training compared with a high-carbohydrate diet. Methods: One pair of male monozygotic twins ran 100 kmon treadmills after 4 wk of training on either a high-carbohydrate or periodized low-carbohydrate, high-fat diet. Muscle biopsies were collected 4 wk before the run, as well as 4 and 52 h postrun. Blood draws were also performed immediately before as well as 4 and 52 h after the run. Results: Four hours postrun, respiratory capacity, citrate synthase activity, and mitochondrial complex protein content were decreased. Two days later, both twins showed signs of rapid recovery in several of these measures. Furthermore, blood levels of creatine phosphokinase, C-reactive protein, and aspartate transaminase were elevated 4 h after the run but partially recovered 2 d later. Conclusion: Although there were some differences between the twins, the primary finding is that there is significant mitochondrial impairment induced by running 100 km, which rapidly recovers within 2 d. These results provide ample rationale for future investigations of the effects of ultraendurance activity on mitochondrial function.

The primary objective of this case study was to investigate the changes in skeletal muscle mitochondria that occur in response to a 100-km ultramarathon in monozygotic twins. Secondarily, we sought to determine if periodically consuming a low-carbohydrate, high-fat diet (LCHFD) during training would affect these changes. To accomplish these objectives, one pair of male monozygotic twins ran 100 km side-by-side on treadmills. During 8 weeks of training leading up to this event, twin 1 (T1) and twin 2 (T2) consumed a calorie and macronutrient-matched, high-carbohydrate diet, with the exception of 2 consecutive days per week for the final 4 weeks where T2 consumed a calorie and protein-matched LCHFD. Training load was matched between the subjects, most of which they completed together. The subjects were extremely well-matched in VO2 max (61.2 and 60.3 ml/kg/min), BMI (20.4 and 20.6 kg/m2), and body mass (62 and 61.9 kg). During the 100-km run, food and fluid intake were matched, as well as the pace of the run. The run took 16 hours and 3 minutes to complete. Blood draws and muscle biopsies from the vastus lateralis were collected 4 weeks prior to the run, as well as 4- and 52-hours post-run. During the last 4 weeks of training, T2 lost an average of ~1 kg of body weight (BW) over the two days of LCHFD each week (comparing BW of 4 consecutive Mondays to Wednesdays, p=0.02), which was regained two days after reintroduction of carbohydrate, consistent with fluctuating glycogen levels. Though T2 trained with periodically low glycogen levels, respiratory-exchange ratio was no different from T1 throughout the 100-km run. Four hours post-run, state 3, uncoupled, and fatty acid-supported mitochondrial respiration were reduced in T1 by 22.3%, 34.1% and 25.7%, respectively, and by 28.4%, 49.4% and 39.9% in T2, which was associated with a decrease of 15-20% in citrate synthase (CS) activity. Both twins exhibited decreases in mitochondrial complex proteins, though this was more marked in T2. Blood CPK, CRP, and AST levels were also elevated post-run. Possibly the most novel finding from this study was that 52 hours post-run, respiratory capacity, mitochondrial proteins, CS activity and blood markers showed signs of rapid recovery. It is currently unclear by what mechanism mitochondrial respiration and content was reduced 4 hours post-run, or how it recovered in 2 days. Interestingly, maximal mitochondrial ROS production (glutamate, malate and succinate-supported) was decreased post-run at both timepoints but was increased compared to baseline with the addition of ADP (12.5 μM – 100 μM). This suggests that the mitochondria were less sensitive to ADP-induced reductions in ROS. Neither diet resulted in better preservation of mitochondrial function 4-hours post-run, though T2 showed some signs of supercompensation 52 hours-post run. This study is valuable because it provides evidence of an acute reduction in mitochondrial content and function following ultra-endurance activity, followed by rapid recovery. This study was approved by the Brigham Young University Institutional Review Board and conformed to the principles of human experimentation set by the Declaration of Helsinki.