Mitochondrial Protein Content Research Articles

Impact of Branched Chain Amino Acid Supplementation on Hepatic Mitochondrial Metabolism in Mice with Non-alcoholic Fatty Liver Disease (P08-136-19)

ObjectivesElevated circulating branched chain amino acid (BCAA) levels are correlated with the development of insulin resistance and type 2 diabetes mellitus in humans. On the other hand, BCAA supplementation has been shown to be beneficial in improving insulin resistance during chronic liver disease. Furthermore, there is recent evidence of significant crosstalk between BCAA and mitochondrial lipid metabolism. Considering the central role of dysfunctional mitochondrial lipid metabolism in diet-induced obesity and non-alcoholic fatty liver disease (NAFLD), our objective was to determine the impact of dietary BCAA supplementation on hepatic mitochondrial function, in a diet-induced mouse model of NAFLD. We hypothesized that the dietary supplementation of BCAA, together with a high fat diet, will exacerbate hepatic mitochondrial dysfunction during NAFLD. MethodsC57BL/6NJ mice were fed with a control (10% kcal fat), high fat (HF; 60% kcal fat), or HF diet supplemented with BCAA (BA; 60% kcal fat; 1.5X BCAA) diet for 16 weeks. Livers from these mice were used for mitochondrial isolation, total liver and mitochondrial protein estimation, determination of amino acids and Kreb’s cycle intermediates by mass spectrometry, and gene expression profiles. ResultsWhile the liver weights (g ± SEM) of HF fed mice (2.6 ± 0.36) were significantly higher than the control mice (1.5 ± 0.19), BCAA supplemented mice had significantly lower liver weights than the HF fed mice (1.8 ± 0.28). Hepatic mitochondrial protein content (μg/g liver ± SEM) was enriched in BCAA supplemented mice compared to their HF diet fed counterparts (BA, 3858 ± 476; HF, 2635 ± 394; P ˂ 0.05). Many of the organic acid intermediates of the Kreb’s cycle were significantly lower in the liver of HF fed mice. Interestingly, BCAA supplementation (BA) with HF feeding restored hepatic organic acid intermediates to similar levels observed in control mice. Further, HF and BA feeding, both downregulated lipogenic gene expression (e.g., Fasn, Scd1, Acly) in the liver. ConclusionsOur results suggest that BCAA supplementation enhanced hepatic mitochondrial biogenesis and oxidative metabolism in the liver of mice with NAFLD. High-fat diets which significantly suppressed the rates of de novo lipogenesis, could have provided the BCAAs, a metabolic milieu favorable for the induction of mitochondrial activity. Funding SourcesNational Institutes of Health (R01).

High-intensity exercise training enhances mitochondrial oxidative phosphorylation efficiency in a temperature-dependent manner in human skeletal muscle: implications for exercise performance.

The purpose of the present study was to investigate whether exercise training-induced adaptations in human skeletal muscle mitochondrial bioenergetics are magnified under thermal conditions resembling sustained intense contractile activity and whether training-induced changes in mitochondrial oxidative phosphorylation (OXPHOS) efficiency influence exercise efficiency. Twenty healthy men performed 6 wk of high-intensity exercise training [i.e., speed endurance training (SET; n = 10)], or maintained their usual lifestyle (n = 10). Before and after the intervention, mitochondrial respiratory function was determined ex vivo in permeabilized muscle fibers under experimentally-induced normothermia (35°C) and hyperthermia (40°C) mimicking in vivo muscle temperature at rest and during intense exercise, respectively. In addition, activity and content of muscle mitochondrial enzymes and proteins were quantified. Exercising muscle efficiency was determined in vivo by measurements of leg hemodynamics and blood parameters during one-legged knee-extensor exercise. SET enhanced maximal OXPHOS capacity and OXPHOS efficiency at 40°C, but not at 35°C, and attenuated hyperthermia-induced decline in OXPHOS efficiency. Furthermore, SET increased expression of markers of mitochondrial content and up-regulated content of MFN2, DRP1, and ANT1. Also, SET improved exercise efficiency and capacity. These findings indicate that muscle mitochondrial bioenergetics adapts to high-intensity exercise training in a temperature-dependent manner and that enhancements in mitochondrial OXPHOS efficiency may contribute to improving exercise performance.-Fiorenza, M., Lemminger, A. K., Marker, M., Eibye, K., Iaia, F. M., Bangsbo, J., Hostrup, M. High-intensity exercise training enhances mitochondrial oxidative phosphorylation efficiency in a temperature-dependent manner in human skeletal muscle: implications for exercise performance.

Expression of Genes that Comprise the Core Molecular Clock are Altered in the Atrophied Skeletal Muscle by Androgen Deprivation

Skeletal muscle atrophy increases the risk of morbidity and mortality during various pathological conditions. In males, a decrease in the production and/or bioavailability of androgens (termed hypogonadism) directly contributes to muscle atrophy during various pathological conditions. While it is known that androgens prevent muscle atrophy, the mechanism(s) by which androgens mediate this effect are largely undefined. Our laboratory previously showed that mitochondrial turnover is enhanced in the tibialis anterior (TA) muscle of mice by androgen deprivation induced by castration surgery, and the magnitude of turnover was related to the degree of muscle atrophy. These data suggest that potentially dysfunctional mitochondria contribute to the muscle atrophy observed following androgen deprivation. To gain a better understanding of that factors that might contribute to changes in mitochondrial quality control during androgen deprived conditions, we subjected total RNA from the TA of sham and castrated mice to microarray analysis. This unbiased approach identified significant changes in expression of genes that comprise the core molecular clock. qRT‐PCR confirmed that expression of Brain and Muscle Arntl 1 (Bmal1) was decreased, while expression of Period 1, Period 2, and Period 3 (Per1, 2, & 3) were increased in the TA of castrated mice. When measured across a diurnal cycle, the change in expression of Bmal1, Per1, and Per2 exhibited reduced amplitude under androgen‐deprived conditions. Interestingly, strong relationships were observed between the castration‐mediated changes of core clock components and the measures of mitochondrial turnover. Specifically, Bmal1 expression was directly related to BCL2/adenovirus E1B 19 kDA protein‐interacting protein 3 (BNIP3) protein content (R2 = 0.88), and the expression of core clock components were also directly related to the content of various mitochondrial proteins. Expression of core clock components were also related to the autophagy marker, p62, and the mass of the TA. Ex post facto analysis of the microarray also identified changes in genes regulating polyamine biosynthesis. As polyamines are known to alter core clock function, we determined whether androgen deprivation altered polyamine content. While castration did not alter Spermine content, there was a significant reduction in the content of Spermidine in the TA of castrated mice. Overall, these data suggest that reduced Spermidine concentrations may contribute to alterations in the core molecular clock in the skeletal muscle under androgen‐deprived conditions, which may in turn contribute to reduced mitochondrial quality control and subsequent muscle atrophySupport or Funding InformationNIH‐CA204345 (RAC) and Institute for Successful Longevity (BSG)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Examining the effects of housing temperature on mitochondrial adaptations to exercise and the browning of white adipose tissue

The thermoneutral zone for small rodents is ~29–31°C and with decreases in ambient temperature below this there are consequent increases in metabolism to promote thermogenesis. Despite this, many studies report housing mice at room temperature for the convenience of the researchers studying them. As such, the conflicting reports between human and rodent models of exercise‐induced mitochondrial biogenesis in adipose tissue and the browning of white adipose tissue may be explained by the often‐overlooked variable, housing temperature. Male 8‐week‐old C57BL/6 mice were housed in cages with (VWR) or without (SED) running wheels (n = 12/group/condition) for 5–6 weeks at room temperature (RT; 22°C) or at thermoneutrality (TN; ~29°C). Mice housed at TN ran a greater distance. VWR protected against weight gain independent of housing temperature however food intake was greater in both VWR and SED mice housed at RT compared to TN. Inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) mass were reduced following VWR independent of housing temperature and there were main effects of both exercise and temperature to reduce brown adipose tissue (BAT) mass. There was no effect of housing temperature on circulating glucose or glycerol concentrations however NEFA, insulin, and the HOMA‐IR were increased at TN. In eWAT there was a main effect of exercise to increase cytochrome c oxidoreductase core I subunit (CORE1), cytochrome c oxidase, and peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) protein content with a main effect of housing temperature for PGC‐1α as well. There was a strong trend for an exercise‐induced increase in pyruvate dehydrogenase (PDH). In iWAT, there was a main effect of exercise and temperature on mitochondrial proteins with VWR leading to the expected increases in PDH, CORE1, COX IV, and PGC‐1α, but reduced markers of mitochondrial content at TN. Most notably protein content of the thermogenic uncoupling protein‐1 (UCP‐1) was undetectable in iWAT at TN. Qualitative histological analysis revealed patches of multilocular adipocytes in iWAT with VWR at RT but not at TN. Together our findings provide novel evidence that while housing mice at TN reduces markers of mitochondrial content in adipose tissue under sedentary conditions, the ability of exercise to increase mitochondrial protein content remains largely intact.Support or Funding InformationOntario Graduate Scholarship Canada Research Chairs ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inclusion of sprints in moderate intensity continuous training leads to muscle oxidative adaptations in trained individuals.

This study examined adaptations in muscle oxidative capacity and exercise performance induced by two work‐ and duration‐matched exercise protocols eliciting different muscle metabolic perturbations in trained individuals. Thirteen male subjects (V˙O2‐max 53.5 ± 7.0 mL·kg−1·min−1) (means ± SD) performed 8 weeks (three sessions/week) of training consisting of 60 min of moderate intensity continuous cycling (157 ± 20 W) either without (C) or with (C+S) inclusion of 30‐s sprints (473 ± 79 W) every 10 min. Total work performed during training was matched between groups. Muscle biopsies and arm venous blood were collected before as well as immediately and 2 h after exercise during the first and last training session. Plasma epinephrine and lactate concentrations after the first and last training session were 2–3‐fold higher in C+S than in C. After the first and last training session, muscle phosphocreatine and pH were lower (12–25 mmol·kg d.w.−1 and 0.2–0.4 units, respectively) and muscle lactate higher (48‐64 mmol·kg d.w.−1) in C+S than in C, whereas exercise‐induced changes in muscle PGC‐1α mRNA levels were similar within‐ and between‐groups. Muscle content of cytochrome c oxidase IV and citrate synthase (CS) increased more in C+S than in C, and content of CS in type II muscle fibers increased in C+S only (9–17%), with no difference between groups. Performance during a 45‐min time‐trial improved by 4 ± 3 and 9 ± 3% in C+S and C, respectively, whereas peak power output at exhaustion during an incremental test increased by 3 ± 3% in C+S only, with no difference between groups. In conclusion, addition of sprints in moderate intensity continuous exercise causes muscle oxidative adaptations in trained male individuals which appear to be independent of the exercise‐induced PGC‐1α mRNA response. Interestingly, time‐trial performance improved similarly between groups, suggesting that changes in content of mitochondrial proteins are of less importance for endurance performance in trained males.

The combined effect of sprint interval training and postexercise blood flow restriction on critical power, capillary growth, and mitochondrial proteins in trained cyclists

Sprint interval training (SIT) combined with postexercise blood flow restriction (BFR) is a novel method to increase maximal oxygen uptake (V̇o2max) in trained individuals and also provides a potent acute stimulus for angiogenesis and mitochondrial biogenesis. The efficacy to enhance endurance performance, however, has yet to be demonstrated. Trained male cyclists ( n = 21) (V̇o2max: 62.8 ± 3.7 ml·min-1·kg-1) undertook 4 wk of SIT (repeated 30-s maximal sprints) either alone (CON; n = 10) or with postexercise BFR ( n = 11). Before and after training V̇o2max, critical power (CP) and curvature constant ( W') were determined and muscle biopsies obtained for determination of skeletal muscle capillarity and mitochondrial protein content. CP increased ( P = 0.001) by a similar extent following CON (287 ± 39 W to 297 ± 43 W) and BFR (296 ± 40 W to 306 ± 36 W). V̇o2max increased following BFR by 5.9% ( P = 0.02) but was unchanged after CON ( P = 0.56). All markers of skeletal muscle capillarity and mitochondrial protein content were unchanged following either training intervention. In conclusion, 4 wk of SIT increased CP; however, this was not enhanced further with BFR. SIT was not sufficient to elicit changes in skeletal muscle capillarity and mitochondrial protein content with or without BFR. However, we further demonstrate the potency of combining BFR with SIT to enhance V̇o2max in trained individuals. NEW & NOTEWORTHY This investigation has demonstrated that 4 wk of sprint interval training (SIT) increased critical power in trained individuals; however, postexercise blood flow restriction (BFR) did not enhance this further. SIT, with or without BFR, did not induce any changes in skeletal muscle capillarity or mitochondrial protein content in our trained population. We do, however, confirm previous findings that SIT combined with BFR is a potent stimulus to enhance maximal oxygen uptake.

(-)-Epicatechin Supplementation Inhibits Aerobic Adaptations to Cycling Exercise in Humans.

The purpose of the study was to determine if cycling exercise combined with (–)-epicatechin supplementation was more effective at increasing training adaptations than cycling combined with a placebo. Blood and muscle samples were obtained at rest before and after training to determine the effects of (–)-epicatechin supplementation on total serum antioxidant capacity, skeletal muscle mitochondrial protein content, and skeletal muscle myostatin gene expression. Participants (n = 20) completed two testing sessions separated by 4 weeks of cycle training, with supplementation of 100 mg (200 mg total daily) of (–)-epicatechin or a placebo, twice daily. Data were analyzed using a two-way mixed model ANOVA for each variable and the alpha level was set at p ≤ 0.05. A significant increase was observed for time for relative peak anaerobic power (p < 0.01), relative anaerobic capacity (p < 0.01), and fatigue index (p < 0.01). A significant increase was observed for time for absolute peak VO2 (p < 0.01) and peak power output obtained during the peak VO2 test (p < 0.01). A significant interaction between group and time for relative peak VO2 was observed (p = 0.04). Relative peak VO2 significantly increased over time in the placebo group (p < 0.01), but not in the (–)-epicatechin group (p = 0.21). A significant increase was observed for time for total serum antioxidant capacity (p = 0.01). No interaction or main effect of time was observed for myostatin (p > 0.05). Likewise, no interaction or main effect of time was observed for cytochrome C or citrate synthase (p > 0.05). A significant interaction effect was observed for succinate dehydrogenase (SDH; p = 0.02). SDH content increased significantly for the placebo group (p = 0.03, partial η2 = 0.59), but not for the (–)-epicatechin group (p = 0.81). Further, whereas no difference existed between the groups for SDH at baseline (p = 0.23), SDH content was significantly greater in the placebo group at the post time point (p = 0.01). Results indicate that (–)-epicatechin supplementation does not affect myostatin gene expression or anaerobic training adaptations but inhibits aerobic and mitochondrial SDH adaptations to cycle exercise training.

Peripheral blood mononuclear cells do not reflect skeletal muscle mitochondrial function or adaptation to high-intensity interval training in healthy young men.

Measurement of skeletal muscle mitochondrial respiration requires invasive biopsy to obtain a muscle sample. Peripheral blood mononuclear cell (PBMC) mitochondrial protein content appears to reflect training status in young men; however, no studies have investigated whether there are training-induced changes in PBMC mitochondrial respiration. Therefore, we determined whether PBMC mitochondrial respiration could be used as a marker of skeletal muscle mitochondrial respiration in young healthy men and whether PBMC mitochondrial respiration responds to short-term training. Skeletal muscle and PBMC samples from 10 healthy young (18-35 yr) male participants were taken before and after a 2-wk high-intensity interval training protocol. High-resolution respirometry was used to determine mitochondrial respiration from muscle and PBMCs, and Western blotting and quantitative PCR were used to assess mitochondrial biogenesis in PBMCs. PBMC mitochondrial respiration was not correlated with muscle mitochondrial respiration at baseline ( R2 = 0.012-0.364, P > 0.05). While muscle mitochondrial respiration increased in response to training (32.1-61.5%, P < 0.05), PBMC respiration was not affected by training. Consequently, PBMCs did not predict training effect on muscle mitochondrial respiration ( R2 = 0.024-0.283, P > 0.05). Similarly, gene and protein markers of mitochondrial biogenesis did not increase in PBMCs following training. This suggests PBMC mitochondrial function does not reflect that of skeletal muscle and does not increase following short-term high-intensity training. PBMCs are therefore not a suitable biomarker for muscle mitochondrial function in young healthy men. It may be useful to study PBMC mitochondrial function as a biomarker of muscle mitochondrial function in pathological populations with different respiration capacities. NEW & NOTEWORTHY Research in primates has suggested that peripheral blood mononuclear cells (PBMCs) may provide a less-invasive alternative to a muscle biopsy for measuring muscle mitochondrial function. Furthermore, trained individuals appear to have greater mitochondrial content in PBMCs. Here we show that in healthy young men, PBMCs do not reflect skeletal muscle mitochondrial function and do not adapt in response to a training intervention that increases muscle mitochondrial function, suggesting PBMCs are a poor marker of muscle mitochondrial function in humans.

Mitochondrial Degeneration, Depletion of NADH, and Oxidative Stress Decrease Color Stability of Wet-Aged Beef Longissimus Steaks.

Interrelationship between mitochondria and myoglobin function influence beef color. NADH level in postmortem muscle is an important determinant of mitochondrial activity and metmyoglobin reduction. Increased aging time promotes discoloration of steaks; however, the mechanism of this effect is not clear. The objective was to characterize the role of wet-aging in beef longissimus lumborum muscle mitochondrial function and to characterize the global metabolome to determine the mechanism of that can regenerate NADH. Beef longissimus lumborum muscles were randomly assigned to 3, 7, 14, 21, and 28 days aging periods. Surface color, biochemical, mitochondrial, and metabolite profiles were determined at each aging period and at the end of 6-day display. During 6-day display, sections aged for 28 days had 30.4% decrease in redness than sections aged for 3 days. Aging time decreased (P <0.05) muscle oxygen consumption, mitochondrial protein content, and antioxidant capacity. Metabolites such as fumaric acid, creatinine, and fructose, that can take part in glycolytic/TCA cycle and regenerate NADH decreased (P <0.05) with aging and display time. In support, NADH levels also decreased (P <0.05) with aging time, but aging time had no effect (P = 0.44) on NADH-dependent reductase activity. The results suggest that decreased color stability in aged beef can be attributed to increased mitochondrial damage, depletion of metabolites that can regenerate NADH, and increased oxidative stress. PRACTICAL APPLICATION: Beef aging time results in increased discoloration of steaks under retail display. The current research determines the fundamental basis of lower color stability in aged beef. The results indicate that mitochondrial degeneration, depletion of metabolites that produce NADH, and increased oxidative stress can limit shelf-life of aged steaks. Hence, application of post-harvest strategies to minimize mitochondrial damage and oxidative changes may have the potential to increase shelf-life of aged beef.

The HDAC3 enzymatic activity regulates skeletal muscle fuel metabolism.

Histone deacetylase 3 (HDAC3) is a major HDAC, whose enzymatic activity is targeted by small molecule inhibitors for treating a variety of conditions. However, its enzymatic activity is largely dispensable for its function in embryonic development and hepatic lipid metabolism. HDAC3 plays a pivotal role in regulating muscle fuel metabolism and contractile function. Here, we address whether these muscular functions of HDAC3 require its enzymatic activity. By mutating the NCoR/SMRT corepressors in a knock-in mouse model named NS-DADm, we ablated the enzymatic activity of HDAC3 without affecting its protein levels. Compared to the control mice, skeletal muscles from NS-DADm mice showed lower force generation, enhanced fatigue resistance, enhanced fatty acid oxidation, reduced glucose uptake during exercise, upregulated expression of metabolic genes involved in branched-chain amino acids catabolism, and reduced muscle mass during aging, without changes in the muscle fiber-type composition or mitochondrial protein content. These muscular phenotypes are similar to those observed in the HDAC3-depleted skeletal muscles, which demonstrates that, unlike that in the liver or embryonic development, the metabolic function of HDAC3 in skeletal muscles requires its enzymatic activity. These results suggest that drugs specifically targeting HDAC3 enzyme activity could be developed and tested to modulate muscle energy metabolism and exercise performance.

Mitochondrial-derived reactive oxygen species influence ADP sensitivity, but not CPT-I substrate sensitivity.

The mechanisms regulating oxidative phosphorylation during exercise remain poorly defined; however, key mitochondrial proteins, including carnitine palmitoyltransferase-I (CPT-I) and adenine nucleotide translocase, have redox-sensitive sites. Interestingly, muscle contraction has recently been shown to increase mitochondrial membrane potential and reactive oxygen species (ROS) production; therefore, we aimed to determine if mitochondrial-derived ROS influences bioenergetic responses to exercise. Specifically, we examined the influence of acute exercise on mitochondrial bioenergetics in WT (wild type) and transgenic mice (MCAT, mitochondrial-targeted catalase transgenic) possessing attenuated mitochondrial ROS. We found that ablating mitochondrial ROS did not alter palmitoyl-CoA (P-CoA) respiratory kinetics or influence the exercise-mediated reductions in malonyl CoA sensitivity, suggesting that mitochondrial ROS does not regulate CPT-I. In contrast, while mitochondrial protein content, maximal coupled respiration, and ADP (adenosine diphosphate) sensitivity in resting muscle were unchanged in the absence of mitochondrial ROS, exercise increased the apparent ADP Km (decreased ADP sensitivity) ∼30% only in WT mice. Moreover, while the presence of P-CoA decreased ADP sensitivity, it did not influence the basic response to exercise, as the apparent ADP Km was increased only in the presence of mitochondrial ROS. This basic pattern was also mirrored in the ability of ADP to suppress mitochondrial H2O2 emission rates, as exercise decreased the suppression of H2O2 only in WT mice. Altogether, these data demonstrate that while exercise-induced mitochondrial-derived ROS does not influence CPT-I substrate sensitivity, it inhibits ADP sensitivity independent of P-CoA. These data implicate mitochondrial redox signaling as a regulator of oxidative phosphorylation.

Long-Term Effects of Metabolic Stress on Human Malignant Melanoma Cell Line

Abstract: Introduction: Tumour is a heterogeneous tissue consisting of cells with different levels of metabolic activity. Often the outer layer cells of a tumour have more optimal conditions to grow/proliferate compared to the inner cells. Objective: The aim of the current study was to study the reactions of malignant melanoma A375 cells in exposure to different levels of metabolic stress and their ability to returning to life upon re-exposure to optimum nutritional conditions. Methods: A375 cells, at early plateau phase, were exposed to media containing 10% (control), 0.5, 0.25 and 0% serum for 1 to 6 days. At 24 h intervals, the cells were tested for morphology, cell cycle distribution, cell size changes, cell count, mitochondrial function and protein content. Also, after each day of starvation, the cells were re-exposed to optimum media (10% serum) and tested again. Results: The results showed that the cells made foci structures at long-term starvation. They primarily accumulated in G1-phase and at long-term starvation, most of them entered the apoptotic state. However, before death, cell count did not decrease, even though the mitochondrial function and protein content were less than the control cells. Releasing the cells in optimum conditions could trigger cells' proliferation ability, mitochondrial function and protein content. Conclusion: The results suggest that this model of malignant melanoma has a resistant feature against sub-optimal nutritional and metabolic status and the cells can return to life with even stronger internal engines, as evidenced by higher levels of mitochondrial function and protein content. Key words: A375, Neoplasm, Serum, Starvation, Metabolic Stress.

The multilevel antibiotic-induced perturbations to biological systems: Early-life exposure induces long-lasting damages to muscle structure and mitochondrial metabolism in flies

Antibiotics have been increasingly used over the past decades for human medicine, food-animal agriculture, aquaculture, and plant production. A significant part of the active molecules of antibiotics can be released into the environment, in turn affecting ecosystem functioning and biogeochemical processes. At lower organizational scales, these substances affect bacterial symbionts of insects, with negative consequences on growth and development of juveniles, and population dynamics. Yet, the multiple alterations of cellular physiology and metabolic processes have remained insufficiently explored in insects. We evaluated the effects of five antibiotics with different mode of action, i.e. ampicillin, cefradine, chloramphenicol, cycloheximide, and tetracycline, on the survival and ultrastructural organization of the flight muscles of newly emerged blow flies Chrysomya albiceps. Then, we examined the effects of different concentrations of antibiotics on mitochondrial protein content, efficiency of oxidative phosphorylation, and activity of transaminases (Glutamate oxaloacetate transaminase and glutamate pyruvate transaminase) and described the cellular metabolic perturbations of flies treated with antibiotics. All antibiotics affected the survival of the insects and decreased the total mitochondrial protein content in a dose-dependent manner. Ultrastructural organization of flight muscles in treated flies differs dramatically compared to the control groups and severe pathological damages/structures disorganization of mitochondria appeared. The activities of mitochondrial transaminases significantly increased with increased antibiotic concentrations. The oxidation rate of pyruvate + proline from isolated mitochondria of the flight muscles of 1-day-old flies was significantly reduced at high doses of antibiotics. In parallel, the level of several metabolites, including TCA cycle intermediates, was reduced in antibiotics-treated flies. Overall, antibiotics provoked a system-wide alteration of the structure and physiology of flight muscles of the blow fly Ch. albiceps, and may have fitness consequences at the organism level. Environmental antibiotic pollution is likely to have unwanted cascading ecological effects of insect population dynamics and community structure.

Perspective on AMD Pathobiology: A Bioenergetic Crisis in the RPE.

AMD is the leading cause of blindness in developed countries. The dry form of AMD, also known as atrophic AMD, is characterized by the death of RPE and photoreceptors. Currently, there are no treatments for this form of the disease due in part to our incomplete understanding of the mechanism causing AMD. Strong experimental evidence from studies of human donors with AMD supports the emerging hypothesis that defects in RPE mitochondria drive AMD pathology. These studies, using different experimental methods, have shown disrupted RPE mitochondrial architecture and decreased mitochondrial number and mass, altered content of multiple mitochondrial proteins, increased mitochondrial DNA damage that correlates with disease severity, and defects in bioenergetics for primary RPE cultures from AMD donors. Herein, we discuss a model of metabolic uncoupling that alters bioenergetics in the diseased retina and drives AMD pathology. These data provide the rationale for targeting the mitochondria in the RPE as the most efficacious intervention strategy if administered early, before vision loss and cell death.

Synthesis of Novel Aza-aromatic Curcuminoids with Improved Biological Activities towards Various Cancer Cell Lines.

Curcumin, a natural compound extracted from the rhizomes of Curcuma longa, displays pronounced anticancer properties but lacks good bioavailability and stability. In a previous study, we initiated structure modification of the curcumin scaffold by imination of the labile β‐diketone moiety to produce novel β‐enaminone derivatives. These compounds showed promising properties for elaborate follow‐up studies. In this work, we focused on another class of nitrogen‐containing curcuminoids with a similar objective: to address the bioavailability and stability issues and to improve the biological activity of curcumin. This paper thus reports on the synthesis of new pyridine‐, indole‐, and pyrrole‐based curcumin analogues (aza‐aromatic curcuminoids) and discusses their water solubility, antioxidant activity, and antiproliferative properties. In addition, multivariate statistics, including hierarchical clustering analysis and principal component analysis, were performed on a broad set of nitrogen‐containing curcuminoids. Compared to their respective mother structures, that is, curcumin and bisdemethoxycurcumin, all compounds, and especially the pyridin‐3‐yl β‐enaminone analogues, showed better water solubility profiles. Interestingly, the pyridine‐, indole‐, and pyrrole‐based curcumin derivatives demonstrated improved biological effects in terms of mitochondrial activity impairment and protein content, in addition to comparable or decreased antioxidant properties. Overall, the biologically active N‐alkyl β‐enaminone aza‐aromatic curcuminoids were shown to offer a desirable balance between good solubility and significant bioactivity.

Adaptation of motor unit contractile properties in rat medial gastrocnemius to treadmill endurance training: Relationship to muscle mitochondrial biogenesis.

This study aimed at investigating the effects of 2, 4 and 8 weeks of endurance training on the contractile properties of slow (S), fast fatigue resistant (FR) and fast fatigable (FF) motor units (MUs) in rat medial gastrocnemius (MG) in relation to the changes in muscle mitochondrial biogenesis. The properties of functionally isolated MUs were examined in vivo. Mitochondrial biogenesis was judged based on the changes in mitochondrial DNA copy number (mtDNA), the content of the electron transport chain (ETC) proteins and PGC-1α in the MG. Moreover, the markers of mitochondria remodeling mitofusins (Mfn1, Mfn2) and dynamin-like protein (Opa1) were studied using qPCR. A proportion of FR MUs increased from 37.9% to 50.8% and a proportion of FF units decreased from 44.7% to 26.6% after 8 weeks of training. The increased fatigue resistance, shortened twitch duration, and increased ability to potentiate force were found as early as after 2 weeks of endurance training, predominantly in FR MUs. Moreover, just after 2 weeks of the training an enhancement of the mitochondrial network remodeling was present as judged by an increase in expression of Mfn1, Opa1 and an increase in PGC-1α in the slow part of MG. Interestingly, no signs of intensification of mitochondrial biogenesis assessed by ETC proteins content and mtDNA in slow and fast parts of gastrocnemius were found at this stage of the training. Nevertheless, after 8 weeks of training an increase in the ETC protein content was observed, but mainly in the slow part of gastrocnemius. Concluding, the functional changes in MUs’ contractile properties leading to the enhancement of muscle performance accompanied by an activation of signalling that controls the muscle mitochondrial network reorganisation and mitochondrial biogenesis belong to an early muscle adaptive responses that precede an increase in mitochondrial ETC protein content.

Effect of chronic lactate administration prior to endurance training on mitochondrial adaptations in mouse skeletal muscle

Lactate is considered as not only an oxidizable fuel, but also a signal molecule inducing mitochondrial biogenesis. In this study, we hypothesized that lactate administration prior to training augments mitochondrial adaptations. ICR male mice were randomly assigned to Con (n=9), Tr (n=7) or La+Tr (n=8) group. Mice in Tr and La+Tr were performed treadmill running (20 m/min, 60 min) immediately after administration of PBS (same volume as lactate) or sodium lactate (1 mg/g BW) via i.p. injection for 21 consecutive days. Mice in Con were administrated same volume of PBS on the same days. All the mice were performed lactate tolerance test (LTT: i.p. injection of 1 mg/g BW of sodium lactate) 24h after final training. Blood samples were collected pre, 5, 15, 30, 60 and 180 minutes after the lactate administration. Soleus and plantaris muscle were taken for the biochemical analysis 24h after LTT. As a result, some mitochondrial enzyme activities in soleus and plantaris muscle were significantly higher in La+Tr than Tr (CS: P&lt;0.05, β‐HAD: P=0.05, COX: P&lt;0.01 in soleus, CS: n.s., β‐HAD: n.s., COX: P&lt;0.05 in plantaris). However, mitochondrial OXPHOS protein content was not different between Tr and La+Tr. MCT1 protein content was significantly higher in La+Tr than Con (+18%; P&lt;0.01) in plantaris muscle, but not MCT4. As a result of LTT, blood lactate concentrations after lactate administration were significantly lower in Tr (60 min: P&lt;0.05 vs. Con) and La+Tr (30 min: p&lt;0.01, 60 min: P&lt;0.01 vs. Con) than Con. These data indicate that lactate upregulates mitochondria functionally, and that lactate administration increased MCT1 protein content leading to lower blood lactate concentration after lactate administration.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Properties of the vastus lateralis muscle in relation to age and physiological function in master cyclists aged 55-79years.

SummaryIn this study, results are reported from the analyses of vastus lateralis muscle biopsy samples obtained from a subset (n = 90) of 125 previously phenotyped, highly active male and female cyclists aged 55–79 years in regard to age. We then subsequently attempted to uncover associations between the findings in muscle and in vivo physiological functions. Muscle fibre type and composition (ATPase histochemistry), size (morphometry), capillary density (immunohistochemistry) and mitochondrial protein content (Western blot) in relation to age were determined in the biopsy specimens. Aside from an age‐related change in capillary density in males (r = −.299; p = .02), no other parameter measured in the muscle samples showed an association with age. However, in males type I fibres and capillarity (p < .05) were significantly associated with training volume, maximal oxygen uptake, oxygen uptake kinetics and ventilatory threshold. In females, the only association observed was between capillarity and training volume (p < .05). In males, both type II fibre proportion and area (p < .05) were associated with peak power during sprint cycling and with maximal rate of torque development during a maximal voluntary isometric contraction. Mitochondrial protein content was not associated with any cardiorespiratory parameter in either males or females (p > .05). We conclude in this highly active cohort, selected to mitigate most of the effects of inactivity, that there is little evidence of age‐related changes in the properties of VL muscle across the age range studied. By contrast, some of these muscle characteristics were correlated with in vivo physiological indices.

MitoQ improves mitochondrial dysfunction in heart failure induced by pressure overload

Heart failure remains a major public-health problem with an increase in the number of patients worsening from this disease. Despite current medical therapy, the condition still has a poor prognosis. Heart failure is complex but mitochondrial dysfunction seems to be an important target to improve cardiac function directly.Our goal was to analyze the effects of MitoQ (100 µM in drinking water) on the development and progression of heart failure induced by pressure overload after 14 weeks. The main findings are that pressure overload-induced heart failure in rats decreased cardiac function in vivo that was not altered by MitoQ. However, we observed a reduction in right ventricular hypertrophy and lung congestion in heart failure animals treated with MitoQ. Heart failure also decreased total mitochondrial protein content, mitochondrial membrane potential in the intermyofibrillar mitochondria. MitoQ restored membrane potential in IFM but did not restore mitochondrial protein content. These alterations are associated with the impairment of basal and stimulated mitochondrial respiration in IFM and SSM induced by heart failure. Moreover, MitoQ restored mitochondrial respiration in heart failure induced by pressure overload. We also detected higher levels of hydrogen peroxide production in heart failure and MitoQ restored the increase in ROS production. MitoQ was also able to improve mitochondrial calcium retention capacity, mainly in the SSM whereas in the IFM we observed a small alteration. In summary, MitoQ improves mitochondrial dysfunction in heart failure induced by pressure overload, by decreasing hydrogen peroxide formation, improving mitochondrial respiration and improving mPTP opening.

Resistance Training with Co-ingestion of Anti-inflammatory Drugs Attenuates Mitochondrial Function.

Aim: The current study aimed to examine the effects of resistance exercise with concomitant consumption of high vs. low daily doses of non-steroidal anti-inflammatory drugs (NSAIDs) on mitochondrial oxidative phosphorylation in skeletal muscle. As a secondary aim, we compared the effects of eccentric overload with conventional training.Methods: Twenty participants were randomized to either a group taking high doses (3 × 400 mg/day) of ibuprofen (IBU; 27 ± 5 year; n = 11) or a group ingesting a low dose (1 × 75 mg/day) of acetylsalicylic acid (ASA; 26 ± 4 year; n = 9) during 8 weeks of supervised knee extensor resistance training. Each of the subject's legs were randomized to complete the training program using either a flywheel (FW) device emphasizing eccentric overload, or a traditional weight stack machine (WS). Maximal mitochondrial oxidative phosphorylation (CI+IIP) from permeabilized skeletal muscle bundles was assessed using high-resolution respirometry. Citrate synthase (CS) activity was assessed using spectrophotometric techniques and mitochondrial protein content using western blotting.Results: After training, CI+IIP decreased (P < 0.05) in both IBU (23%) and ASA (29%) with no difference across medical treatments. Although CI+IIP decreased in both legs, the decrease was greater (interaction p = 0.015) in WS (33%, p = 0.001) compared with FW (19%, p = 0.078). CS activity increased (p = 0.027) with resistance training, with no interactions with medical treatment or training modality. Protein expression of ULK1 increased with training in both groups (p < 0.001). The increase in quadriceps muscle volume was not correlated with changes in CI+IIP (R = 0.16).Conclusion: These results suggest that 8 weeks of resistance training with co-ingestion of anti-inflammatory drugs reduces mitochondrial function but increases mitochondrial content. The observed changes were not affected by higher doses of NSAIDs consumption, suggesting that the resistance training intervention was the prime mediator of the decreased mitochondrial phosphorylation. Finally, we noted that flywheel resistance training, emphasizing eccentric overload, rescued some of the reduction in mitochondrial function seen with conventional resistance training.