Intramuscular diacylglycerol accumulates with acute hyperinsulinemia in insulin-resistant phenotypes.

Elevated skeletal muscle diacylglycerols (DAG) and ceramides can impair insulin signaling, and acylcarnitines (acylCN) reflect impaired fatty acid oxidation, thus the intramuscular lipid profile is indicative of insulin resistance. Acute (i.e., postprandial) hyperinsulinemia has been shown to elevate lipids in healthy muscle and is an independent risk factor for type 2 diabetes (T2D). It is unclear how the relationship between acute hyperinsulinemia and the muscle lipidome interacts, thus contributing to or exacerbating insulin resistance. We investigated the impact of acute hyperinsulinemia on the muscle lipidome in order to help characterize the physiological basis in which hyperinsulinemia elevates T2D risk. Endurance athletes (n=12), sedentary lean adults (n=12), and individuals with obesity (n=13) and T2D (n=7) underwent a hyperinsulinemic-euglycemic clamp with muscle biopsies. While there were no significant differences in total 1,2-DAG fluctuations, there was a 2% decrease in athletes versus a 53% increase in T2D. C18 1,2-DAGs increased during the clamp with T2D only, which negatively correlated with insulin sensitivity. Basal muscle C18:0 ceramides were elevated with T2D, but not altered by clamp. Acylcarnitines were universally lowered during hyperinsulinemia, with more robust reductions of 80% in athletes compared to only 46% with T2D. Similar fluctuations with acute hyperinsulinemia increasing 1,2 DAGs in insulin-resistant phenotypes and universally lowering acylcarnitines were observed in male mice. In conclusion, acute hyperinsulinemia elevates muscle 1,2-DAG levels with insulin-resistant phenotypes. This suggests a possible dysregulation of intramuscular lipid metabolism in the fed state in individuals with low insulin sensitivity, which may exacerbate insulin resistance.

Recent advances in understanding the mechanisms in skeletal muscle of interaction between exercise and frontline antihyperglycemic drugs.

Regular exercise and antihyperglycemic drugs are front-line treatments for type-2 diabetes and related metabolic disorders. Leading drugs are metformin, sodium-glucose cotransporter-2 inhibitors, and glucagon-like peptide 1 receptor agonists. Each class has strong individual efficacy to treat hyperglycemia, yet the combination with exercise can yield varied results, some of which include blunting of expected metabolic benefits. Skeletal muscle insulin resistance contributes to the development of type-2 diabetes while improvements in skeletal muscle insulin signaling are among key adaptations to exercise training. The current review identifies recent advances into the mechanisms, with an emphasis on skeletal muscle, of the interaction between exercise and these common antihyperglycemic drugs. The review is written toward researchers and thus highlights specific gaps in knowledge and considerations for future study directions.

Divergent Skeletal Muscle Metabolomic Signatures of Different Exercise Training Modes Independently Predict Cardiometabolic Risk Factors.

We investigated the link between enhancement of SI (by hyperinsulinemic-euglycemic clamp) and muscle metabolites after 12 weeks of aerobic (high-intensity interval training [HIIT]), resistance training (RT), or combined training (CT) exercise in 52 lean healthy individuals. Muscle RNA sequencing revealed a significant association between SI after both HIIT and RT and the branched-chain amino acid (BCAA) metabolic pathway. Concurrently with increased expression and activity of branched-chain ketoacid dehydrogenase enzyme, many muscle amino metabolites, including BCAAs, glutamate, phenylalanine, aspartate, asparagine, methionine, and γ-aminobutyric acid, increased with HIIT, supporting the substantial impact of HIIT on amino acid metabolism. Short-chain C3 and C5 acylcarnitines were reduced in muscle with all three training modes, but unlike RT, both HIIT and CT increased tricarboxylic acid metabolites and cardiolipins, supporting greater mitochondrial activity with aerobic training. Conversely, RT and CT increased more plasma membrane phospholipids than HIIT, suggesting a resistance exercise effect on cellular membrane protection against environmental damage. Sex and age contributed modestly to the exercise-induced changes in metabolites and their association with cardiometabolic parameters. Integrated transcriptomic and metabolomic analyses suggest various clusters of genes and metabolites are involved in distinct effects of HIIT, RT, and CT. These distinct metabolic signatures of different exercise modes independently link each type of exercise training to improved SI and cardiometabolic risk. ARTICLE HIGHLIGHTS: We aimed to understand the link between skeletal muscle metabolites and cardiometabolic health after exercise training. Although aerobic, resistance, and combined exercise training each enhance muscle insulin sensitivity as well as other cardiometabolic parameters, they disparately alter amino and citric acid metabolites as well as the lipidome, linking these metabolomic changes independently to the improvement of cardiometabolic risks with each exercise training mode. These findings reveal an important layer of the unique exercise mode-dependent changes in muscle metabolism, which may eventually lead to more informed exercise prescription for improving SI.

Impact of biological sex and sex hormones on molecular signatures of skeletal muscle at rest and in response to distinct exercise training modes.

Substantial divergence in cardio-metabolic risk, muscle size, and performance exists between men and women. Considering the pivotal role of skeletal muscle in human physiology, we investigated and found, based on RNA sequencing (RNA-seq), that differences in the muscle transcriptome between men and women are largely related to testosterone and estradiol and much less related to genes located on the Y chromosome. We demonstrate inherent unique, sex-dependent differences in muscle transcriptional responses to aerobic, resistance, and combined exercise training in young and older cohorts. The hormonal changes with age likely explain age-related differential expression of transcripts. Furthermore, in primary human myotubes we demonstrate the profound but distinct effects of testosterone and estradiol on amino acid incorporation to multiple individual proteins with specific functions. These results clearly highlight the potential of designing exercise programs tailored specifically to men and women and have implications for people who change gender by altering their hormone profile.

High-fat diet increases electron transfer flavoprotein synthesis and lipid respiration in skeletal muscle during exercise training in female mice.

High-fat diet (HFD) and exercise remodel skeletal muscle mitochondria. The electron transfer flavoproteins (ETF) transfer reducing equivalents from ß-oxidation into the electron transfer system. Exercise may stimulate the synthesis of ETF proteins to increase lipid respiration. We determined mitochondrial remodeling for lipid respiration through ETF in the context of higher mitochondrial abundance/capacity seen in female mice. We hypothesized HFD would be a greater stimulus than exercise to remodel ETF and lipid pathways through increased protein synthesis alongside increased lipid respiration. Female C57BL/6J mice (n = 15 per group) consumed HFD or low-fat diet (LFD) for 4 weeks then remained sedentary (SED) or completed 8 weeks of treadmill training (EX). We determined mitochondrial lipid respiration, RNA abundance, individual protein synthesis, and abundance for ETFα, ETFß, and ETF dehydrogenase (ETFDH). HFD increased absolute and relative lipid respiration (p = 0.018 and p = 0.034) and RNA abundance for ETFα (p = 0.026), ETFß (p = 0.003), and ETFDH (p = 0.0003). HFD increased synthesis for ETFα and ETFDH (p = 0.0007 and p = 0.002). EX increased synthesis of ETFß and ETFDH (p = 0.008 and p = 0.006). Higher synthesis rates of ETF were not always reflected in greater protein abundance. Greater synthesis of ETF during HFD indicates mitochondrial remodeling which may contribute higher mitochondrial lipid respiration through enhanced ETF function.

High-intensity aerobic, but not resistance or combined, exercise training improves both cardiometabolic health and skeletal muscle mitochondrial dynamics.

This study investigated how different exercise training modalities influence skeletal muscle mitochondrial dynamics. Healthy [average body mass index (BMI): 25.8 kg/m2], sedentary younger and older participants underwent 12 wk of supervised high-intensity aerobic interval training (HIIT; n = 13), resistance training (RT; n = 14), or combined training (CT; n = 11). Mitochondrial structure was assessed using transmission electron microscopy (TEM). Regulators of mitochondrial fission and fusion, cardiorespiratory fitness (V̇o2peak), insulin sensitivity via a hyperinsulinemic-euglycemic clamp, and muscle mitochondrial respiration were assessed. TEM showed increased mitochondrial volume, number, and perimeter following HIIT (P < 0.01), increased mitochondrial number following CT (P < 0.05), and no change in mitochondrial abundance after RT. Increased mitochondrial volume associated with increased mitochondrial respiration and insulin sensitivity following HIIT (P < 0.05). Increased mitochondrial perimeter associated with increased mitochondrial respiration, insulin sensitivity, and V̇o2peak following HIIT (P < 0.05). No such relationships were observed following CT or RT. OPA1, a regulator of fusion, was increased following HIIT (P < 0.05), whereas FIS1, a regulator of fission, was decreased following HIIT and CT (P < 0.05). HIIT also increased the ratio of OPA1/FIS1 (P < 0.01), indicative of the balance between fission and fusion, which positively correlated with improvements in respiration, insulin sensitivity, and V̇o2peak (P < 0.05). In conclusion, HIIT induces a larger, more fused mitochondrial tubular network. Changes indicative of increased fusion following HIIT associate with improvements in mitochondrial respiration, insulin sensitivity, and V̇o2peak supporting the idea that enhanced mitochondrial fusion accompanies notable health benefits of HIIT.NEW & NOTEWORTHY We assessed the effects of 12 wk of supervised high-intensity interval training (HIIT), resistance training, and combined training (CT) on skeletal muscle mitochondrial abundance and markers of fission and fusion. HIIT increased mitochondrial area and size and promoted protein changes indicative of increased mitochondrial fusion, whereas lessor effects were observed after CT and no changes were observed after RT. Furthermore, increased mitochondrial area and size after HIIT associated with improved mitochondrial respiration, cardiorespiratory fitness, and insulin sensitivity.

Novel whole blood transcriptome signatures of changes in maximal aerobic capacity in response to endurance exercise training in healthy women.

Maximal aerobic exercise capacity [maximal oxygen consumption (V̇o2max)] is one of the strongest predictors of morbidity and mortality. Aerobic exercise training can increase V̇o2max, but inter-individual variability is marked and unexplained physiologically. The mechanisms underlying this variability have major clinical implications for extending human healthspan. Here, we report a novel transcriptome signature related to ΔV̇o2max with exercise training detected in whole blood RNA. We used RNA-Seq to characterize transcriptomic signatures of ΔV̇o2max in healthy women who completed a 16-wk randomized controlled trial comparing supervised, higher versus lower aerobic exercise training volume and intensity (4 training groups, fully crossed). We found significant baseline gene expression differences in subjects who responded to aerobic exercise training with robust versus little/no ΔV̇o2max, and differentially expressed genes/transcripts were mostly related to inflammatory signaling and mitochondrial function/protein translation. Baseline gene expression signatures associated with robust versus little/no ΔV̇o2max were also modulated by exercise training in a dose-dependent manner, and they predicted ΔV̇o2max in this and a separate dataset. Collectively, our data demonstrate the potential utility of using whole blood transcriptomics to study the biology of inter-individual variability in responsiveness to the same exercise training stimulus.

Two weeks of high-intensity interval training increases skeletal muscle mitochondrial respiration via complex-specific remodeling in sedentary humans.

Aerobic training remodels the quantity and quality (function per unit) of skeletal muscle mitochondria to promote substrate oxidation, however, there remain key gaps in understanding the underlying mechanisms during initial training adaptations. We used short-term high-intensity interval training (HIIT) to determine changes to mitochondrial respiration and regulatory pathways that occur early in remodeling. Fifteen normal-weight sedentary adults started seven sessions of HIIT over 14 days and 14 participants completed the intervention. We collected vastus lateralis biopsies before and 48 h after HIIT to determine mitochondrial respiration, RNA sequencing, and Western blotting for proteins of mitochondrial respiration and degradation via autophagy. HIIT increased respiration per mitochondrial protein for lipid (+23% P = 0.020), complex I (+18%, P = 0.0015), complex I + II (+14%, P < 0.0001), and complex II (+24% P < 0.0001). Transcripts that increased with HIIT identified several gene sets of mitochondrial respiration, particularly for complex I, whereas transcripts that decreased identified pathways of DNA and chromatin remodeling. HIIT lowered protein abundance of autophagy markers for p62 (-19%, P = 0.012) and LC3 II/I (-20%, P = 0.004) in whole tissue lysates but not isolated mitochondria. Meal tolerance testing revealed HIIT increased the change in whole body respiratory exchange ratio and lowered cumulative plasma insulin concentrations. Gene transcripts and respiratory function indicate remodeling of mitochondria within 2 wk of HIIT. Overall changes are consistent with increased protein quality driving rapid improvements in substrate oxidation.NEW & NOTEWORTHY Aerobic training stimulates mitochondrial metabolism in skeletal muscle that is linked to improvements to whole body fuel metabolism. The mechanisms driving changes to the quantity and quality (function per unit) of mitochondria are less known. We used seven sessions of high-intensity interval training (HIIT) to determine functional changes and mechanisms of mitochondrial remodeling in skeletal muscle. HIIT increased mitochondrial respiration per mass for fatty acids, complex I, and complex II substrates. HIIT-induced remodeling pathways including gene transcripts for mitochondrial respiration (via RNA sequencing of muscle tissue) and proteins related to complex I respiration. We conclude that an early feature of aerobic training is increased mitochondrial protein quality via improved respiration and induction of mitochondrial transcriptional patterns.

Impact of 4 weeks of western diet and aerobic exercise training on whole-body phenotype and skeletal muscle mitochondrial respiration in male and female mice.

High dietary fat intake induces significant whole-body and skeletal muscle adaptations in mice, including increased capacity for fat oxidation and mitochondrial biogenesis. The impact of a diet that is high in fat and simple sugars (i.e., western diet [WD]), particularly on regulation of skeletal muscle mitochondrial function, is less understood. The purpose of the current study was to determine physiologic adaptations in mitochondrial respiratory capacity in skeletal muscle during short-term consumption of WD, including if adaptive responses to WD-feeding are modified by concurrent exercise training or may be sex-specific. Male and female C57BL/6J mice were randomized to consume low-fat diet (LFD) or WD for 4 weeks, with some WD-fed mice also performing concurrent treadmill training (WD + Ex). Group sizes were n = 4-7. Whole-body metabolism was measured using in-cage assessment of food intake and energy expenditure, DXA body composition analysis and insulin tolerance testing. High-resolution respirometry of mitochondria isolated from quadriceps muscle was used to determine skeletal muscle mitochondrial respiratory function. Male mice fed WD gained mass (p < 0.001), due to increased fat mass (p < 0.001), and displayed greater respiratory capacity for both lipid and non-lipid substrates compared with LFD mice (p < 0.05). There was no effect of concurrent treadmill training on maximal respiration (WD + Ex vs. WD). Female mice had non-significant changes in body mass and composition as a function of the interventions, and no differences in skeletal muscle mitochondrial oxidative capacity. These findings indicate 4 weeks of WD feeding can increase skeletal muscle mitochondrial oxidative capacity among male mice; whereas WD, with or without exercise, had minimal impact on mass gain and skeletal muscle respiratory capacity among female mice. The translational relevance is that mitochondrial adaptation to increases in dietary fat intake that model WD may be related to differences in weight gain among male and female mice.

Characterization of cellular senescence in aging skeletal muscle.

Senescence is a cell fate that contributes to multiple aging-related pathologies. Despite profound age-associated changes in skeletal muscle (SkM), whether its constituent cells are prone to senesce has not been methodically examined. Herein, using single cell and bulk RNA-sequencing and complementary imaging methods on SkM of young and old mice, we demonstrate that a subpopulation of old fibroadipogenic progenitors highly expresses p16 Ink4a together with multiple senescence-related genes and, concomitantly, exhibits DNA damage and chromatin reorganization. Through analysis of isolated myofibers, we also detail a senescence phenotype within a subset of old cells, governed instead by p2 Cip1 . Administration of a senotherapeutic intervention to old mice countered age-related molecular and morphological changes and improved SkM strength. Finally, we found that the senescence phenotype is conserved in SkM from older humans. Collectively, our data provide compelling evidence for cellular senescence as a hallmark and potentially tractable mediator of SkM aging.

Use of c-peptide as a measure of cephalic phase insulin release in humans.

Cephalic phase insulin release (CPIR) is a rapid pulse of insulin secreted within minutes of food-related sensory stimulation. Understanding the mechanisms underlying CPIR in humans has been hindered by its small observed effect size and high variability within and between studies. One contributing factor to these limitations may be the use of peripherally measured insulin as an indicator of secreted insulin, since a substantial portion of insulin is metabolized by the liver before delivery to peripheral circulation. Here, we investigated the use of c-peptide, which is co-secreted in equimolar amounts to insulin from pancreatic beta cells, as a proxy for insulin secretion during the cephalic phase period. Changes in insulin and c-peptide were monitored in 18 adults over two repeated sessions following oral stimulation with a sucrose-containing gelatin stimulus. We found that, on average, insulin and c-peptide release followed a similar time course over the cephalic phase period, but that c-peptide showed a greater effect size. Importantly, when insulin and c-peptide concentrations were compared across sessions, we found that changes in c-peptide were significantly correlated at the 2 min (r = 0.50, p = 0.03) and 4 min (r = 0.65, p = 0.003) time points, as well as when participants' highest c-peptide concentrations were considered (r = 0.64, p = 0.004). In contrast, no significant correlations were observed for changes in insulin measured from the sessions (r = -0.06-0.35, p > 0.05). Herein, we detail the individual variability of insulin and c-peptide concentrations measured during the cephalic phase period, and identify c-peptide as a valuable metric for insulin secretion alongside insulin concentrations when investigating CPIR.

Enhancement of anaerobic glycolysis - a role of PGC-1α4 in resistance exercise.

Resistance exercise training (RET) is an effective countermeasure to sarcopenia, related frailty and metabolic disorders. Here, we show that an RET-induced increase in PGC-1α4 (an isoform of the transcriptional co-activator PGC-1α) expression not only promotes muscle hypertrophy but also enhances glycolysis, providing a rapid supply of ATP for muscle contractions. In human skeletal muscle, PGC-1α4 binds to the nuclear receptor PPARß following RET, resulting in downstream effects on the expressions of key glycolytic genes. In myotubes, we show that PGC-1α4 overexpression increases anaerobic glycolysis in a PPARß-dependent manner and promotes muscle glucose uptake and fat oxidation. In contrast, we found that an acute resistance exercise bout activates glycolysis in an AMPK-dependent manner. These results provide a mechanistic link between RET and improved glucose metabolism, offering an important therapeutic target to counteract aging and inactivity-induced metabolic diseases benefitting those who cannot exercise due to many reasons.

Substrate-Specific Respiration of Isolated Skeletal Muscle Mitochondria after 1 h of Moderate Cycling in Sedentary Adults.

INTRODUCTION: Skeletal muscle mitochondria have dynamic shifts in oxidative metabolism to meet energy demands of aerobic exercise. Specific complexes oxidize lipid and nonlipid substrates. It is unclear if aerobic exercise stimulates intrinsic oxidative metabolism of mitochondria or varies between substrates. METHODS: We studied mitochondrial metabolism in sedentary male and female adults (n = 11F/4M) who were free of major medical conditions with mean ± SD age of 28 ± 7 yr, peak aerobic capacity of 2.0 ± 0.4 L·min-1, and body mass index of 22.2 ± 2 kg·m-2. Biopsies were collected from the vastus lateralis muscle on separate study days at rest or 15 min after exercise (1 h cycling at 65% peak aerobic capacity). Isolated mitochondria were analyzed using high-resolution respirometry of separate titration protocols for lipid (palmitoylcarnitine, F-linked) and nonlipid substrates (glutamate-malate, N-linked; succinate S-linked). Titration protocols distinguished between oxidative phosphorylation and leak respiration and included the measurement of reactive oxygen species emission (H2O2). Western blotting determined the protein abundance of electron transfer flavoprotein (ETF) subunits, including inhibitory methylation site on ETF-ß. RESULTS: Aerobic exercise induced modest increases in mitochondrial respiration because of increased coupled respiration across F-linked (+13%, P = 0.08), N(S)-linked (+14%, P = 0.09), and N-linked substrates (+17%, P = 0.08). Prior exercise did not change P:O ratio. Electron leak to H2O2 increased 6% increased after exercise (P = 0.06) for lipid substrates but not for nonlipid. The protein abundance of ETF-α or ETF-ß subunit or inhibitory methylation on ETF-ß was not different between rest and after exercise. CONCLUSION: In sedentary adults, the single bout of moderate-intensity cycling induced modest increases for intrinsic mitochondrial oxidative phosphorylation that was consistent across multiple substrates.

Tetrahydroxanthohumol, a xanthohumol derivative, attenuates high-fat diet-induced hepatic steatosis by antagonizing PPARγ.

We previously reported xanthohumol (XN), and its synthetic derivative tetrahydro-XN (TXN), attenuates high-fat diet (HFD)-induced obesity and metabolic syndrome in C57Bl/6J mice. The objective of the current study was to determine the effect of XN and TXN on lipid accumulation in the liver. Non-supplemented mice were unable to adapt their caloric intake to 60% HFD, resulting in obesity and hepatic steatosis; however, TXN reduced weight gain and decreased hepatic steatosis. Liver transcriptomics indicated that TXN might antagonize lipogenic PPARγ actions in vivo. XN and TXN inhibited rosiglitazone-induced 3T3-L1 cell differentiation concomitant with decreased expression of lipogenesis-related genes. A peroxisome proliferator activated receptor gamma (PPARγ) competitive binding assay showed that XN and TXN bind to PPARγ with an IC50 similar to pioglitazone and 8-10 times stronger than oleate. Molecular docking simulations demonstrated that XN and TXN bind in the PPARγ ligand-binding domain pocket. Our findings are consistent with XN and TXN acting as antagonists of PPARγ.

Nitrate-induced improvements in exercise performance are coincident with exuberant changes in metabolic genes and the metabolome in zebrafish (Danio rerio) skeletal muscle.

Dietary nitrate supplementation improves exercise performance by reducing the oxygen cost of exercise and enhancing skeletal muscle function. However, the mechanisms underlying these effects are not well understood. The purpose of this study was to assess changes in skeletal muscle energy metabolism associated with exercise performance in a zebrafish model. Fish were exposed to sodium nitrate (60.7 mg/L, 303.5 mg/L, 606.9 mg/L), or control water, for 21 days and analyzed at intervals (5, 10, 20, 30, 40 cm/s) during a 2-h strenuous exercise test. We measured oxygen consumption during an exercise test and assessed muscle nitrate concentrations, gene expression, and the muscle metabolome before, during, and after exercise. Nitrate exposure reduced the oxygen cost of exercise and increased muscle nitrate concentrations at rest, which were reduced with increasing exercise duration. In skeletal muscle, nitrate treatment upregulated expression of genes central to nutrient sensing (mtor), redox signaling (nrf2a), and muscle differentiation (sox6). In rested muscle, nitrate treatment increased phosphocreatine (P = 0.002), creatine (P = 0.0005), ATP (P = 0.0008), ADP (P = 0.002), and AMP (P = 0.004) compared with rested-control muscle. Following the highest swimming speed, concentration of phosphocreatine (P = 8.0 × 10-5), creatine (P = 6.0 × 10-7), ATP (P = 2.0 × 10-6), ADP (P = 0.0002), and AMP (P = 0.004) decreased compared with rested nitrate muscle. Our data suggest nitrate exposure in zebrafish lowers the oxygen cost of exercise by changing the metabolic programming of muscle prior to exercise and increasing availability of energy-rich metabolites required for exercise.NEW & NOTEWORTHY We show that skeletal muscle nitrate concentration is higher with supplementation at rest and was lower in groups with increasing exercise duration in a zebrafish model. The higher availability of nitrate at rest is associated with upregulation of key nutrient-sensing genes and greater availability of energy-producing metabolites (i.e., ATP, phosphocreatine, glycolytic intermediates). Overall, nitrate supplementation may lower oxygen cost of exercise through improved fuel availability resulting from metabolic programming of muscle prior to exercise.

Short-Term High-Fat Feeding Does Not Alter Mitochondrial Lipid Respiratory Capacity but Triggers Mitophagy Response in Skeletal Muscle of Mice.

Lipid overload of the mitochondria is linked to the development of insulin resistance in skeletal muscle which may be a contributing factor to the progression of type 2 diabetes during obesity. The targeted degradation of mitochondria through autophagy, termed mitophagy, contributes to the mitochondrial adaptive response to changes in dietary fat. Our previous work demonstrates long-term (2-4 months) consumption of a high-fat diet increases mitochondrial lipid oxidation capacity but does not alter markers of mitophagy in mice. The purpose of this study was to investigate initial stages of mitochondrial respiratory adaptations to high-fat diet and the activation of mitophagy. C57BL/6J mice consumed either a low-fat diet (LFD, 10% fat) or high-fat diet (HFD, 60% fat) for 3 or 7 days. We measured skeletal muscle mitochondrial respiration and protein markers of mitophagy in a mitochondrial-enriched fraction of skeletal muscle. After 3 days of HFD, mice had lower lipid-supported oxidative phosphorylation alongside greater electron leak compared with the LFD group. After 7 days, there were no differences in mitochondrial respiration between diet groups. HFD mice had greater autophagosome formation potential (Beclin-1) and greater activation of mitochondrial autophagy receptors (Bnip3, p62) in isolated mitochondria, but no difference in downstream autophagosome (LC3II) or lysosome (Lamp1) abundance after both 3 and 7 days compared with the LFD groups. In cultured myotubes, palmitate treatment decreased mitochondrial membrane potential and hydrogen peroxide treatment increased accumulation of upstream mitophagy markers. We conclude that several days of high-fat feeding stimulated upstream activation of skeletal muscle mitophagy, potentially through lipid-induced oxidative stress, without downstream changes in respiration.

Skeletal Muscle ACSL Isoforms Relate to Measures of Fat Metabolism in Humans.

INTRODUCTION: Evidence from model systems implicates long-chain acyl-coenzyme A synthetase (ACSL) as key regulators of skeletal muscle fat oxidation and fat storage; however, such roles remain underexplored in humans. PURPOSE: We sought to determine the protein expression of ACSL isoforms in skeletal muscle at rest and in response to acute exercise and identify relationships between skeletal muscle ACSL and measures of fat metabolism in humans. METHODS: Sedentary adults (n = 14 [4 males and 10 females], body mass index = 22.2 ± 2.1 kg·m-2, V˙O2max = 32.2 ± 4.5 mL·kg-1⋅min-1) completed two study visits. Trials were identical other than completing 1 h of cycling exercise (65% V˙O2max) or remaining sedentary. Vastus lateralis biopsies were obtained 15-min postexercise (or rest) and 2-h postexercise to determine ACSL protein abundance. Whole-body fat oxidation was assessed at rest and during exercise using indirect calorimetry. Skeletal muscle triacylglycerol (TAG) was measured via lipidomic analysis. RESULTS: We detected protein expression for four of the five known ACSL isoforms in human skeletal muscle. ACSL protein abundances were largely unaltered in the hours after exercise aside from a transient increase in ACSL5 15-min postexercise (P = 0.01 vs rest). Skeletal muscle ACSL1 protein abundance tended to be positively related with whole-body fat oxidation during exercise (P = 0.07, r = 0.53), when skeletal muscle accounts for the majority of energy expenditure. No such relationship between ACSL1 and fat oxidation was observed at rest. Skeletal muscle ACSL6 protein abundance was positively associated with muscle TAG content at rest (P = 0.05, r = 0.57). CONCLUSION: Most ACSL protein isoforms can be detected in human skeletal muscle, with minimal changes in abundance after acute exercise. Our findings agree with those from model systems implicating ACSL1 and ACSL6 as possible determinants of fat oxidation and fat storage within skeletal muscle.

Comparative Analysis of Skeletal Muscle Transcriptional Signatures Associated With Aerobic Exercise Capacity or Response to Training in Humans and Rats.

Increasing exercise capacity promotes healthy aging and is strongly associated with lower mortality rates. In this study, we analyzed skeletal muscle transcriptomics coupled to exercise performance in humans and rats to dissect the inherent and response components of aerobic exercise capacity. Using rat models selected for intrinsic and acquired aerobic capacity, we determined that the high aerobic capacity muscle transcriptome is associated with pathways for tissue oxygenation and vascularization. Conversely, the low capacity muscle transcriptome indicated immune response and metabolic dysfunction. Low response to training was associated with an inflammatory signature and revealed a potential link to circadian rhythm. Next, we applied bioinformatics tools to predict potential secreted factors (myokines). The predicted secretome profile for exercise capacity highlighted circulatory factors involved in lipid metabolism and the exercise response secretome was associated with extracellular matrix remodelling. Lastly, we utilized human muscle mitochondrial respiration and transcriptomics data to explore molecular mediators of exercise capacity and response across species. Human transcriptome comparison highlighted epigenetic mechanisms linked to exercise capacity and the damage repair for response. Overall, our findings from this cross-species transcriptome analysis of exercise capacity and response establish a foundation for future studies on the mechanisms that link exercise and health.

Mitochondrial adaptations to exercise do not require Bcl2-mediated autophagy but occur with BNIP3/Parkin activation.

Understanding the mechanisms regulating mitochondrial respiratory function and adaptations to metabolic challenges, such as exercise and high dietary fat, is necessary to promote skeletal muscle health and attenuate metabolic disease. Autophagy is a constitutively active degradation pathway that promotes mitochondrial turnover and transiently increases postexercise. Recent evidence indicates Bcl2 mediates exercise-induced autophagy and skeletal muscle adaptions to training during high-fat diet. We determined if improvements in mitochondrial respiration due to exercise training required Bcl2-mediated autophagy using a transgenic mouse model of impaired inducible autophagy (Bcl2AAA ). Mitochondrial adaptations to a treadmill exercise training protocol, in either low-fat or high-fat diet fed mice, did not require Bcl2-mediated autophagy activation. Instead, training increased protein synthesis rates and basal autophagy in the Bcl2AAA mice, while acute exercise activated BNIP3 and Parkin autophagy. High-fat diet stimulated lipid-specific mitochondrial adaptations. These data demonstrate increases in basal mitochondrial turnover, not transient activation with exercise, mediate adaptations to exercise and high-fat diet.