Cardiac glycogen-autophagy (‘glycophagy’) is disturbed in cardiometabolic pathologies. The physiological role of cardiac glycophagy is unclear. Exercise induces transient cardiac glycogen accumulation. Thus, this study experimentally examined glycophagy involvement during recovery from an exhaustive exercise protocol. Peak myocardial glycogen accumulation in mice was evident at 2 h post-exercise, preceded by transient activation of glycogen synthase. At 4 and 16 h post-exercise, glycogen degradation was associated with decreased STBD1 (glycophagy tagging protein) and increased GABARAPL1 (Atg8 protein), suggesting that glycophagy activity was increased. These findings provide the first evidence that glycophagy is involved in cardiac glycogen physiologic homeostasis post-exercise.

Neutrophils accumulate in peripheral tissues during obesity and are critical coordinators of tissue inflammatory responses. Here, we provide evidence that inhibition of the primary neutrophil protease, neutrophil elastase, with α1-antitrypsin A (A1AT) can improve insulin sensitivity and glucose homeostasis of mice fed a high-fat diet. This was attributed to improved insulin-induced IRS-1 phosphorylation in white adipose tissue and provides further support for a role of neutrophils in mediating diet-induced peripheral tissue insulin resistance.

Mitochondrial-derived peptides (MDPs) are encoded by the mitochondrial genome and hypothesised to form part of a retrograde signalling network that modulates adaptive responses to metabolic stress. To understand how metabolic stress regulates MDPs in humans we assessed the association between circulating MOTS-c and SHLP2 and components of metabolic syndrome (MS), as well as depot-specific fat mass in participants without overt type 2 diabetes or cardiovascular disease. One-hundred and twenty-five Chinese participants (91 male, 34 female) had anthropometry, whole body dual-energy X-ray absorptiometry scans and fasted blood samples analysed. Chinese female participants and an additional 34 European Caucasian female participants also underwent magnetic resonance imaging and spectroscopy (MRI/S) for visceral, pancreatic and liver fat quantification. In Chinese participants (age = 41 ± 1 years, BMI = 27.8 ± 3.9 kg/m2), plasma MOTS-c (315 ± 27 pg/ml) and SHLP2 (1393 ± 82 pg/ml) were elevated in those with MS (n = 26). While multiple components of the MS sequelae positively associated with both MOTS-c and SHLP2, including blood pressure, fasting plasma glucose and triglycerides, the most significant of these was waist circumference (p < 0.0001). Android fat had a greater effect on increasing plasma MOTS-c (p < 0.004) and SHLP2 (p < 0.009) relative to whole body fat. Associations with MRI/S parameters corrected for total body fat mass revealed that liver fat positively associated with plasma MOTS-c and SHLP2 and visceral fat with SHLP2. Consistent with hepatic stress being a driver of circulating MDP concentrations, plasma MOTS-c and SHLP2 were higher in participants with elevated liver damage markers and in male C57Bl/6j mice fed a diet that induces hepatic lipid accumulation and damage. Our findings provide evidence that in the absence of overt type 2 diabetes, components of the MS positively associated with levels of MOTS-c and SHLP2 and that android fat, in particular liver fat, is a primary driver of these associations. MOTS-c and SHLP2 have previously been shown to have cyto- and metabolo-protective properties, therefore we suggest that liver stress may be a mitochondrial peptide signal, and that mitochondrial peptides are part of a hepatic centric-hormetic response intended to restore metabolic balance.

Healthy aging can be promoted by enhanced metabolic fitness and physical capacity. Mitochondria are chief metabolic organelles with strong implications in aging that also coordinate broad physiological functions, in part, using peptides that are encoded within their independent genome. However, mitochondrial-encoded factors that actively regulate aging are unknown. Here, we report that mitochondrial-encoded MOTS-c can significantly enhance physical performance in young (2 mo.), middle-age (12 mo.), and old (22 mo.) mice. MOTS-c can regulate (i) nuclear genes, including those related to metabolism and proteostasis, (ii) skeletal muscle metabolism, and (iii) myoblast adaptation to metabolic stress. We provide evidence that late-life (23.5 mo.) initiated intermittent MOTS-c treatment (3x/week) can increase physical capacity and healthspan in mice. In humans, exercise induces endogenous MOTS-c expression in skeletal muscle and in circulation. Our data indicate that aging is regulated by genes encoded in both of our co-evolved mitochondrial and nuclear genomes.

Reactive oxygen species (ROS) generated during exercise are considered integral for the health-promoting effects of exercise. However, the precise mechanisms by which exercise and ROS promote metabolic health remain unclear. Here, we demonstrate that skeletal muscle NADPH oxidase 4 (NOX4), which is induced after exercise, facilitates ROS-mediated adaptive responses that promote muscle function, maintain redox balance, and prevent the development of insulin resistance. Conversely, reductions in skeletal muscle NOX4 in aging and obesity contribute to the development of insulin resistance. NOX4 deletion in skeletal muscle compromised exercise capacity and antioxidant defense and promoted oxidative stress and insulin resistance in aging and obesity. The abrogated adaptive mechanisms, oxidative stress, and insulin resistance could be corrected by deleting the H2O2-detoxifying enzyme GPX-1 or by treating mice with an agonist of NFE2L2, the master regulator of antioxidant defense. These findings causally link NOX4-derived ROS in skeletal muscle with adaptive responses that promote muscle function and insulin sensitivity.

Key points Loss of β‐catenin impairs in vivo and isolated muscle exercise/contraction‐stimulated glucose uptake. β‐Catenin is required for exercise‐induced skeletal muscle actin cytoskeleton remodelling. β‐Catenin675 phosphorylation during exercise may be intensity dependent. AbstractThe conserved structural protein β‐catenin is an emerging regulator of vesicle trafficking in multiple tissues and supports insulin‐stimulated glucose transporter 4 (GLUT4) translocation in skeletal muscle by facilitating cortical actin remodelling. Actin remodelling may be a convergence point between insulin and exercise/contraction‐stimulated glucose uptake. Here we investigated whether β‐catenin is involved in regulating exercise/contraction‐stimulated glucose uptake. We report that the muscle‐specific deletion of β‐catenin induced in adult mice (BCAT‐mKO) impairs both exercise‐ and contraction (isolated muscle)‐induced glucose uptake without affecting running performance or canonical exercise signalling pathways. Furthermore, high intensity exercise in mice and contraction of myotubes and isolated muscles led to the phosphorylation of β‐cateninS675, and this was impaired by Rac1 inhibition. Moderate intensity exercise in control and Rac1 muscle‐specific knockout mice did not induce muscle β‐cateninS675 phosphorylation, suggesting exercise intensity‐dependent regulation of β‐cateninS675. Introduction of a non‐phosphorylatable S675A mutant of β‐catenin into myoblasts impaired GLUT4 translocation and actin remodelling stimulated by carbachol, a Rac1 and RhoA activator. Exercise‐induced increases in cross‐sectional phalloidin staining (F‐actin marker) of gastrocnemius muscle was impaired in muscle from BCAT‐mKO mice. Collectively our findings suggest that β‐catenin is required for optimal glucose transport in muscle during exercise/contraction, potentially via facilitating actin cytoskeleton remodelling.

Acute exercise, and in particular aerobic exercise, increases skeletal muscle energy demand causing mitochondrial stress, and mitochondrial-related adaptations which are a hallmark of exercise training. Given that mitochondria are central players in the exercise response, it is imperative that they have networks that can communicate their status both intra- and inter-cellularly. Peptides encoded by short open-reading frames within mitochondrial DNA, mitochondrial-derived peptides (MDPs), have been suggested to form a newly recognised branch of this retrograde signalling cascade that contribute to coordinating the adaptive response to regular exercise. Here we summarise the recent evidence that acute high intensity exercise in humans can increase concentrations of the MDPs humanin and MOTS-c in skeletal muscle and plasma, and speculate on the mechanisms controlling MDP responses to exercise stress. Evidence that exercise training results in chronic changes in MDP expression within tissues and the circulation is conflicting and may depend on the mode, duration, intensity of training plan and participant characteristics. Further research is required to define the effect of these variables on MDPs and to determine whether MDPs other than MOTS-c have exercise mimetic properties. MOTS-c treatment of young and aged mice improves exercise capacity/performance and leads to adaptions that are similar to that of being physically active (weight loss, increased antioxidant capacity and improved insulin sensitivity), however, studies utilising a MOTS-c inactivating genetic variant or combination of exercise + MOTS-c treatment in mice suggest that there are distinct and overlapping pathways through which exercise and MOTS-c evoke metabolic benefits. Overall, MOTS-c, and potentially other MDPs, may be exercise-sensitive myokines and further work is required to define inter- and intra-tissue targets in an exercise context.

Mitochondrial-derived peptides (MDPs) are small bioactive peptides encoded by short open-reading frames (sORF) in mitochondrial DNA that do not necessarily have traditional hallmarks of protein-coding genes. To date, eight MDPs have been identified, all of which have been shown to have various cyto- or metaboloprotective properties. The 12S ribosomal RNA (MT-RNR1) gene harbors the sequence for MOTS-c, whereas the other seven MDPs [humanin and small humanin-like peptides (SHLP) 1-6] are encoded by the 16S ribosomal RNA gene. Here, we review the evidence that endogenous MDPs are sensitive to changes in metabolism, showing that metabolic conditions like obesity, diabetes, and aging are associated with lower circulating MDPs, whereas in humans muscle MDP expression is upregulated in response to stress that perturbs the mitochondria like exercise, some mtDNA mutation-associated diseases, and healthy aging, which potentially suggests a tissue-specific response aimed at restoring cellular or mitochondrial homeostasis. Consistent with this, treatment of rodents with humanin, MOTS-c, and SHLP2 can enhance insulin sensitivity and offer protection against a range of age-associated metabolic disorders. Furthermore, assessing how mtDNA variants alter the functions of MDPs is beginning to provide evidence that MDPs are metabolic signal transducers in humans. Taken together, MDPs appear to form an important aspect of a retrograde signaling network that communicates mitochondrial status with the wider cell and to distal tissues to modulate adaptative responses to metabolic stress. It remains to be fully determined whether the metaboloprotective properties of MDPs can be harnessed into therapies for metabolic disease.

Neutrophil elastase (NE) is a serine protease expressed by neutrophils which is inhibited endogenously by α1-Antitrypsin A (AAT). Obesity is associated with increased plasma NE:AAT ratios and adipose tissue NE accumulation, which may drive obesity-associated metabolic dysfunction. Exogenous AAT is now an FDA approved treatment for AAT deficiency, therefore we investigated whether AAT treatment may also have potential therapeutic benefits for diet induced metabolic dysfunction. Standard Chow and high-fat diet (HFD) fed male C57Bl6 mice were randomized to receive 3x weekly IP injections of either AAT (2mg) or vehicle (PBS) for 10 weeks. Chow fed mice treated with recombinant AAT showed no differences in plasma NE, body weight or metabolic phenotype compared to vehicle treated control mice. However, during HFD feeding AAT treatment attenuated increases in plasma NE and white adipose tissue (WAT) NE and neutrophil accumulation without affecting circulatory neutrophil levels or body weight. Consistent with NE knockout mice being partially protected from HFD glucose intolerance, treatment of HFD fed mice with AAT enhanced whole body insulin sensitivity which was attributed to higher insulin-dependent p-AktSer473 and reduced inflammation markers in WAT but no other peripheral tissues. Treatment of 3T3L1 adipocytes with recombinant NE impaired insulin-stimulated glucose uptake andp-AktSer473, suggesting that inhibition of NE in WAT is the primary mechanism through which AAT treatment enhances insulin sensitivity. Collectively, our data suggests AAT may play a potential role in mitigating diet-induced neutrophil infiltration in WAT insulin resistance. Disclosure R.F. D’Souza: None. J.S.T. Woodhead: None. S.W. Masson: None. S.L. James: None. C. Hedges: None. T.L. Merry: None. Funding Health Research Council of New Zealand

Healthy aging can be promoted by enhancing metabolic fitness and physical capacity (1, 2). Mitochondria are chief metabolic organelles with strong implications in aging (3–8). In addition to their prominent role in bioenergetics, mitochondria also coordinate broad physiological functions by communicating to other cellular compartments or distal cells using multiple factors (9, 10), including peptides that are encoded within their own independent genome (11, 12). However, it is unknown if aging is actively regulated by factors encoded in the mitochondrial genome. MOTS-c is a mitochondrial-encoded peptide that regulates metabolic homeostasis (13, 14), in part, by translocating to the nucleus to regulate adaptive nuclear gene expression in response to cellular stress (15–17). Here, we report that MOTS-c is an exercise-induced mitochondrial-encoded peptide that significantly enhanced physical performance when administered to young (2 mo.), middle-aged (12 mo.), and old (22 mo.) mice. In humans, we found that endogenous MOTS-c levels significantly increased in response to exercise in skeletal muscle (11.9-fold) and in circulation (1.5-fold). Systemic MOTS-c treatment in mice significantly enhanced the performance on a treadmill of all age groups (~2-fold). MOTS-c regulated (i) nuclear genes, including those related to metabolism and protein homeostasis, (ii) glucose and amino acid metabolism in skeletal muscle, and (iii) myoblast adaptation to metabolic stress. Late-life (23.5 mo.) initiated intermittent MOTS-c treatment (3x/week) improved physical capacity and trended towards increasing lifespan. Our data indicate that aging is regulated by genes that are encoded not only in the nuclear genome (18, 19), but also in the mitochondrial genome. Considering that aging is the major risk factor for multiple chronic diseases (20, 21), our study provides new grounds for further investigation into mitochondrial-encoded regulators of healthy lifespan.

Mitochondria putatively regulate the aging process, in part, through the small regulatory peptide, mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) that is encoded by the mitochondrial genome. Here we investigated the regulation of MOTS-c in the plasma and skeletal muscle of healthy aging men. Circulating MOTS-c reduced with age, but older (70-81 y) and middle-aged (45-55 y) men had ~1.5-fold higher skeletal muscle MOTS-c expression than young (18-30 y). Plasma MOTS-c levels only correlated with plasma in young men, was associated with markers of slow-type muscle, and associated with improved muscle quality in the older group (maximal leg-press load relative to thigh cross-sectional area). Using small mRNA assays we provide evidence that MOTS-c transcription may be regulated independently of the full length 12S rRNA gene in which it is encoded, and expression is not associated with antioxidant response element (ARE)-related genes as previously seen in culture. Our results suggest that plasma and muscle MOTS-c are differentially regulated with aging, and the increase in muscle MOTS-c expression with age is consistent with fast-to-slow type muscle fiber transition. Further research is required to determine the molecular targets of endogenous MOTS-c in human muscle but they may relate to factors that maintain muscle quality.

Small regulatory peptides encoded within the mitochondrial genome (mitochondrial-derived peptides) have been shown to have cellular cyto- and metabolo-protective roles that parallel those of exercise. Here we provide evidence that humanin and SHLP6 are exercise-sensitive mitochondrial derived peptides. Studies to determine whether mitochondrial-derived peptides play a role in regulating exercise-induced adaptations are warranted.

Aging is the leading risk factor for multiple non-communicable chronic diseases. Age-related physiological deterioration is underlined by a progressive loss of cellular homeostasis and physical capacity. Healthy aging can be promoted by preemptively enhancing physical capacity and metabolic fitness. Mitochondria are chief metabolic organelles with strong implications in aging. In addition to their prominent role in bioenergetics, mitochondria also coordinate broad physiological functions by communicating to other cellular compartments or distal cells, using multiple factors including peptides that are encoded within their own independent genome. However, it is unknown if aging is actively regulated by factors encoded in the mitochondrial genome. MOTS-c is a mitochondrial-encoded peptide that regulates metabolic homeostasis, in part, by translocating to the nucleus to regulate adaptive nuclear gene expression in response to cellular stress. Here, we report that MOTS-c is an exercise-induced mitochondrial-encoded peptide that significantly enhanced physical performance when administered to young (2 mo.), middle-aged (12 mo.), and old (22 mo.) mice. In humans, we found that endogenous MOTS-c levels significantly increased in response to exercise in skeletal muscle (11.9-fold) and in circulation (1.5-fold). Systemic MOTS-c treatment in mice significantly enhanced the performance on a treadmill of all age groups (∼2-fold). MOTS-c regulated (i) nuclear genes, including those related to metabolism and protein homeostasis, (ii) glucose and amino acid metabolism in skeletal muscle, and (iii) myoblast adaptation to metabolic stress. Notably, a statistical enrichment analysis on our RNA-seq data, from both mouse skeletal muscle and myoblasts, revealed heat shock factor 1 (HSF1) as a putative transcriptional factor that could regulate gene expression upon MOTS-c treatment. Indeed, siRNA-mediated HSF1 knockdown reversed MOTS-c-dependent stress resistance against glucose restriction/serum deprivation. Ultimately, late-life initiated intermittent MOTS-c treatment (23.5 months; 3x/week) improved overall physical capacity and trended towards increasing lifespan. Our data indicate that aging is regulated by genes that are encoded not only in the nuclear genome, but also in the mitochondrial genome. Considering that aging is the major risk factor for multiple chronic diseases, our study provides new grounds for further investigation into mitochondrial-encoded regulators of healthy lifespan that could also provide novel therapeutic targets of mitochondrial basis.

Neutrophil elastase (NE) is a serine protease expressed by neutrophils which is inhibited endogenously by α1-Antitrypsin A (AAT). Obesity is associated with increased plasma NE:AAT ratios and adipose tissue NE accumulation, which may drive obesity-associated metabolic dysfunction. Exogenous AAT is now an FDA approved treatment for AAT deficiency, therefore we investigated whether AAT treatment may also have potential therapeutic benefits for diet induced metabolic dysfunction. Standard Chow and high-fat diet (HFD) fed male C57Bl6 mice were randomized to receive 3x weekly IP injections of either AAT (2mg) or vehicle (PBS) for 10 weeks. Chow fed mice treated with recombinant AAT showed no differences in plasma NE, body weight or metabolic phenotype compared to vehicle treated control mice. However, during HFD feeding AAT treatment attenuated increases in plasma NE and white adipose tissue (WAT) NE and neutrophil accumulation without affecting circulatory neutrophil levels or body weight. Consistent with NE knockout mice being partially protected from HFD glucose intolerance, treatment of HFD fed mice with AAT enhanced whole body insulin sensitivity which was attributed to higher insulin-dependent p-AktSer473 and reduced inflammation markers in WAT but no other peripheral tissues. Treatment of 3T3L1 adipocytes with recombinant NE impaired insulin-stimulated glucose uptake andp-AktSer473, suggesting that inhibition of NE in WAT is the primary mechanism through which AAT treatment enhances insulin sensitivity. Collectively, our data suggests AAT may play a potential role in mitigating diet-induced neutrophil infiltration in WAT insulin resistance. Disclosure R.F. D’Souza: None. J.S.T. Woodhead: None. S.W. Masson: None. S.L. James: None. C. Hedges: None. T.L. Merry: None. Funding Health Research Council of New Zealand

MicroRNAs (miRNAs) regulate gene expression via transcript degradation and translational inhibition, and they may also function as long distance signaling molecules. Circulatory miRNAs are either protein-bound or packaged within vesicles (exosomes). Ten young men (24.6 ± 4.0 yr) underwent a single bout of high-intensity interval cycling exercise. Vastus lateralis biopsies and plasma were collected immediately before and after exercise, as well as 4 h following the exercise bout. Twenty-nine miRNAs previously reported to be regulated by acute exercise were assessed within muscle, venous plasma, and enriched circulatory exosomes via qRT-PCR. Of the 29 targeted miRNAs, 11 were altered in muscle, 8 in plasma, and 9 in the exosome fraction. Although changes in muscle and plasma expression were bidirectional, all regulated exosomal miRNAs increased following exercise. Three miRNAs were altered in all three sample pools (miR-1-3p, -16-5p, and -222-3p), three in both muscle and plasma (miR-21-5p, -134-3p, and -107), three in both muscle and exosomes (miR-23a-3p, -208a-3p, and -150-5p), and three in both plasma and exosomes (miR-486-5p, -126-3p, and -378a-5p). There was a marked discrepancy between the observed alterations between sample pools. A subset of exosomal miRNAs increased in abundance following exercise, suggesting an exercise-induced release of exosomes enriched in specific miRNAs. The uniqueness of the exosomal miRNA response suggests its relevance as a sample pool that needs to be further explored in better understanding biological functions.

One of the main sources of reactive oxygen species (ROS) in skeletal muscle is the mitochondria. Prolonged or very high ROS exposure causes oxidative damage, which can be deleterious to muscle function, and as such, there is growing interest in targeting antioxidants to the mitochondria in an effort to prevent or treat muscle dysfunction and damage associated with disease and injury. Paradoxically, however, ROS also act as important signalling molecules in controlling cellular homeostasis, and therefore caution must be taken when supplementing with antioxidants. It is possible that mitochondria-targeted antioxidants may limit oxidative stress without suppressing ROS from non-mitochondrial sources that might be important for cell signalling. Therefore, in this review, we summarise literature relating to the effect of mitochondria-targeted antioxidants on skeletal muscle function. Overall, mitochondria-targeted antioxidants appear to exert beneficial effects on mitochondrial capacity and function, insulin sensitivity and age-related declines in muscle function. However, it seems that this is dependent on the type of mitochondrial-trageted antioxidant employed, and its specific mechanism of action, rather than simply targeting to the mitochondria.

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.