Physical activity, inactivity and sleep during the Diabetes Remission Clinical Trial (DiRECT).

AIMS: As sustained weight loss is vital for achieving remission of type 2 diabetes, we explored whether randomisation to weight loss plus maintenance in the DiRECT trial was associated with physical activity, inactivity or sleep. METHODS: Participants were randomised to either a dietary weight management programme or best-practice care. The weight management group were encouraged to increase daily physical activity to their sustainable maximum. Objective measurement was achieved using a wrist-worn GENEActiv accelerometer for 7 days at baseline, 12 and 24 months in both groups. RESULTS: Despite average weight loss of 10 kg at 12 months in the intervention (n = 66) group, there were no differences in total physical activity or inactivity compared with the control (n = 104) at any time point. However, in our exploratory analysis, those who lost more than 10% of their baseline body weight performed on average 11 mins/day more light activity than the <10% group at 24 months (p = 0.033) and had significantly lower bouts of Inactivity30min (interaction, p = 0.005) across 12 and 24 months. At 24 months, the ≥10% group had higher daily acceleration (38.5 ± 12.1 vs. 33.2 ± 11.1 mg, p = 0.020), and higher accelerations in the most active 5-hour period (59.4 ± 21.8 vs. 50.6 ± 18.3 mg, p = 0.023). Wakefulness after sleep onset decreased in the intervention group compared with the control group and also in the ≥10% weight loss group at 12 and 24 months. CONCLUSIONS: Randomisation to a successful intensive weight loss intervention, including regular physical activity encouragement, was not associated with increased physical activity although sleep parameters improved. Physical activity was greater, and night-time waking reduced in those who maintained >10% weight loss at 12 and 24 months. TRIAL REGISTRATION ISRCTN03267836.

Acipimox in Mitochondrial Myopathy (AIMM): study protocol for a randomised, double-blinded, placebo-controlled, adaptive design trial of the efficacy of acipimox in adult patients with mitochondrial myopathy.

BACKGROUND: Mitochondrial disease is a heterogenous group of rare, complex neurometabolic disorders. Despite their individual rarity, collectively mitochondrial diseases represent the most common cause of inherited metabolic disorders in the UK; they affect 1 in every 4300 individuals, up to 15,000 adults (and a similar number of children) in the UK. Mitochondrial disease manifests multisystem and isolated organ involvement, commonly affecting those tissues with high energy demands, such as skeletal muscle. Myopathy manifesting as fatigue, muscle weakness and exercise intolerance is common and debilitating in patients with mitochondrial disease. Currently, there are no effective licensed treatments and consequently, there is an urgent clinical need to find an effective drug therapy. AIM: To investigate the efficacy of 12-week treatment with acipimox on the adenosine triphosphate (ATP) content of skeletal muscle in patients with mitochondrial disease and myopathy. METHODS: AIMM is a single-centre, double blind, placebo-controlled, adaptive designed trial, evaluating the efficacy of 12 weeks' administration of acipimox on skeletal muscle ATP content in patients with mitochondrial myopathy. Eligible patients will receive the trial investigational medicinal product (IMP), either acipimox or matched placebo. Participants will also be prescribed low dose aspirin as a non-investigational medical product (nIMP) in order to protect the blinding of the treatment assignment. Eighty to 120 participants will be recruited as required, with an interim analysis for sample size re-estimation and futility assessment being undertaken once the primary outcome for 50 participants has been obtained. Randomisation will be on a 1:1 basis, stratified by Fatigue Impact Scale (FIS) (dichotomised as < 40, ≥ 40). Participants will take part in the trial for up to 20 weeks, from screening visits through to follow-up at 16 weeks post randomisation. The primary outcome of change in ATP content in skeletal muscle and secondary outcomes relating to quality of life, perceived fatigue, disease burden, limb function, balance and walking, skeletal muscle analysis and symptom-limited cardiopulmonary fitness (optional) will be assessed between baseline and 12 weeks. DISCUSSION: The AIMM trial will investigate the effect of acipimox on modulating muscle ATP content and whether it can be repurposed as a new treatment for mitochondrial disease with myopathy. TRIAL REGISTRATION: EudraCT2018-002721-29 . Registered on 24 December 2018, ISRCTN 12895613. Registered on 03 January 2019, https://www.isrctn.com/search?q=aimm.

l-Arginine in Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like Episodes: A Systematic Review.

BACKGROUND AND OBJECTIVES: Stroke management in the context of primary mitochondrial disease is clinically challenging, and the best treatment options for patients with stroke-like episodes remain uncertain. We sought to perform a systematic review of the safety and efficacy of l-arginine use in the acute and prophylactic management of stroke-like episodes in patients with mitochondrial disease. METHODS: The systematic review was registered in PROSPERO (CRD42020181230). We searched 6 databases from inception to January 15, 2021: MEDLINE, Embase, Scopus, Web of Science, CINAHL, and ClinicalTrials.gov. Original articles and registered trials available, in English, reporting l-arginine use in the acute or prophylactic management of stroke-like episodes in patients with genetically confirmed mitochondrial disease were eligible for inclusion. Data on safety and treatment response were extracted and summarized by multiple observers. Risk of bias was assessed by the methodologic quality of case reports, case series, and a risk-of-bias checklist for nonrandomized studies. Quality of evidence was synthesized with the Oxford Centre for Evidence-Based Medicine Levels of Evidence and Grade of Recommendations. The predetermined main outcome measures were clinical response to l-arginine treatment, adverse events, withdrawals, and deaths (on treatment and/or during follow-up), as defined by the author. RESULTS: Thirty-seven articles met inclusion criteria (0 randomized controlled trials; 3 open-label; 1 retrospective cohort; 33 case reports/case series) (N = 91 patients; 86% m.3243A>G). In the case reports, 54% of patients reported a positive clinical response to acute l-arginine, of which 40% were concomitantly treated with antiepileptic drugs. Improved headache at 24 hours was the greatest reported benefit in response to IV l-arginine in the open-label trials (31 of 39, 79%). In 15 of 48 patients (31%) who positively responded to prophylactic l-arginine, antiepileptic drugs were either used (7 of 15) or unreported (8 of 15). Moderate adverse events were reported in the follow-up of both IV and oral l-arginine treatment, and 11 patients (12%) died during follow-up or while on prophylactic treatment. DISCUSSION: The available evidence is of poor methodologic quality and classified as Level 5. IV and oral l-arginine confers no demonstrable clinical benefit in either the acute or prophylactic treatment of mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes, with more robust controlled trials required to assess its efficacy and safety profile.

Current and Emerging Clinical Treatment in Mitochondrial Disease.

Primary mitochondrial disease (PMD) is a group of complex genetic disorders that arise due to pathogenic variants in nuclear or mitochondrial genomes. Although PMD is one of the most prevalent inborn errors of metabolism, it often exhibits marked phenotypic variation and can therefore be difficult to recognise. Current treatment for PMD revolves around supportive and preventive approaches, with few disease-specific therapies available. However, over the last decade there has been considerable progress in our understanding of both the genetics and pathophysiology of PMD. This has resulted in the development of a plethora of new pharmacological and non-pharmacological therapies at varying stages of development. Many of these therapies are currently undergoing clinical trials. This review summarises the latest emerging therapies that may become mainstream treatment in the coming years. It is distinct from other recent reviews in the field by comprehensively addressing both pharmacological non-pharmacological therapy from both a bench and a bedside perspective. We highlight the current and developing therapeutic landscape in novel pharmacological treatment, dietary supplementation, exercise training, device use, mitochondrial donation, tissue replacement gene therapy, hypoxic therapy and mitochondrial base editing.

Measuring the effects of exercise in neuromuscular disorders: a systematic review and meta-analyses.

Background: The benefit and safety of exercise training for patients with neuromuscular disorders (NMDs) has long been a contentious topic. This is, in part, due to recognised challenges associated with rare diseases including small and heterogenous patient populations. We performed a systematic review and meta-analyses to evaluate the effectiveness and safety of interventional exercise and establish minimal clinically important differences (MCID) in outcomes to facilitate clinical interpretation. Methods: We searched six databases from inception to Mar 2018. Aerobic, strength, and combined (aerobic and strength) intervention were eligible. Meta-analyses compared outcomes at baseline with those after at least six weeks (before-after exercise within individuals). A further meta-analysis compared outcomes before-after exercise between groups (exercise training versus usual care). Disease heterogeneity was explored using a random effect model. This study was registered (PROSPERO, CRD42018102183). An interactive database was developed to facilitate full interrogations of data. Results: We identified 130 articles describing 1,805 participants with 35 different forms of NMD. Of these studies, 76 were suitable for meta-analyses. Within group and between group meta-analyses detected an increase in peak aerobic capacity (p=0·04), and peak power (p=0·01). Six-minute walk test (p=0·04), sit-to-stand (STS) (repetitions) (p=0·03), STS (seconds) (p=0·04), rise from supine (p=0·008), SF-36 (p=0·0003), fatigue severity (p=<0·0001), citrate synthase (p=0·0002), central nuclei (p=0·04), type 1 (p=0·002) and type II muscle fibre area (p=0·003), were only able to detect change within group meta-analyses. Substantial I 2 statistic heterogeneity was revealed for STS (seconds) ( I²=58·5%; p=0·04) and citrate synthase ( I²=70·90%; p=0·002), otherwise heterogeneity for all outcomes was low. No study-related serious adverse events were reported nor significant increases in creatine kinase. Conclusions: Exercise training in patients with NMDs appears to cause no harm across a range of outcomes. With the emergence of new therapeutic strategies, defining MCID is vital in informing future clinical trial design.

Metabolic effects of bezafibrate in mitochondrial disease.

Mitochondrial disorders affect 1/5,000 and have no cure. Inducing mitochondrial biogenesis with bezafibrate improves mitochondrial function in animal models, but there are no comparable human studies. We performed an open-label observational experimental medicine study of six patients with mitochondrial myopathy caused by the m.3243A>G MTTL1 mutation. Our primary aim was to determine the effects of bezafibrate on mitochondrial metabolism, whilst providing preliminary evidence of safety and efficacy using biomarkers. The participants received 600-1,200 mg bezafibrate daily for 12 weeks. There were no clinically significant adverse events, and liver function was not affected. We detected a reduction in the number of complex IV-immunodeficient muscle fibres and improved cardiac function. However, this was accompanied by an increase in serum biomarkers of mitochondrial disease, including fibroblast growth factor 21 (FGF-21), growth and differentiation factor 15 (GDF-15), plus dysregulation of fatty acid and amino acid metabolism. Thus, although potentially beneficial in short term, inducing mitochondrial biogenesis with bezafibrate altered the metabolomic signature of mitochondrial disease, raising concerns about long-term sequelae.

Recent advances in understanding the role of FOXO3.

The forkhead box O3 (FOXO3, or FKHRL1) protein is a member of the FOXO subclass of transcription factors. FOXO proteins were originally identified as regulators of insulin-related genes; however, they are now established regulators of genes involved in vital biological processes, including substrate metabolism, protein turnover, cell survival, and cell death. FOXO3 is one of the rare genes that have been consistently linked to longevity in in vivo models. This review provides an update of the most recent research pertaining to the role of FOXO3 in (i) the regulation of protein turnover in skeletal muscle, the largest protein pool of the body, and (ii) the genetic basis of longevity. Finally, it examines (iii) the role of microRNAs in the regulation of FOXO3 and its impact on the regulation of the cell cycle.

A novel cardiac output response to stress test developed to improve diagnosis and monitoring of heart failure in primary care.

AIMS: Primary care physicians lack access to an objective cardiac function test. This study for the first time describes a novel cardiac output response to stress (CORS) test developed to improve diagnosis and monitoring of heart failure in primary care and investigates its reproducibility. METHODS AND RESULTS: Prospective observational study recruited 32 consecutive primary care patients (age, 63 ± 9 years; female, n = 18). Cardiac output was measured continuously using the bioreactance method in supine and standing positions and during two 3 min stages of a step-exercise protocol (10 and 15 steps per minute) using a 15 cm height bench. The CORS test was performed on two occasions, i.e. Test 1 and Test 2. There was no significant difference between repeated measures of cardiac output and stroke volume at supine standing and Stage 1 and Stage 2 step exercises (all P > 0.3). There was a significant positive relationship between Test 1 and Test 2 cardiac outputs (r = 0.92, P = 0.01 with coefficient of variation of 7.1%). The mean difference in cardiac output (with upper and lower limits of agreement) between Test 1 and Test 2 was 0.1 (-1.9 to 2.1) L/min, combining supine, standing, and step-exercise data. CONCLUSIONS: The CORS, as a novel test for objective evaluation of cardiac function, demonstrates acceptable reproducibility and can potentially be implemented in primary care.

Regulation of ubiquitin proteasome pathway molecular markers in response to endurance and resistance exercise and training.

Knowledge on the effects of divergent exercise on ostensibly protein degradation pathways may be valuable for counteracting muscle wasting and for understanding muscle remodelling. This study examined mRNA and/or protein levels of molecular markers of the ubiquitin proteasome pathway (UPP), including FBXO32 (atrogin-1), MURF-1, FBXO40, FOXO1 and FOXO3. Protein substrates of atrogin-1-including EIF3F, MYOG and MYOD1-and of MURF-1-including PKM and MHC-were also measured. Subjects completed 10 weeks of endurance training (ET) or resistance training (RT) followed by a single-bout of endurance exercise (EE) or resistance exercise (RE). Following training, atrogin-1, FBXO40, FOXO1 and FOXO3 mRNA increased independently of exercise mode, whereas MURF-1 mRNA and FOXO3 protein increased following ET only. No change in other target proteins occurred post-training. In the trained state, single-bout EE, but not RE, increased atrogin-1, MURF-1, FBXO40, FOXO1, FOXO3 mRNA and FOXO3 protein. In contrast to EE, FBXO40 mRNA and protein decreased following single-bout RE. MURF-1 and FOXO1 protein levels as well as the protein substrates of atrogin-1 and MURF-1 were unchanged following training and single-bout exercise. This study demonstrates that the intracellular signals elicited by ET and RT result in an upregulation of UPP molecular markers, with a greater increase following ET. However, in the trained state, the expression levels of UPP molecular markers are increased following single-bout EE, but are less responsive to single-bout RE. This suggests that adaptations following endurance exercise training are more reliant on protein UPP degradation processes than adaptations following resistance exercise training.

Ageing has no effect on the regulation of the ubiquitin proteasome-related genes and proteins following resistance exercise.

Skeletal muscle atrophy is a critical component of the ageing process. Age-related muscle wasting is due to disrupted muscle protein turnover, a process mediated in part by the ubiquitin proteasome pathway (UPP). Additionally, older subjects have been observed to have an attenuated anabolic response, at both the molecular and physiological levels, following a single-bout of resistance exercise (RE). We investigated the expression levels of the UPP-related genes and proteins involved in muscle protein degradation in 10 older (60-75 years) vs. 10 younger (18-30 years) healthy male subjects at basal as well as 2 h after a single-bout of RE. MURF1, atrogin-1 and FBXO40, their substrate targets PKM2, myogenin, MYOD, MHC and EIF3F as well as MURF1 and atrogin-1 transcriptional regulators FOXO1 and FOXO3 gene and/or protein expression levels were measured via real time PCR and western blotting, respectively. At basal, no age-related difference was observed in the gene/protein levels of atrogin-1, MURF1, myogenin, MYOD and FOXO1/3. However, a decrease in FBXO40 mRNA and protein levels was observed in older subjects, while PKM2 protein was increased. In response to RE, MURF1, atrogin-1 and FBXO40 mRNA were upregulated in both the younger and older subjects, with changes observed in protein levels. In conclusion, UPP-related gene/protein expression is comparably regulated in healthy young and old male subjects at basal and following RE. These findings suggest that UPP signaling plays a limited role in the process of age-related muscle wasting. Future studies are required to investigate additional proteolytic mechanisms in conjunction with skeletal muscle protein breakdown (MPB) measurements following RE in older vs. younger subjects.

Influence of divergent exercise contraction mode and whey protein supplementation on atrogin-1, MuRF1, and FOXO1/3A in human skeletal muscle.

Knowledge from human exercise studies on regulators of muscle atrophy is lacking, but it is important to understand the underlying mechanisms influencing skeletal muscle protein turnover and net protein gain. This study examined the regulation of muscle atrophy-related factors, including atrogin-1 and MuRF1, their upstream transcription factors FOXO1 and FOXO3A and the atrogin-1 substrate eIF3-f, in response to unilateral isolated eccentric (ECC) vs. concentric (CONC) exercise and training. Exercise was performed with whey protein hydrolysate (WPH) or isocaloric carbohydrate (CHO) supplementation. Twenty-four subjects were divided into WPH and CHO groups and completed both single-bout exercise and 12 wk of training. Single-bout ECC exercise decreased atrogin-1 and FOXO3A mRNA compared with basal and CONC exercise, while MuRF1 mRNA was upregulated compared with basal. ECC exercise downregulated FOXO1 and phospho-FOXO1 protein compared with basal, and phospho-FOXO3A was downregulated compared with CONC. CONC single-bout exercise mediated a greater increase in MuRF1 mRNA and increased FOXO1 mRNA compared with basal and ECC. CONC exercise downregulated FOXO1, FOXO3A, and eIF3-f protein compared with basal. Following training, an increase in basal phospho-FOXO1 was observed. While WPH supplementation with ECC and CONC training further increased muscle hypertrophy, it did not have an additional effect on mRNA or protein levels of the targets measured. In conclusion, atrogin-1, MuRF1, FOXO1/3A, and eIF3-f mRNA, and protein levels, are differentially regulated by exercise contraction mode but not WPH supplementation combined with hypertrophy-inducing training. This highlights the complexity in understanding the differing roles these factors play in healthy muscle adaptation to exercise.

Effect of resistance exercise contraction mode and protein supplementation on members of the STARS signalling pathway.

The striated muscle activator of Rho signalling (STARS) pathway is suggested to provide a link between external stress responses and transcriptional regulation in muscle. However, the sensitivity of STARS signalling to different mechanical stresses has not been investigated. In a comparative study, we examined the regulation of the STARS signalling pathway in response to unilateral resistance exercise performed as either eccentric (ECC) or concentric (CONC) contractions as well as prolonged training; with and without whey protein supplementation. Skeletal muscle STARS, myocardian-related transcription factor-A (MRTF-A) and serum response factor (SRF) mRNA and protein, as well as muscle cross-sectional area and maximal voluntary contraction, were measured. A single-bout of exercise produced increases in STARS and SRF mRNA and decreases in MRTF-A mRNA with both ECC and CONC exercise, but with an enhanced response occurring following ECC exercise. A 31% increase in STARS protein was observed exclusively after CONC exercise (P < 0.001), while pSRF protein levels increased similarly by 48% with both CONC and ECC exercise (P < 0.001). Prolonged ECC and CONC training equally stimulated muscle hypertrophy and produced increases in MRTF-A protein of 125% and 99%, respectively (P < 0.001). No changes occurred for total SRF protein. There was no effect of whey protein supplementation. These results show that resistance exercise provides an acute stimulation of the STARS pathway that is contraction mode dependent. The responses to acute exercise were more pronounced than responses to accumulated training, suggesting that STARS signalling is primarily involved in the initial phase of exercise-induced muscle adaptations.

Thiol-based antioxidant supplementation alters human skeletal muscle signaling and attenuates its inflammatory response and recovery after intense eccentric exercise.

BACKGROUND: The major thiol-disulfide couple of reduced glutathione (GSH) and oxidized glutathione is a key regulator of major transcriptional pathways regulating aseptic inflammation and recovery of skeletal muscle after aseptic injury. Antioxidant supplementation may hamper exercise-induced cellular adaptations. OBJECTIVE: The objective was to examine how thiol-based antioxidant supplementation affects skeletal muscle's performance and redox-sensitive signaling during the inflammatory and repair phases associated with exercise-induced microtrauma. DESIGN: In a double-blind, crossover design, 10 men received placebo or N-acetylcysteine (NAC; 20 mg · kg(-1) · d(-1)) after muscle-damaging exercise (300 eccentric contractions). In each trial, muscle performance was measured at baseline, after exercise, 2 h after exercise, and daily for 8 consecutive days. Muscle biopsy samples from vastus lateralis and blood samples were collected before exercise and 2 h, 2 d, and 8 d after exercise. RESULTS: NAC attenuated the elevation of inflammatory markers of muscle damage (creatine kinase activity, C-reactive protein, proinflammatory cytokines), nuclear factor κB phosphorylation, and the decrease in strength during the first 2 d of recovery. NAC also blunted the increase in phosphorylation of protein kinase B, mammalian target of rapamycin, p70 ribosomal S6 kinase, ribosomal protein S6, and mitogen activated protein kinase p38 at 2 and 8 d after exercise. NAC also abolished the increase in myogenic determination factor and reduced tumor necrosis factor-α 8 d after exercise. Performance was completely recovered only in the placebo group. CONCLUSION: Although thiol-based antioxidant supplementation enhances GSH availability in skeletal muscle, it disrupts the skeletal muscle inflammatory response and repair capability, potentially because of a blunted activation of redox-sensitive signaling pathways. This trial was registered at clinicaltrials.gov as NCT01778309.

Regulation of the STARS signaling pathway in response to endurance and resistance exercise and training.

The striated muscle activator of Rho signaling (STARS) protein and members of its downstream signaling pathway, including myocardin-related transcription factor-A (MRTF-A) and SRF, are increased in response to prolonged resistance exercise training but also following a single bout of endurance cycling. The aim of the present study was to measure and compare the regulation of STARS, MRTF-A and SRF mRNA and protein following 10 weeks of endurance training (ET) versus resistance training (RT), as well as before and following a single bout of endurance (EE) versus resistance exercise (RE). Following prolonged training, STARS, MRTF-A and SRF mRNA levels were all increased by similar magnitude, irrespective of training type. In the training-habituated state, STARS mRNA increased following a single-bout RE when measured 2.5 and 5 h post-exercise and had returned to resting level by 22 h following exercise. MRTF-A and SRF mRNA levels were decreased by 2.5, 5, and 22 h following a single bout of RE and EE exercise when compared to their respective basal levels, with no significant difference seen between the groups at any of the time points. No changes in protein levels were observed following the two modes of exercise training or a single bout of exercise. This study demonstrates that the stress signals elicited by ET and RT result in a comparable regulation of members of the STARS pathway. In contrast, a single bout of EE and RE, performed in the trained state, elicit different responses. These observations suggest that in the trained state, the acute regulation of the STARS pathway following EE or RE may be responsible for exercise-specific muscle adaptations.