Pharmacological Activation of PDC Flux Reverses Lipid-Induced Inhibition of Insulin Action in Muscle During Recovery From Exercise.

Insulin resistance is a risk factor for type 2 diabetes, and exercise can improve insulin sensitivity. However, following exercise, high circulating fatty acid (FA) levels might counteract this. We hypothesized that such inhibition would be reduced by forcibly increasing carbohydrate oxidation through pharmacological activation of the pyruvate dehydrogenase complex (PDC). Insulin-stimulated glucose uptake was examined with a crossover design in healthy young men (n = 8) in a previously exercised and a rested leg during a hyperinsulinemic-euglycemic clamp 5 h after one-legged exercise with 1) infusion of saline, 2) infusion of intralipid imitating circulating FA levels during recovery from whole-body exercise, and 3) infusion of intralipid + oral PDC activator, dichloroacetate (DCA). Intralipid infusion reduced insulin-stimulated glucose uptake by 19% in the previously exercised leg, which was not observed in the contralateral rested leg. Interestingly, this effect of intralipid in the exercised leg was abolished by DCA, which increased muscle PDC activity (130%) and flux (acetylcarnitine 130%) and decreased inhibitory phosphorylation of PDC on Ser293 (∼40%) and Ser300 (∼80%). Novel insight is provided into the regulatory interaction between glucose and lipid metabolism during exercise recovery. Coupling exercise and PDC flux activation upregulated the capacity for both glucose transport (exercise) and oxidation (DCA), which seems necessary to fully stimulate insulin-stimulated glucose uptake during recovery.

Bed-rest and exercise remobilization: Concurrent adaptations in muscle glucose and protein metabolism.

BACKGROUND: Bed-rest (BR) of only a few days duration reduces muscle protein synthesis and induces skeletal muscle atrophy and insulin resistance, but the scale and juxtaposition of these events have not been investigated concurrently in the same individuals. Moreover, the impact of short-term exercise-supplemented remobilization (ESR) on muscle volume, protein turnover and leg glucose uptake (LGU) in humans is unknown. METHODS: Ten healthy males (24 ± 1 years, body mass index 22.7 ± 0.6 kg/m2) underwent 3 days of BR, followed immediately by 3 days of ESR consisting of 5 × 30 maximal voluntary single-leg isokinetic knee extensions at 90°/s each day. An isoenergetic diet was maintained throughout the study (30% fat, 15% protein and 55% carbohydrate). Resting LGU was calculated from arterialized-venous versus venous difference across the leg and leg blood flow during the steady-state of a 3-h hyperinsulinaemic-euglycaemic clamp (60 mU/m2/min) measured before BR, after BR and after remobilization. Glycogen content was measured in vastus lateralis muscle biopsy samples obtained before and after each clamp. Leg muscle volume (LMV) was measured using magnetic resonance imaging before BR, after BR and after remobilization. Cumulative myofibrillar protein fractional synthetic rate (FSR) and whole-body muscle protein breakdown (MPB) were measured over the course of BR and remobilization using deuterium oxide and 3-methylhistidine stable isotope tracers that were administered orally. RESULTS: Compared with before BR, there was a 45% decline in insulin-stimulated LGU (P < 0.05) after BR, which was paralleled by a reduction in insulin-stimulated leg blood flow (P < 0.01) and removal of insulin-stimulated muscle glycogen storage. These events were accompanied by a 43% reduction in myofibrillar protein FSR (P < 0.05) and a 2.5% decrease in LMV (P < 0.01) during BR, along with a 30% decline in whole-body MPB after 2 days of BR (P < 0.05). Myofibrillar protein FSR and LMV were restored by 3 days of ESR (P < 0.01 and P < 0.01, respectively) but not by ambulation alone. However, insulin-stimulated LGU and muscle glycogen storage were not restored by ESR. CONCLUSIONS: Three days of BR caused concurrent reductions in LMV, myofibrillar protein FSR, myofibrillar protein breakdown and insulin-stimulated LGU, leg blood flow and muscle glycogen storage in healthy, young volunteers. Resistance ESR restored LMV and myofibrillar protein FSR, but LGU and muscle glycogen storage remained depressed, highlighting divergences in muscle fuel and protein metabolism. Furthermore, ambulation alone did not restore LMV and myofibrillar protein FSR in the non-exercised contralateral limb, emphasizing the importance of exercise rehabilitation following even short-term BR.

Human adaptation to immobilization: Novel insights of impacts on glucose disposal and fuel utilization.

BACKGROUND: Bed rest (BR) reduces whole-body insulin-stimulated glucose disposal (GD) and alters muscle fuel metabolism, but little is known about metabolic adaptation from acute to chronic BR nor the mechanisms involved, particularly when volunteers are maintained in energy balance. METHODS: Healthy males (n = 10, 24.0 ± 1.3 years), maintained in energy balance, underwent 3-day BR (acute BR). A second cohort matched for sex and body mass index (n = 20, 34.2 ± 1.8 years) underwent 56-day BR (chronic BR). A hyperinsulinaemic euglycaemic clamp (60 mU/m2 /min) was performed to determine rates of whole-body insulin-stimulated GD before and after BR (normalized to lean body mass). Indirect calorimetry was performed before and during steady state of each clamp to calculate rates of whole-body fuel oxidation. Muscle biopsies were taken to determine muscle glycogen, metabolite and intramyocellular lipid (IMCL) contents, and the expression of 191 mRNA targets before and after BR. Two-way repeated measures analysis of variance was used to detect differences in endpoint measures. RESULTS: Acute BR reduced insulin-mediated GD (Pre 11.5 ± 0.7 vs. Post 9.3 ± 0.6 mg/kg/min, P < 0.001), which was unchanged in magnitude following chronic BR (Pre 10.2 ± 0.4 vs. Post 7.9 ± 0.3 mg/kg/min, P < 0.05). This reduction in GD was paralleled by the elimination of the 35% increase in insulin-stimulated muscle glycogen storage following both acute and chronic BR. Acute BR had no impact on insulin-stimulated carbohydrate (CHO; Pre 3.69 ± 0.39 vs. Post 4.34 ± 0.22 mg/kg/min) and lipid (Pre 1.13 ± 0.14 vs. Post 0.59 ± 0.11 mg/kg/min) oxidation, but chronic BR reduced CHO oxidation (Pre 3.34 ± 0.18 vs. Post 2.72 ± 0.13 mg/kg/min, P < 0.05) and blunted the magnitude of insulin-mediated inhibition of lipid oxidation (Pre 0.60 ± 0.07 vs. Post 0.85 ± 0.06 mg/kg/min, P < 0.05). Neither acute nor chronic BR increased muscle IMCL content. Plentiful mRNA abundance changes were detected following acute BR, which waned following chronic BR and reflected changes in fuel oxidation and muscle glycogen storage at this time point. CONCLUSIONS: Acute BR suppressed insulin-stimulated GD and storage, but the extent of this suppression increased no further in chronic BR. However, insulin-mediated inhibition of fat oxidation after chronic BR was less than acute BR and was accompanied by blunted CHO oxidation. The juxtaposition of these responses shows that the regulation of GD and storage can be dissociated from substrate oxidation. Additionally, the shift in substrate oxidation after chronic BR was not explained by IMCL accumulation but reflected by muscle mRNA and pyruvate dehydrogenase kinase 4 protein abundance changes, pointing to lack of muscle contraction per se as the primary signal for muscle adaptation.

Whole-body and muscle responses to aerobic exercise training and withdrawal in ageing and COPD.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) patients exhibit lower peak oxygen uptake (V'O2 peak), altered muscle metabolism and impaired exercise tolerance compared with age-matched controls. Whether these traits reflect muscle-level deconditioning (impacted by ventilatory constraints) and/or dysfunction in mitochondrial ATP production capacity is debated. By studying aerobic exercise training (AET) at a matched relative intensity and subsequent exercise withdrawal period we aimed to elucidate the whole-body and muscle mitochondrial responsiveness of healthy young (HY), healthy older (HO) and COPD volunteers to whole-body exercise. METHODS: HY (n=10), HO (n=10) and COPD (n=20) volunteers were studied before and after 8 weeks of AET (65% V'O2 peak) and after 4 weeks of exercise withdrawal. V'O2 peak, muscle maximal mitochondrial ATP production rate (MAPR), mitochondrial content, mitochondrial DNA (mtDNA) copy number and abundance of 59 targeted fuel metabolism mRNAs were determined at all time-points. RESULTS: Muscle MAPR (normalised for mitochondrial content) was not different for any substrate combination in HO, HY and COPD at baseline, but mtDNA copy number relative to a nuclear-encoded housekeeping gene (mean±sd) was greater in HY (804±67) than in HO (631±69; p=0.041). AET increased V'O2 peak in HO (17%; p=0.002) and HY (21%; p<0.001), but not COPD (p=0.603). Muscle MAPR for palmitate increased with training in HO (57%; p=0.041) and HY (56%; p=0.003), and decreased with exercise withdrawal in HO (-45%; p=0.036) and HY (-30%; p=0.016), but was unchanged in COPD (p=0.594). mtDNA copy number increased with AET in HY (66%; p=0.001), but not HO (p=0.081) or COPD (p=0.132). The observed changes in muscle mRNA abundance were similar in all groups after AET and exercise withdrawal. CONCLUSIONS: Intrinsic mitochondrial function was not impaired by ageing or COPD in the untrained state. Whole-body and muscle mitochondrial responses to AET were robust in HY, evident in HO, but deficient in COPD. All groups showed robust muscle mRNA responses. Higher relative exercise intensities during whole-body training may be needed to maximise whole-body and muscle mitochondrial adaptation in COPD.

Molecular Mechanisms of Muscle Fatigue.

Muscle fatigue (MF) declines the capacity of muscles to complete a task over time at a constant load. MF is usually short-lasting, reversible, and is experienced as a feeling of tiredness or lack of energy. The leading causes of short-lasting fatigue are related to overtraining, undertraining/deconditioning, or physical injury. Conversely, MF can be persistent and more serious when associated with pathological states or following chronic exposure to certain medication or toxic composites. In conjunction with chronic fatigue, the muscle feels floppy, and the force generated by muscles is always low, causing the individual to feel frail constantly. The leading cause underpinning the development of chronic fatigue is related to muscle wasting mediated by aging, immobilization, insulin resistance (through high-fat dietary intake or pharmacologically mediated Peroxisome Proliferator-Activated Receptor (PPAR) agonism), diseases associated with systemic inflammation (arthritis, sepsis, infections, trauma, cardiovascular and respiratory disorders (heart failure, chronic obstructive pulmonary disease (COPD))), chronic kidney failure, muscle dystrophies, muscle myopathies, multiple sclerosis, and, more recently, coronavirus disease 2019 (COVID-19). The primary outcome of displaying chronic muscle fatigue is a poor quality of life. This type of fatigue represents a significant daily challenge for those affected and for the national health authorities through the financial burden attached to patient support. Although the origin of chronic fatigue is multifactorial, the MF in illness conditions is intrinsically linked to the occurrence of muscle loss. The sequence of events leading to chronic fatigue can be schematically denoted as: trigger (genetic or pathological) -> molecular outcome within the muscle cell -> muscle wasting -> loss of muscle function -> occurrence of chronic muscle fatigue. The present review will only highlight and discuss current knowledge on the molecular mechanisms that contribute to the upregulation of muscle wasting, thereby helping us understand how we could prevent or treat this debilitating condition.

PPARα, δ and FOXO1 Gene Silencing Overturns Palmitate-Induced Inhibition of Pyruvate Oxidation Differentially in C2C12 Myotubes.

The molecular mechanisms by which free fatty acids (FFA) inhibit muscle glucose oxidation is still elusive. We recently showed that C2C12 myotubes treated with palmitate (PAL) presented with greater protein expression levels of PDK4 and transcription factors PPARα and PPARδ and lower p-FOXO/t-FOXO protein ratios when compared to control. This was complemented with the hallmarks of metabolic inflexibility (MI), i.e., reduced rates of glucose uptake, PDC activity and maximal pyruvate-derived ATP production rates (MAPR). However, the relative contribution of these transcription factors to the increase in PDK4 and reduced glucose oxidation could not be established. Therefore, by using a similar myotube model, a series of individual siRNA gene silencing experiments, validated at transcriptional and translation levels, were performed in conjunction with measurements of glucose uptake, PDC activity, MAPR and concentrations of metabolites reflecting PDC flux (lactate and acetylcarnitine). Gene silencing of PPARα, δ and FOXO1 individually reduced PAL-mediated inhibition of PDC activity and increased glucose uptake, albeit by different mechanisms as only PPARδ and FOXO1 silencing markedly reduced PDK4 protein content. Additionally, PPARα and FOXO1 silencing, but not PPARδ, increased MAPR with PAL. PPARδ silencing also decreased FOXO1 protein. Since FOXO1 silencing did not alter PPARδ protein, this suggests that FOXO1 might be a PPARδ downstream target. In summary, this study suggests that the molecular mechanisms by which PAL reduces PDC-mediated glucose-derived pyruvate oxidation in muscle occur primarily through increased PPARδ and FOXO1 mediated increases in PDK4 protein expression and secondarily through PPARα mediated allosteric inhibition of PDC flux. Furthermore, since PPARδ seems to control FOXO1 expression, this may reflect an important role for PPARδ in preventing glucose oxidation under conditions of increased lipid availability.

The Regulatory Roles of PPARs in Skeletal Muscle Fuel Metabolism and Inflammation: Impact of PPAR Agonism on Muscle in Chronic Disease, Contraction and Sepsis.

The peroxisome proliferator-activated receptor (PPAR) family of transcription factors has been demonstrated to play critical roles in regulating fuel selection, energy expenditure and inflammation in skeletal muscle and other tissues. Activation of PPARs, through endogenous fatty acids and fatty acid metabolites or synthetic compounds, has been demonstrated to have lipid-lowering and anti-diabetic actions. This review will aim to provide a comprehensive overview of the functions of PPARs in energy homeostasis, with a focus on the impacts of PPAR agonism on muscle metabolism and function. The dysregulation of energy homeostasis in skeletal muscle is a frequent underlying characteristic of inflammation-related conditions such as sepsis. However, the potential benefits of PPAR agonism on skeletal muscle protein and fuel metabolism under these conditions remains under-investigated and is an area of research opportunity. Thus, the effects of PPARγ agonism on muscle inflammation and protein and carbohydrate metabolism will be highlighted, particularly with its potential relevance in sepsis-related metabolic dysfunction. The impact of PPARδ agonism on muscle mitochondrial function, substrate metabolism and contractile function will also be described.

Potential role for pyruvate kinase M2 in the regulation of murine cardiac glycolytic flux during in vivo chronic hypoxia.

Carbohydrate metabolism in heart failure shares similarities to that following hypoxic exposure, and is thought to maintain energy homoeostasis in the face of reduced O2 availability. As part of these in vivo adaptations during sustained hypoxia, the heart up-regulates and maintains a high glycolytic flux, but the underlying mechanism is still elusive. We followed the cardiac glycolytic responses to a chronic hypoxic (CH) intervention using [5-3H]-glucose labelling in combination with detailed and extensive enzymatic and metabolomic approaches to provide evidence of the underlying mechanism that allows heart survivability. Following 3 weeks of in vivo hypoxia (11% oxygen), murine hearts were isolated and perfused in a retrograde mode with function measured via an intraventricular balloon and glycolytic flux quantified using [5-3H]-glucose labelling. At the end of perfusion, hearts were flash-frozen and central carbon intermediates determined via liquid chromatography tandem mass spectrometry (LC-MS/MS). The maximal activity of glycolytic enzymes considered rate-limiting was assessed enzymatically, and protein abundance was determined using Western blotting. Relative to normoxic hearts, CH increased ex vivo cardiac glycolytic flux 1.7-fold with no effect on cardiac function. CH up-regulated cardiac pyruvate kinase (PK) flux 3.1-fold and cardiac pyruvate kinase muscle isoenzyme M2 (PKM2) protein content 1.4-fold compared with normoxic hearts. CH also augmented cardiac pentose phosphate pathway (PPP) flux, reflected by higher ribose-5-phosphate (R5P) content. These findings support an increase in the covalent (protein expression) and allosteric (flux) control of PKM2 as being central to the sustained up-regulation of the glycolytic flux in the chronically hypoxic heart.

Major elective abdominal surgery acutely impairs lower limb muscle pyruvate dehydrogenase complex activity and mitochondrial function.

BACKGROUND & AIMS: This post hoc study aimed to determine whether major elective abdominal surgery had any acute impact on mitochondrial pyruvate dehydrogenase complex (PDC) activity and maximal mitochondrial ATP production rates (MAPR) in a large muscle group (vastus lateralis -VL) distant to the site of surgical trauma. METHODS: Fifteen patients undergoing major elective open abdominal surgery were studied. Muscle biopsies were obtained after the induction of anesthesia from the VL immediately before and after surgery for the determination of PDC and maximal MAPR (utilizing a variety of energy substrates). RESULTS: Muscle PDC activity was reduced by >50% at the end of surgery compared with pre-surgery (p < 0.05). Muscle MAPR were comprehensively suppressed by surgery for the substrate combinations: glutamate + succinate; glutamate + malate; palmitoylcarnitine + malate; and pyruvate + malate (all p < 0.05), and could not be explained by a lower mitochondrial yield. CONCLUSIONS: PDC activity and mitochondrial ATP production capacity were acutely impaired in muscle distant to the site of surgical trauma. In keeping with the limited data available, we surmise these events resulted from the general anesthesia procedures employed and the surgery related trauma. These findings further the understanding of the acute dysregulation of mitochondrial function in muscle distant to the site of major surgical trauma in patients, and point to the combination of general anesthesia and trauma related inflammation as being drivers of muscle metabolic insult that warrants further investigation. CLINICAL TRIAL REGISTRATION: Registered at (NCT01134809).

Spinal neuronal excitability and neuroinflammation in a model of chemotherapeutic neuropathic pain: targeting the resolution pathways.

BACKGROUND: Neuroinflammation is a critical feature of sensitisation of spinal nociceptive processing in chronic pain states. We hypothesised that the resolvin pathways, a unique endogenous control system, may ameliorate aberrant spinal processing of somatosensory inputs associated with chemotherapy-induced neuropathic pain (CINP). METHOD: The paclitaxel (PCX) model of CINP was established in male Sprague-Dawley rats and compared to control rats (n = 23 and 22, respectively). Behavioural pain responses were measured, and either single unit electrophysiological recordings of dorsal horn wide dynamic range (WDR) neurones were performed, or mRNA microarray analysis of the dorsal horn of the spinal cord was undertaken. RESULTS: PCX rats exhibited significant changes in behavioural responses to mechanical and cold stimuli. A higher proportion of WDR neurones in PCX rats were polymodal (generating post-discharge following a non-noxious mechanical stimulus, responding to non-noxious cold and exhibiting spontaneous activity) compared to control (p < 0.05). Microarray analysis revealed changes in proinflammatory pathways (Tlr, Tnfrsf1a, Nlrp1a, Cxcr1, Cxcr5, Ccr1, Cx3cr1) and anti-inflammatory lipid resolvin pathways (Alox5ap, Cyp2j4 and Ptgr1) compared to control (p < 0.05). Ingenuity pathway analysis predicted changes in glutamatergic and astrocyte signaling in the PCX group. Activation of the resolvin system via the spinal administration of aspirin-triggered resolvin D1 (AT-RvD1) markedly inhibited (73 ± 7% inhibition) normally non-noxious mechanically (8 g) evoked responses of WDR neurones only in PCX rats, whilst leaving responses to noxious mechanically induced stimuli intact. Inhibitory effects of AT-RvD1were comparable in magnitude to spinal morphine (84 ± 4% inhibition). CONCLUSION: The PCX model of CINP was associated with mechanical allodynia, altered neuronal responses and dysregulation of pro- and anti-inflammatory signalling in the spinal dorsal horn. The resolvin AT-RvD1 selectively inhibited low weight mechanical-evoked responses of WDR neurones in PCX rats, but not in controls. Our data support the targeting of spinal neuroinflammation via the activation of the resolvin system as a new therapeutic approach for CINP.

Longitudinal hypertrophic and transcriptional responses to high-load eccentric-concentric vs concentric training in males.

High-load eccentric training reputedly produces greater muscle hypertrophy than concentric training, possibly due to greater loading and/or inflammation. We quantified the temporal impact of combined maximal concentric-eccentric training vs maximal concentric training on muscle cross-sectional area (CSA), volume, and targeted mRNA expression (93 transcripts). Eight recreationally active males (24 ± 5 years, BMI 23.5 ± 2.5 kg/m2 ) performed 3 x 30 maximal eccentric isokinetic knee extensions and 2 x 30 maximal concentric knee extensions in dominant limb (ECC + CON) and 5 x 30 maximal concentric contractions (CON) in the non-dominant limb for 12 weeks (all 90°/s, 3x/wk). Quadriceps muscle CSA and volume were measured at baseline, 28 days (d), and 84 d in both limbs (3T MRI). Resting vastus lateralis biopsies were obtained from both limbs at baseline, 24 hours (h), 7, 28, and 84 d for mRNA abundance measurements (RT-PCR microfluidic cards). Work output was greater throughout training in ECC + CON vs CON (20.8 ± 9.7%, P < .001). Muscle CSA increased from baseline in both limbs at 28 d (CON 4.3 ± 2.6%, ECC + CON 4.0 ± 1.9%, both P < .001) and 84d (CON 3.9 ± 2.3%, ECC + CON 4.0 ± 3.1%, both P < .001), and muscle volume and isometric strength at 84 d (CON 44.8 ± 40.0%, P < .001; ECC + CON 36.9 ± 40.0%, P < .01), but no between-limb differences existed in any parameter. Ingenuity Pathway Analysis identified several cellular functions associated with regulation of muscle mass and metabolism as altered by both modalities at 24 h and 7 d, but particularly with ECC + CON. However, mRNA responses waned thereafter, regardless of modality. Initial muscle mRNA responses to training did not reflect chronic training-induced hypertrophy. Moreover, ECC + CON did not produce greater hypertrophy than CON, despite greater loading throughout and a differential mRNA response during the initial training week.

PPARδ and FOXO1 Mediate Palmitate-Induced Inhibition of Muscle Pyruvate Dehydrogenase Complex and CHO Oxidation, Events Reversed by Electrical Pulse Stimulation.

The mechanisms behind the reduction in muscle pyruvate dehydrogenase complex (PDC)-controlled carbohydrate (CHO) oxidation during chronic high-fat dietary intake are poorly understood, as is the basis of CHO oxidation restoration during muscle contraction. C2C12 myotubes were treated with (300 µM) palmitate or without (control) for 16 h in the presence and absence of electrical pulse stimulation (EPS, 11.5 V, 1 Hz, 2 ms). Compared to control, palmitate reduced cell glucose uptake (p < 0.05), PDC activity (p < 0.01), acetylcarnitine accumulation (p < 0.05) and glucose-derived mitochondrial ATP production (p < 0.01) and increased pyruvate dehydrogenase kinase isoform 4 (PDK4) (p < 0.01), peroxisome proliferator-activated receptor alpha (PPARα) (p < 0.01) and peroxisome proliferator-activated receptor delta (PPARδ) (p < 0.01) proteins, and reduced the whole-cell p-FOXO1/t-FOXO1 (Forkhead Box O1) ratio (p < 0.01). EPS rescued palmitate-induced inhibition of CHO oxidation, reflected by increased glucose uptake (p < 0.01), PDC activity (p < 0.01) and glucose-derived mitochondrial ATP production (p < 0.01) compared to palmitate alone. EPS was also associated with less PDK4 (p < 0.01) and PPARδ (p < 0.01) proteins, and lower nuclear p-FOXO1/t-FOXO1 ratio normalised to the cytoplasmic ratio, but with no changes in PPARα protein. Collectively, these data suggest PPARδ, and FOXO1 transcription factors increased PDK4 protein in the presence of palmitate, which limited PDC activity and flux, and blunted CHO oxidation and glucose uptake. Conversely, EPS rescued these metabolic events by modulating the same transcription factors.

Skeletal muscle dysregulation in rheumatoid arthritis: Metabolic and molecular markers in a rodent model and patients.

Rheumatoid arthritis (RA) is accompanied by pain, inflammation and muscle weakness. Skeletal muscle inflammation and inactivity are independently associated with muscle insulin resistance and atrophy. Our objective was to identify early molecular and biochemical markers in muscle from a rodent model of RA relative to control and subsequently identify commonality in muscle gene expression between this model and muscle from RA patients. Pain behaviour and locomotor activity were measured in a collagen-induced arthritis (CIA) model of RA (n = 9) and control (n = 9) rats. Energy substrates and metabolites, total alkaline-soluble protein:DNA ratio and mRNA abundance of 46 targeted genes were also determined in Extensor digitorum longus muscle. Expression of targeted mRNAs was quantified in Vastus Lateralis muscle from RA patients (n = 7) and healthy age-matched control volunteers (n = 6). CIA rats exhibited pain behaviour (p<0.01) and reduced activity (p<0.05) compared to controls. Muscle glycogen content was less (p<0.05) and muscle lactate content greater (p<0.01) in CIA rats. The bioinformatics analysis of muscle mRNA abundance differences from the control, predicted the activation of muscle protein metabolism and inhibition of muscle carbohydrate and fatty acid metabolism in CIA rats. Compared to age-matched control volunteers, RA patients exhibited altered muscle mRNA expression of 8 of the transcripts included as targets in the CIA model of RA. In conclusion, muscle energy metabolism and metabolic gene expression were altered in the CIA model, which was partly corroborated by targeted muscle mRNA measurements in RA patients. This research highlights the negative impact of RA on skeletal muscle metabolic homeostasis.

Mitochondrial DNA copy number associates with insulin sensitivity and aerobic capacity, and differs between sedentary, overweight middle-aged males with and without type 2 diabetes.

BACKGROUND/OBJECTIVES: Increased risk of type 2 diabetes mellitus (T2DM) is linked to impaired muscle mitochondrial function and reduced mitochondrial DNA copy number (mtDNAnum). However, studies have failed to control for habitual physical activity levels, which directly influences both mtDNA copy number and insulin sensitivity. We, therefore, examined whether physical conditioning status (maximal oxygen uptake, V̇O2max) was associated with skeletal muscle mitochondrial volume and mtDNAnum, and was predictive of T2DM in overweight, middle-aged men. METHODS: Whole-body physiological (ISI-insulin sensitivity index, HOMA-IR, V̇O2max) and muscle biochemical/molecular (vastus lateralis; mtDNAnum, mitochondrial and glycolytic enzymes activity, lipid content and markers of lipid peroxidation) measurements were performed in three groups of overweight, middle-aged male volunteers (n = 10 per group): sedentary T2DM (ST2DM); sedentary control (SC) and non-sedentary control (NSC), who differed in aerobic capacity (ST2DM < SC < NSC). RESULTS: mtDNAnum was greater in NSC versus SC and ST2DM (P < 0.001; P < 0.001), and less in ST2DM versus SC (P < 0.01). Across all groups, mtDNAnum positively correlated with ISI (P < 0.001; r = 0.688) and V̇O2max (normalised to free fat mass; r = 0.684, P < 0.001), and negatively correlated to HOMA-IR (r = -0.544, P < 0.01). The activity of mitochondrial enzymes (GluDH, CS and ß-HAD) was greater in NSC than ST2DM (P < 0.01, P < 0.001 and P < 0.05) and SC (P < 0.05, P < 0.01 and P < 0.05), but similar between ST2DM and SC. Intramuscular-free fatty acids, triglycerides and malondialdehyde contents were similar between ST2DM and SC. CONCLUSIONS: Body composition and indices of muscle mitochondrial volume/function were similar between SC and ST2DM. However, mtDNAnum differed and was positively associated with ISI, HOMA-IR and V̇O2max across all groups. Collectively, the findings support the contention that habitual physical activity is a key component of T2DM development, possibly by influencing mtDNAnum.

Inorganic nitrate and nitrite supplementation fails to improve skeletal muscle mitochondrial efficiency in mice and humans.

BACKGROUND: Inorganic nitrate, abundant in leafy green vegetables and beetroot, is thought to have protective health benefits. Adherence to a Mediterranean diet reduces the incidence and severity of coronary artery disease, whereas supplementation with nitrate can improve submaximal exercise performance. Once ingested, oral commensal bacteria may reduce nitrate to nitrite, which may subsequently be reduced to nitric oxide during conditions of hypoxia and in the presence of "nitrite reductases" such as heme- and molybdenum-containing enzymes. OBJECTIVE: We aimed to explore the putative effects of inorganic nitrate and nitrite on mitochondrial function in skeletal muscle. METHODS: Mice were subjected to a nitrate/nitrite-depleted diet for 2 wk, then supplemented with sodium nitrate, sodium nitrite, or sodium chloride (1 g/L) in drinking water ad libitum for 7 d before killing. Skeletal muscle mitochondrial function and expression of uncoupling protein (UCP) 3, ADP/ATP carrier protein (AAC) 1 and AAC2, and pyruvate dehydrogenase (PDH) were assessed by respirometry and Western blotting. Studies were also undertaken in human skeletal muscle biopsies from a cohort of coronary artery bypass graft patients treated with either sodium nitrite (30-min infusion of 10 µmol/min) or vehicle [0.9% (wt:vol) saline] 24 h before surgery. RESULTS: Neither sodium nitrate nor sodium nitrite supplementation altered mitochondrial coupling efficiency in murine skeletal muscle, and expression of UCP3, AAC1, or AAC2, and PDH phosphorylation status did not differ between the nitrite and saline groups. Similar results were observed in human samples. CONCLUSIONS: Sodium nitrite failed to improve mitochondrial metabolic efficiency, rendering this mechanism implausible for the purported exercise benefits of dietary nitrate supplementation. This trial was registered at clinicaltrials.gov as NCT04001283.

Testosterone therapy induces molecular programming augmenting physiological adaptations to resistance exercise in older men.

BACKGROUND: The andropause is associated with declines in serum testosterone (T), loss of muscle mass (sarcopenia), and frailty. Two major interventions purported to offset sarcopenia are anabolic steroid therapies and resistance exercise training (RET). Nonetheless, the efficacy and physiological and molecular impacts of T therapy adjuvant to short-term RET remain poorly defined. METHODS: Eighteen non-hypogonadal healthy older men, 65-75 years, were assigned in a random double-blinded fashion to receive, biweekly, either placebo (P, saline, n = 9) or T (Sustanon 250 mg, n = 9) injections over 6 week whole-body RET (three sets of 8-10 repetitions at 80% one-repetition maximum). Subjects underwent dual-energy X-ray absorptiometry, ultrasound of vastus lateralis (VL) muscle architecture, and knee extensor isometric muscle force tests; VL muscle biopsies were taken to quantify myogenic/anabolic gene expression, anabolic signalling, muscle protein synthesis (D2 O), and breakdown (extrapolated). RESULTS: Testosterone adjuvant to RET augmented total fat-free mass (P=0.007), legs fat-free mass (P=0.02), and appendicular fat-free mass (P=0.001) gains while decreasing total fat mass (P=0.02). Augmentations in VL muscle thickness, fascicle length, and quadriceps cross-section area with RET occured to a greater extent in T (P < 0.05). Sum strength (P=0.0009) and maximal voluntary contract (e.g. knee extension at 70°) (P=0.002) increased significantly more in the T group. Mechanistically, both muscle protein synthesis rates (T: 2.13 ± 0.21%·day-1 vs. P: 1.34 ± 0.13%·day-1 , P=0.0009) and absolute breakdown rates (T: 140.2 ± 15.8 g·day-1 vs. P: 90.2 ± 11.7 g·day-1 , P=0.02) were elevated with T therapy, which led to higher net turnover and protein accretion in the T group (T: 8.3 ± 1.4 g·day-1 vs. P: 1.9 ± 1.2 g·day-1 , P=0.004). Increases in ribosomal biogenesis (RNA:DNA ratio); mRNA expression relating to T metabolism (androgen receptor: 1.4-fold; Srd5a1: 1.6-fold; AKR1C3: 2.1-fold; and HSD17ß3: two-fold); insulin-like growth factor (IGF)-1 signalling [IGF-1Ea (3.5-fold) and IGF-1Ec (three-fold)] and myogenic regulatory factors; and the activity of anabolic signalling (e.g. mTOR, AKT, and RPS6; P < 0.05) were all up-regulated with T therapy. Only T up-regulated mitochondrial citrate synthase activity (P=0.03) and transcription factor A (1.41 ± 0.2-fold, P=0.0002), in addition to peroxisome proliferator-activated receptor-γ co-activator 1-α mRNA (1.19 ± 0.21-fold, P=0.037). CONCLUSIONS: Administration of T adjuvant to RET enhanced skeletal muscle mass and performance, while up-regulating myogenic gene programming, myocellular translational efficiency and capacity, collectively resulting in higher protein turnover, and net protein accretion. T coupled with RET is an effective short-term intervention to improve muscle mass/function in older non-hypogonadal men.