Postexercise muscle glycogen synthesis with glucose, galactose, and combined galactose-glucose ingestion.

Ingested galactose can enhance postexercise liver glycogen repletion when combined with glucose but effects on muscle glycogen synthesis are unknown. In this double-blind randomized study participants [7 men and 2 women; V̇o2max: 51.1 (8.7) mL·kg-1·min-1] completed three trials of exhaustive cycling exercise followed by a 4-h recovery period, during which carbohydrates were ingested at the rate of 1.2 g·kg-1·h-1 comprising glucose (GLU), galactose (GAL) or galactose + glucose (GAL + GLU; 1:2 ratio). The increase in vastus lateralis skeletal-muscle glycogen concentration during recovery was higher with GLU relative to GAL + GLU [contrast: +50 mmol·(kg DM)-1; 95%CL 10, 89; P = 0.021] and GAL [+46 mmol·(kg DM)-1; 95%CL 8, 84; P = 0.024] with no difference between GAL + GLU and GAL [-3 mmol·(kg DM)-1; 95%CL -44, 37; P = 0.843]. Plasma glucose concentration in GLU was not significantly different vs. GAL + GLU (+ 0.41 mmol·L-1; 95%CL 0.13, 0.94) but was significantly lower than GAL (-0.75 mmol·L-1; 95%CL -1.34, -0.17) and also lower in GAL vs. GAL + GLU (-1.16 mmol·-1; 95%CL -1.80, -0.53). Plasma insulin was higher in GLU + GAL and GLU compared with GAL but not different between GLU + GAL and GLU. Plasma galactose concentration was higher in GAL compared with GLU (3.35 mmol·L-1; 95%CL 3.07, 3.63) and GAL + GLU (3.22 mmol·L-1; 95%CL 3.54, 2.90) with no difference between GLU + GAL (0.13 mmol·L-1; 95%CL -0.11, 0.37) and GLU. Compared with galactose or a galactose + glucose blend, glucose feeding was more effective in postexercise muscle glycogen synthesis. Comparable muscle glycogen synthesis was observed with galactose-glucose coingestion and exclusive galactose-only ingestion.NEW & NOTEWORTHY Postexercise galactose-glucose coingestion or exclusive galactose-only ingestion resulted in a lower rate of skeletal-muscle glycogen replenishment compared with exclusive glucose-only ingestion. Comparable muscle glycogen synthesis was observed with galactose-glucose coingestion and exclusive galactose-only ingestion.

Single vesicle analysis reveals the release of tetraspanin positive extracellular vesicles into circulation with high intensity intermittent exercise.

Small extracellular vesicles (sEVs) are released from all cell types and participate in the intercellular exchange of proteins, lipids, metabolites and nucleic acids. Proteomic, flow cytometry and nanoparticle tracking analyses suggest sEVs are released into circulation with exercise. However, interpretation of these data may be influenced by sources of bias introduced by different analytical approaches. Seven healthy participants carried out a high intensity intermittent training (HIIT) cycle protocol consisting of 4 × 30 s at a work-rate corresponding to 200% of individual max power (watts) interspersed by 4.5 min of active recovery. EDTA-treated blood was collected before and immediately after the final effort. Platelet-poor (PPP) and platelet-free (PFP) plasma was derived by one or two centrifugal spins at 2500 g, respectively (15 min, room temperature). Platelets were counted on an automated haemocytometer. Plasma samples were assessed with the Exoview R100 platform, which immobilises sEVs expressing common tetraspanin markers CD9, CD63, CD81 and CD41a on microfluidic chips and with the aid of fluorescence imaging, counts their abundance at a single sEV resolution, importantly, without a pre-isolation step. There was a lower number of platelets in the PFP than PPP, which was associated with a lower number of CD9, CD63 and CD41a positive sEVs. HIIT induced an increase in fluorescence counts in CD9, CD63 and CD81 positive sEVs in both PPP and PFP. These data support the concept that sEVs are released into circulation with exercise. Furthermore, platelet-free plasma is the preferred, representative analyte to study sEV dynamics and phenotype during exercise. KEY POINTS: Small extracellular vesicles (sEV) are nano-sized particles containing protein, metabolites, lipid and RNA that can be transferred from cell to cell. Previous findings implicate that sEVs are released into circulation with exhaustive, aerobic exercise, but since there is no gold standard method to isolate sEVs, these findings may be subject to bias introduced by different approaches. Here, we use a novel method to immobilise and image sEVs, at single-vesicle resolution, to show sEVs are released into circulation with high intensity intermittent exercise. Since platelet depletion of plasma results in a reduction in sEVs, platelet-free plasma is the preferred analyte to examine sEV dynamics and phenotype in the context of exercise.

No effect of five days of bed rest or short-term resistance exercise prehabilitation on markers of skeletal muscle mitochondrial content and dynamics in older adults.

Bed rest (BR) results in significant impairments in skeletal muscle metabolism. Mitochondrial metabolism is reportedly highly sensitive to disuse, with dysregulated fission-fusion events and impaired oxidative function previously reported. The effects of clinically relevant short-term BR (≤5 days) on mitochondrial protein expression are presently unclear, as are the effects of exercise prehabilitation as a potential counteractive intervention. The present study examined the effects of a 5-day period of BR and short-term resistance exercise prehabilitation (ST-REP) on mitochondrial-protein content. Ten older men (71 ± 4 years) underwent 5 days of BR, completing four sessions of high-volume unilateral resistance exercise prehabilitation over 7 days beforehand. Muscle biopsies were obtained from the vastus lateralis in the non-exercised control and exercised legs, both pre- and post-prehabilitation and pre- and post-BR, to determine changes in citrate synthase enzyme activity and the expression of key proteins in the mitochondrial electron transport chain and molecular regulators of fission-fusion dynamics, biosynthesis, and mitophagy. We observed no significant effect of either BR or ST-REP on citrate synthase protein content, enzyme activity, or ETC complex I-V protein content. Moreover, we observed no significant changes in markers of mitochondrial fission and fusion (p-DRP1S616 , p-DRP1S637 , p-DRP1S616/S637 ratio, p-MFFS146 , Mitofillin, OPA1, or MFN2 (p > 0.05 for all). Finally, we observed no differences in markers of biosynthesis (p-AMPKT172 , p-ACCS79 , PGC1a, TFAM) or mitophagy-related signaling (ULK-1, BNIP3/NIX, LC3B I/II) (p > 0.05 for all). In contrast to previous longer-term periods of musculoskeletal disuse (i.e., 7-14 days), a clinically relevant, 5-day period of BR resulted in no significant perturbation in muscle mitochondrial protein signaling in healthy older adults, with no effect of ST-REP in the week prior to BR. Accordingly, disuse-induced muscle atrophy may precede alterations in mitochondrial content.

The Effect of Ex Vivo Human Serum from Liver Disease Patients on Cellular Protein Synthesis and Growth.

Sarcopenia is a common complication affecting liver disease patients, yet the underlying mechanisms remain unclear. We aimed to elucidate the cellular mechanisms that drive sarcopenia progression using an in vitro model of liver disease. C2C12 myotubes were serum and amino acid starved for 1-h and subsequently conditioned with fasted ex vivo serum from four non-cirrhotic non-alcoholic fatty liver disease patients (NAFLD), four decompensated end-stage liver disease patients (ESLD) and four age-matched healthy controls (CON) for 4- or 24-h. After 4-h C2C12 myotubes were treated with an anabolic stimulus (5 mM leucine) for 30-min. Myotube diameter was reduced following treatment with serum from ESLD compared with CON (−45%) and NAFLD (−35%; p < 0.001 for both). A reduction in maximal mitochondrial respiration (24% and 29%, respectively), coupling efficiency (~12%) and mitophagy (~13%) was identified in myotubes conditioned with NAFLD and ESLD serum compared with CON (p < 0.05 for both). Myostatin (43%, p = 0.04) and MuRF-1 (41%, p = 0.03) protein content was elevated in myotubes treated with ESLD serum compared with CON. Here we highlight a novel, experimental platform to further probe changes in circulating markers associated with liver disease that may drive sarcopenia and develop targeted therapeutic interventions.

Global Deletion of 11ß-HSD1 Prevents Muscle Wasting Associated with Glucocorticoid Therapy in Polyarthritis.

Glucocorticoids provide indispensable anti-inflammatory therapies. However, metabolic adverse effects including muscle wasting restrict their use. The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) modulates peripheral glucocorticoid responses through pre-receptor metabolism. This study investigates how 11ß-HSD1 influences skeletal muscle responses to glucocorticoid therapy for chronic inflammation. We assessed human skeletal muscle biopsies from patients with rheumatoid arthritis and osteoarthritis for 11ß-HSD1 activity ex vivo. Using the TNF-α-transgenic mouse model (TNF-tg) of chronic inflammation, we examined the effects of corticosterone treatment and 11ß-HSD1 global knock-out (11ßKO) on skeletal muscle, measuring anti-inflammatory gene expression, muscle weights, fiber size distribution, and catabolic pathways. Muscle 11ß-HSD1 activity was elevated in patients with rheumatoid arthritis and correlated with inflammation markers. In murine skeletal muscle, glucocorticoid administration suppressed IL6 expression in TNF-tg mice but not in TNF-tg11ßKO mice. TNF-tg mice exhibited reductions in muscle weight and fiber size with glucocorticoid therapy. In contrast, TNF-tg11ßKO mice were protected against glucocorticoid-induced muscle atrophy. Glucocorticoid-mediated activation of catabolic mediators (FoxO1, Trim63) was also diminished in TNF-tg11ßKO compared to TNF-tg mice. In summary, 11ß-HSD1 knock-out prevents muscle atrophy associated with glucocorticoid therapy in a model of chronic inflammation. Targeting 11ß-HSD1 may offer a strategy to refine the safety of glucocorticoids.

Regulatory Roles of PINK1-Parkin and AMPK in Ubiquitin-Dependent Skeletal Muscle Mitophagy.

The selective removal of damaged mitochondria, also known as mitophagy, is an important mechanism that regulates mitochondrial quality control. Evidence suggests that mitophagy is adversely affected in aged skeletal muscle, and this is thought to contribute toward the age-related decline of muscle health. While our knowledge of the molecular mechanisms that regulate mitophagy are derived mostly from work in non-muscle cells, whether these mechanisms are conferred in muscle under physiological conditions has not been thoroughly investigated. Recent findings from our laboratory and those of others have made several novel contributions to this field. Herein, we consolidate current literature, including our recent work, while evaluating how ubiquitin-dependent mitophagy is regulated both in muscle and non-muscle cells through the steps of mitochondrial fission, ubiquitylation, and autophagosomal engulfment. During ubiquitin-dependent mitophagy in non-muscle cells, mitochondrial depolarization activates PINK1-Parkin signaling to elicit mitochondrial ubiquitylation. TANK-binding kinase 1 (TBK1) then activates autophagy receptors, which in turn, tether ubiquitylated mitochondria to autophagosomes prior to lysosomal degradation. In skeletal muscle, evidence supporting the involvement of PINK1-Parkin signaling in mitophagy is lacking. Instead, 5'-AMP-activated protein kinase (AMPK) is emerging as a critical regulator. Mechanistically, AMPK activation promotes mitochondrial fission before enhancing autophagosomal engulfment of damaged mitochondria possibly via TBK1. While TBK1 may be a point of convergence between PINK1-Parkin and AMPK signaling in muscle, the critical question that remains is: whether mitochondrial ubiquitylation is required for mitophagy. In future, improving understanding of molecular processes that regulate mitophagy in muscle will help to develop novel strategies to promote healthy aging.

AMPK activation induces mitophagy and promotes mitochondrial fission while activating TBK1 in a PINK1-Parkin independent manner.

Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1101-152 ). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.