Anabolic signalling in skeletal muscle : impact of diet and disease

This thesis examines the role of dietary proteins on the maintenance of skeletal muscle mass in men who may or may not be insulin-resistant. It identified that dairy foods are powerful stimulators of muscle growth however this response is reduced during insulin-resistance.

Aging and its effects on inflammation in skeletal muscle at rest and following exercise-induced muscle injury

The world's elderly population is expanding rapidly, and we are now faced with the significant challenge of maintaining or improving physical activity, independence, and quality of life in the elderly. Counteracting the progressive loss of muscle mass that occurs in the elderly, known as sarcopenia, represents a major hurdle in achieving these goals. Indirect evidence for a role of inflammation in sarcopenia is that markers of systemic inflammation correlate with the loss of muscle mass and strength in the elderly. More direct evidence is that compared with skeletal muscle of young people, the number of macrophages is lower, the gene expression of several cytokines is higher, and stress signaling proteins are activated in skeletal muscle of elderly people at rest. Sarcopenia may also result from inadequate repair and chronic maladaptation following muscle injury in the elderly. Macrophage infiltration and the gene expression of certain cytokines are reduced in skeletal muscle of elderly people compared with young people following exercise-induced muscle injury. Further research is required to identify the cause(s) of inflammation in skeletal muscle of elderly people. Additional work is also needed to expand our understanding of the cells, proteins, and transcription factors that regulate inflammation in the skeletal muscle of elderly people at rest and after exercise. This knowledge is critical for devising strategies to restrict sarcopenia, and improve the health of today's elderly population.

Contribution of stretch to the change of activation properties of muscle fibers in the diaphragm at the transition from fetal to neonatal life

The transition from fetal to postnatal life involves clearance of liquid from the lung and airways, and rapid formation of a functional residual capacity. Despite the importance of the diaphragm in this process, the impact of birth on the mechanical and functional activity of its muscle fibers is not known. This study determined the contractile characteristics of individual “skinned” diaphragm fibers from 70 days (0.47) gestation to after birth in sheep. Based on differential sensitivity to the divalent ions calcium (Ca²⁺) and strontium (Sr²⁺), all fibers in the fetal diaphragm were classified as “fast,” whereas fibers from the adult sheep diaphragm exhibited a “hybrid” phenotype where both “fast” and “slow” characteristics were present within each single fiber. Transition to the hybrid phenotype occurred at birth, was evident after only 40 min of spontaneous breathing, and could be induced by simple mechanical stretch of diaphragm fibers from near-term fetuses (∼147 days gestation). Both physical stretch of isolated fibers, and mechanical ventilation of the fetal diaphragm in situ, significantly increased sensitivity to Ca²⁺ and Sr²⁺, maximum force generating capacity, and decreased passive tension in near-term and preterm fetuses; however, only fibers from near-term fetuses showed a complete transition to a “hybrid” activation profile. These findings suggest that stretch associated with the transition from a liquid to air-filled lung at birth induces physical changes of proteins determining the activation and elastic properties of the diaphragm. These changes may allow the diaphragm to meet the increased mechanical demands of breathing immediately after birth.

cGMP phosphodiesterase inhibition improves the vascular and metabolic actions of insulin in skeletal muscle

There is considerable support for the concept that insulin-mediated increases in microvascular blood flow to muscle impact significantly on muscle glucose uptake. Since the microvascular blood flow increases with insulin have been shown to be nitric oxide-dependent inhibition of cGMP-degrading phosphodiesterases (cGMP PDEs) is predicted to enhance insulin-mediated increases in microvascular perfusion and muscle glucose uptake. Therefore, we studied the effects of the pan-cGMP PDE inhibitor zaprinast on the metabolic and vascular actions of insulin in muscle. Hyperinsulinemic euglycemic clamps (3 mU·min−1·kg−1) were performed in anesthetized rats and changes in microvascular blood flow assessed from rates of 1-methylxanthine metabolism across the muscle bed by capillary xanthine oxidase in response to insulin and zaprinast. We also characterized cGMP PDE isoform expression in muscle by real-time PCR and immunostaining of frozen muscle sections. Zaprinast enhanced insulin-mediated microvascular perfusion by 29% and muscle glucose uptake by 89%, while whole body glucose infusion rate during insulin infusion was increased by 33% at 2 h. PDE2, -9, and -10 were the major isoforms expressed at the mRNA level in muscle, while PDE1B, -9A, -10A, and -11A proteins were expressed in blood vessels. Acute administration of the cGMP PDE inhibitor zaprinast enhances muscle microvascular blood flow and glucose uptake response to insulin. The expression of a number of cGMP PDE isoforms in skeletal muscle suggests that targeting specific cGMP PDE isoforms may provide a promising avenue for development of a novel class of therapeutics for enhancing muscle insulin sensitivity.

Xanthine oxidase inhibition attenuates skeletal muscle signaling following acute exercise but does not impair mitochondrial adaptations to endurance training.

The aim of this research was to examine the impact of the xanthine oxidase (XO) inhibitor allopurinol on the skeletal muscle activation of cell signaling kinases' and adaptations to mitochondrial proteins and antioxidant enzymes following acute endurance exercise and endurance training. Male Sprague-Dawley rats performed either acute exercise (60 min of treadmill running, 27 m/min, 5% incline) or 6 wk of endurance training (5 days/wk) while receiving allopurinol or vehicle. Allopurinol treatment reduced XO activity to 5% of the basal levels (P < 0.05), with skeletal muscle uric acid levels being almost undetectable. Following acute exercise, skeletal muscle oxidized glutathione (GSSG) significantly increased in allopurinol- and vehicle-treated groups despite XO activity and uric acid levels being unaltered by acute exercise (P < 0.05). This suggests that the source of ROS was not from XO. Surprisingly, muscle GSSG levels were significantly increased following allopurinol treatment. Following acute exercise, allopurinol treatment prevented the increase in p38 MAPK and ERK phosphorylation and attenuated the increase in mitochondrial transcription factor A (mtTFA) mRNA (P < 0.05) but had no effect on the increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor-2, GLUT4, or superoxide dismutase mRNA. Allopurinol also had no impact on the endurance training-induced increases in PGC-1α, mtTFA, and mitochondrial proteins including cytochrome c, citrate synthase, and β-hydroxyacyl-CoA dehydrogenase. In conclusion, although allopurinol inhibits cell signaling pathways in response to acute exercise, the inhibitory effects of allopurinol appear unrelated to exercise-induced ROS production by XO. Allopurinol also has little effect on increases in mitochondrial proteins following endurance training.

Maternal overnutrition programs changes in the expression of skeletal muscle genes that are associated with insulin resistance and defects of oxidative phosphorylation in adult male rat offspring.

Children of obese mothers have increased risk of metabolic syndrome as adults. Here we report the effects of a high-fat diet in the absence of maternal obesity at conception on skeletal muscle metabolic and transcriptional profiles of adult male offspring. Female Sprague Dawley rats were fed a diet rich in saturated fat and sucrose [high-fat diet (HFD): 23.5% total fat, 9.83% saturated fat, 20% sucrose wt:wt] or a normal control diet [(CD) 7% total fat, 0.5% saturated fat, 10% sucrose wt:wt] for the 3 wk prior to mating and throughout pregnancy and lactation. Maternal weights were not different at conception; however, HFD-fed dams were 22% heavier than controls during pregnancy. On a normal diet, the male offspring of HFD-fed dams were not heavier than controls but demonstrated features of insulin resistance, including elevated plasma insulin concentration [40.1 ± 2.5 (CD) vs 56.2 ± 6.1 (HFD) mU/L; P = 0.023]. Next-generation mRNA sequencing was used to identify differentially expressed genes in the offspring soleus muscle, and gene set enrichment analysis (GSEA) was used to detect coordinated changes that are characteristic of a biological function. GSEA identified 15 upregulated pathways, including cytokine signaling (P < 0.005), starch and sucrose metabolism (P < 0.017), inflammatory response (P < 0.024), and cytokine-cytokine receptor interaction (P < 0.037). A further 8 pathways were downregulated, including oxidative phosphorylation (P < 0.004), mitochondrial matrix (P < 0.006), and electron transport/uncoupling (P < 0.022). Phosphorylation of the insulin signaling protein kinase B was reduced [2.86 ± 0.63 (CD) vs 1.02 ± 0.27 (HFD); P = 0.027] and mitochondrial complexes I, II, and V protein were downregulated by 50-68% (P < 0.005). On a normal diet, the male offspring of HFD-fed dams did not become obese adults but developed insulin resistance, with transcriptional evidence of muscle cytokine activation, inflammation, and mitochondrial dysfunction. These data indicate that maternal overnutrition, even in the absence of prepregnancy obesity, can promote metabolic dysregulation and predispose offspring to type 2 diabetes.

Expression and regulation of Homer in human skeletal muscle during neuromuscular junction adaptation to disuse and exercise

Protein calcium sensors of the Homer family have been proposed to modulate the activity of various ion channels and nuclear factor of activated T cells (NFAT), the transcription factor modulating skeletal muscle differentiation. We monitored Homer expression and subcellular localization in human skeletal muscle biopsies following 60 d of bedrest [Second Berlin Bedrest Study (BBR2-2)]. Soleus (SOL) and vastus lateralis (VL) biopsies were taken at start (pre) and at end (end) of bedrest from healthy male volunteers of a control group without exercise (CTR; n=9), a resistive-only exercise group (RE; n=7), and a combined resistive/vibration exercise group (RVE; n=7). Confocal analysis showed Homer immunoreactivity at the postsynaptic microdomain of the neuromuscular junction (NMJ) at bedrest start. After bedrest, Homer immunoreactivity decreased (CTR), remained unchanged (RE), or increased (RVE) at the NMJ. Homer2 mRNA and protein were differently regulated in a muscle-specific way. Activated NFATc1 translocates from cytoplasm to nucleus; increased amounts of NFATc1-immunopositive slow-type myonuclei were found in RVE myofibers of both muscles. Pulldown assays identified NFATc1 and Homer as molecular partners in skeletal muscle. A direct motor nerve control of Homer2 was confirmed in rat NMJs by in vivo denervation. Homer2 is localized at the NMJ and is part of the calcineurin-NFATc1 signaling pathway. RVE has additional benefit over RE as countermeasure preventing disuse-induced neuromuscular maladaptation during bedrest.

Evaluation of follistatin as a therapeutic in models of skeletal muscle atrophy associated with denervation and tenotomy

Follistatin is an inhibitor of TGF-β superfamily ligands that repress skeletal muscle growth and promote muscle wasting. Accordingly, follistatin has emerged as a potential therapeutic to ameliorate the deleterious effects of muscle atrophy. However, it remains unclear whether the anabolic effects of follistatin are conserved across different modes of non-degenerative muscle wasting. In this study, the delivery of a recombinant adeno-associated viral vector expressing follistatin (rAAV:Fst) to the hind-limb musculature of mice two weeks prior to denervation or tenotomy promoted muscle hypertrophy that was sufficient to preserve muscle mass comparable to that of untreated sham-operated muscles. However, administration of rAAV:Fst to muscles at the time of denervation or tenotomy did not prevent subsequent muscle wasting. Administration of rAAV:Fst to innervated or denervated muscles increased protein synthesis, but markedly reduced protein degradation only in innervated muscles. Phosphorylation of the signalling proteins mTOR and S6RP, which are associated with protein synthesis, was increased in innervated muscles administered rAAV:Fst, but not in treated denervated muscles. These results demonstrate that the anabolic effects of follistatin are influenced by the interaction between muscle fibres and motor nerves. These findings have important implications for understanding the potential efficacy of follistatin-based therapies for non-degenerative muscle wasting.

Effects of aging, exercise, and disease on force transfer in skeletal muscle

The loss of muscle strength and increased injury rate in aging skeletal muscle has previously been attributed to loss of muscle protein (cross-sectional area) and/or decreased neural activation. However, it is becoming clear that force transfer within and between fibers plays a significant role in this process as well. Force transfer involves a secondary matrix of proteins that align and transmit the force produced by the thick and thin filaments along muscle fibers and out to the extracellular matrix. These specialized networks of cytoskeletal proteins aid in passing force through the muscle and also serve to protect individual fibers from injury. This review discusses the cytoskeleton proteins that have been identified as playing a role in muscle force transmission, both longitudinally and laterally, and where possible highlights how disease, aging, and exercise influence the expression and function of these proteins.

MicroRNA-194 modulates glucose metabolism and its skeletal muscle expression is reduced in diabetes

BACKGROUND: The regulation of microRNAs (miRNAs) at different stages of the progression of type 2 diabetes mellitus (T2DM) and their role in glucose homeostasis was investigated. METHODS: Microarrays were used to assess miRNA expression in skeletal muscle biopsies taken from healthy individuals and patients with pre-diabetes or T2DM, and insulin resistant offspring of rat dams fed a high fat diet during pregnancy. RESULTS: Twenty-three miRNAs were differentially expressed in patients with T2DM, and 7 in the insulin resistant rat offspring compared to their controls. Among these, only one miRNA was similarly regulated: miR-194 expression was significantly reduced by 25 to 50% in both the rat model and in human with pre-diabetes and established diabetes. Knockdown of miR-194 in L6 skeletal muscle cells induced an increase in basal and insulin-stimulated glucose uptake and glycogen synthesis. This occurred in conjunction with an increased glycolysis, indicated by elevated lactate production. Moreover, oxidative capacity was also increased as we found an enhanced glucose oxidation in presence of the mitochondrial uncoupler FCCP. When miR-194 was down-regulated in vitro, western blot analysis showed an increased phosphorylation of AKT and GSK3β in response to insulin, and an increase in expression of proteins controlling mitochondrial oxidative phosphorylation. CONCLUSIONS: Type 2 diabetes mellitus is associated with regulation of several miRNAs in skeletal muscle. Interestingly, miR-194 was a unique miRNA that appeared regulated across different stages of the disease progression, from the early stages of insulin resistance to the development of T2DM. We have shown miR-194 is involved in multiple aspects of skeletal muscle glucose metabolism from uptake, through to glycolysis, glycogenesis and glucose oxidation, potentially via mechanisms involving AKT, GSK3 and oxidative phosphorylation. MiR-194 could be down-regulated in patients with early features of diabetes as an adaptive response to facilitate tissue glucose uptake and metabolism in the face of insulin resistance.

Immunofluorescence microscopy of SNAP23 in human skeletal muscle reveals colocalization with plasma membrane, lipid droplets, and mitochondria

Synaptosomal-associated protein 23 (SNAP23) is a SNARE protein expressed abundantly in human skeletal muscle. Its established role is to mediate insulin-stimulated docking and fusion of glucose transporter 4 (GLUT4) with the plasma membrane. Recent in vitro research has proposed that SNAP23 may also play a role in the fusion of growing lipid droplets (LDs) and the channeling of LD-derived fatty acids (FAs) into neighboring mitochondria for β-oxidation. This study investigates the subcellular distribution of SNAP23 in human skeletal muscle using immunofluorescence microscopy to confirm that SNAP23 localization supports the three proposed metabolic roles. Percutaneous biopsies were obtained from the m. vastus lateralis of six lean, healthy males in the rested, overnight fasted state. Cryosections were stained with antibodies targeting SNAP23, the mitochondrial marker cytochrome c oxidase and the plasma membrane marker dystrophin, whereas intramuscular LDs were stained using the neutral lipid dye oil red O. SNAP23 displayed areas of intense punctate staining in the intracellular regions of all muscle fibers and continuous intense staining in peripheral regions of the cell. Quantitation of confocal microscopy images showed colocalization of SNAP23 with the plasma membrane marker dystrophin (Pearson's correlation coefficient r = 0.50 ± 0.01). The intense punctate intracellular staining colocalized primarily with the mitochondrial marker cytochrome C oxidase (r = 0.50 ± 0.012) and to a lesser extent with LDs (r = 0.21 ± 0.01) visualized with oil red O. We conclude that the observed subcellular distribution of SNAP23 in human skeletal muscle supports the three aforementioned metabolic roles.

Muscle damage and inflammation during recovery from exercise

Unaccustomed exercise consisting of eccentric (i.e., lengthening) muscle contractions often results in muscle damage characterized by ultrastructural alterations in muscle tissue, clinical signs and symptoms (e.g., reduced muscle strength and range of motion, increased muscle soreness and swelling, efflux of myocellular proteins). The time course of recovery following exercise-induced muscle damage depends on the extent of initial muscle damage, which in turn is influenced by the intensity and duration of exercise, joint angle/muscle length and muscle groups used during exercise. The effects of these factors on muscle strength, soreness and swelling are well characterized. By contrast, much less is known about how they affect intramuscular inflammation and molecular aspects of muscle adaptation/remodeling. Although inflammation has historically been viewed as detrimental for recovery from exercise, it is now generally accepted that inflammatory responses-if tightly regulated-are integral to muscle repair and regeneration. Animal studies have revealed that other cell types including mast cells, eosinophils, CD8 and T regulatory lymphocytes, fibro-adipogenic progenitors and pericytes also help to facilitate muscle tissue regeneration. However, more research is required to determine whether these cells respond to exercise-induced muscle damage. A large body of research has investigated the efficacy of physicotherapeutic, pharmacological and nutritional interventions for reducing the signs and symptoms of exercise-induced muscle damage, with mixed results. More research is needed to examine if/how these treatments influence inflammation and muscle remodeling during recovery from exercise.