Loss of a gluconeogenic muscle enzyme contributed to adaptive metabolic traits in hummingbirds.

Hummingbirds possess distinct metabolic adaptations to fuel their energy-demanding hovering flight, but the underlying genomic changes are largely unknown. Here, we generated a chromosome-level genome assembly of the long-tailed hermit and screened for genes that have been specifically inactivated in the ancestral hummingbird lineage. We discovered that FBP2 (fructose-bisphosphatase 2), which encodes a gluconeogenic muscle enzyme, was lost during a time period when hovering flight evolved. We show that FBP2 knockdown in an avian muscle cell line up-regulates glycolysis and enhances mitochondrial respiration, coincident with an increased mitochondria number. Furthermore, genes involved in mitochondrial respiration and organization have up-regulated expression in hummingbird flight muscle. Together, these results suggest that FBP2 loss was likely a key step in the evolution of metabolic muscle adaptations required for true hovering flight.

Retrograde labeling correlates with motor unit number estimation in rapid-stretch nerve injury.

INTRODUCTION/AIMS: Rapid-stretch nerve injuries represent a substantial treatment challenge. No study has examined motor neuron connection after rapid-stretch injury. Our objective in this study was to characterize the electrophysiological properties of graded rapid-stretch nerve injury and assess motor neuron health using retrograde labeling and muscle adenosine triphosphatase (ATPase) histology. METHODS: Male C57BL/6 mice (n = 6 per group) were rapid-stretch injured at four levels of severity: sham injury, stretch within elastic modulus, inelastic deformation, and stretch rupture. Serial compound muscle action potential (CMAP) and motor unit number estimation (MUNE) measurements were made for 48 days, followed by retrograde labeling and muscle ATPase histology. RESULTS: Elastic injuries showed no durable abnormalities. Inelastic injury demonstrated profound initial reduction in CMAP and MUNE (P < .036) on day 2, with partial recovery by day 14 after injury (CMAP: 40% baseline, P = .003; MUNE: 55% baseline, P = .033). However, at the experimental endpoint, CMAP had recovered to baseline with only limited improvement in MUNE. Inelastic injury led to reduced retrograde-labeled neurons and grouped fiber type histology. Rupture injury had severe and nonrecovering electrophysiological impairment, dramatically reducing labeled neurons (P = .005), and atrophic or type 1 muscle fibers. There was an excellent correlation between MUNE and retrograde-labeled tibial motor neurons across injury severities (R2  = 0.96). DISCUSSION: There was no significant electrophysiological derangement in low-severity injuries but there was recoverable conduction block in inelastic injury with slow recovery, potentially due to collateral sprouting. Rupture injuries yielded permanent failure of injured axons to reinnervate. These results provide insight into the pathophysiology of clinical injuries and recovery.

The effects of neurogranin knockdown on SERCA pump efficiency in soleus muscles of female mice fed a high fat diet.

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump is responsible for the transport of Ca2+ from the cytosol into the sarcoplasmic reticulum at the expense of ATP, making it a regulator of both muscle relaxation and muscle-based energy expenditure. Neurogranin (Ng) is a small protein that negatively regulates calcineurin signaling. Calcineurin is Ca2+/calmodulin dependent phosphatase that promotes the oxidative fibre type in skeletal muscle and regulates muscle-based energy expenditure. A recent study has shown that calcineurin activation reduces SERCA Ca2+ transport efficiency, ultimately raising energy expenditure. Since the biomedical view of obesity states that it arises as an imbalance between energy intake and expenditure which favors the former, we questioned whether heterozygous Ng deletion (Ng+/- ) would reduce SERCA efficiency and increase energy expenditure in female mice fed a high-fat diet (HFD). Young (3-4-month-old) female wild type (WT) and Ng+/- mice were fed a HFD for 12 weeks with their metabolic profile being analyzed using metabolic cages and DXA scanning, while soleus SERCA efficiency was measured using SERCA specific Ca2+ uptake and ATPase activity assays. Ng+/- mice showed significantly less cage ambulation compared to WT mice but this did not lead to any added weight gain nor changes in daily energy expenditure, glucose or insulin tolerance despite a similar level of food intake. Furthermore, we observed significant reductions in SERCA's apparent coupling ratio which were associated with significant reductions in SERCA1 and phospholamban content. Thus, our results show that Ng regulates SERCA pump efficiency, and future studies should further investigate the potential cellular mechanisms.

G6PD Deficiency Is Crucial for Insulin Signaling Activation in Skeletal Muscle.

Glucose 6-P dehydrogenase (G6PD) is the first rate-limiting enzyme in pentose phosphate pathway (PPP), and it is proverbial that G6PD is absent in skeletal muscle. However, how and why G6PD is down-regulated during skeletal muscle development is unclear. In this study, we confirmed the expression of G6PD was down-regulated during myogenesis in vitro and in vivo. G6PD was absolutely silent in adult skeletal muscle. Histone H3 acetylation and DNA methylation act together on the expression of G6PD. Neither knock-down of G6PD nor over-expression of G6PD affects myogenic differentiation. Knock-down of G6PD significantly promotes the sensitivity and response of skeletal muscle cells to insulin; over-expression of G6PD significantly injures the sensitivity and response of skeletal muscle cells to insulin. High-fat diet treatment impairs insulin signaling by up-regulating G6PD, and knock-down of G6PD rescues the impaired insulin signaling and glucose uptake caused by high-fat diet treatment. Taken together, this study explored the importance of G6PD deficiency during myogenic differentiation, which provides new sight to treat insulin resistance and type-2 diabetes.

Gene Transfer of Skeletal Muscle-Type Myosin Light Chain Kinase via Adeno-Associated Virus 6 Improves Muscle Functions in an Amyotrophic Lateral Sclerosis Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that shows progressive muscle weakness. A few treatments exist including symptomatic therapies, which can prolong survival or reduce a symptom; however, no fundamental therapies have been found. As a therapeutic strategy, enhancing muscle force is important for patients' quality of life. In this study, we focused on skeletal muscle-specific myosin regulatory light chain kinase (skMLCK), which potentially enhances muscle contraction, as overexpression of skMLCK was thought to improve muscle function. The adeno-associated virus serotype 6 encoding skMLCK (AAV6/skMLCK) and eGFP (control) was produced and injected intramuscularly into the lower limbs of SOD1G37R mice, which are a familial ALS model. AAV6/skMLCK showed the successful expression of skMLCK in the muscle tissues. Although the control did not affect the muscle force in both of the WT and SOD1G37R mice, AAV6/skMLCK enhanced the twitch force of SOD1G37R mice and the tetanic force of WT and SOD1G37R mice. These results indicate that overexpression of skMLCK can enhance the tetanic force of healthy muscle as well as rescue weakened muscle function. In conclusion, the gene transfer of skMLCK has the potential to be a new therapy for ALS as well as for other neuromuscular diseases.

Role of SERCA and sarcolipin in adaptive muscle remodeling.

Sarcolipin (SLN) is a small regulatory protein that inhibits the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump. When bound to SERCA, SLN reduces the apparent Ca2+ affinity of SERCA and uncouples SERCA Ca2+ transport from its ATP consumption. As such, SLN plays a direct role in altering skeletal muscle relaxation and energy expenditure. Interestingly, the expression of SLN is dynamic during times of muscle adaptation, in that large increases in SLN content are found in response to development, atrophy, overload, and disease. Several groups have suggested that increases in SLN, especially in dystrophic muscle, are deleterious as it may reduce muscle function and exacerbate already abhorrent intracellular Ca2+ levels. However, there is also significant evidence to show that increased SLN content is a beneficial adaptive mechanism that protects the SERCA pump and activates Ca2+ signaling and adaptive remodeling during times of cell stress. In this review, we first discuss the role for SLN in healthy muscle during both development and overload, where SLN has been shown to activate Ca2+ signaling to promote mitochondrial biogenesis, fiber-type shifts, and muscle hypertrophy. Then, with respect to muscle disease, we summarize the discrepancies in the literature as to whether SLN upregulation is adaptive or maladaptive in nature. This review is the first to offer the concept of SLN hormesis in muscle disease, wherein both too much and too little SLN are detrimental to muscle health. Finally, the underlying mechanisms which activate SLN upregulation are discussed, specifically acknowledging a potential positive feedback loop between SLN and Ca2+ signaling molecules.

Sirtuin 3 overexpression preserves maximal sarco(endo)plasmic reticulum calcium ATPase activity in the skeletal muscle of mice subjected to high fat - high sucrose feeding.

Sarco(endo)plasmic reticulum calcium (Ca2+) ATPase (SERCA) transports Ca2+ in muscle. Impaired SERCA activity may contribute to diabetic myopathy. Sirtuin (SIRT) 3 regulates muscle metabolism and function; however, it is unknown if SIRT3 regulates muscle SERCA activity or acetylation. We determined if SIRT3 overexpression enhances SERCA activity in mouse gastrocnemius muscle and if SIRT3 overexpression preserves gastrocnemius SERCA activity in a model of type 2 diabetes, induced by high fat - high sucrose (HFHS) feeding. We also determined if the acetylation status of SERCA proteins in mouse gastrocnemius is altered by SIRT3 overexpression or HFHS feeding. Wild-type (WT) and SIRT3 transgenic (SIRT3TG) mice, overexpressing SIRT3 in skeletal muscle, were fed a standard or HFHS diet for 4 months. SIRT3TG and WT mice developed obesity and glucose intolerance after 4 months of HFHS feeding. SERCA Vmax was higher in gastrocnemius of SIRT3TG mice compared with WT mice. HFHS-fed mice had lower SERCA1a protein levels and lower SERCA Vmax in their gastrocnemius than control-fed mice. The decrease in SERCA Vmax in gastrocnemius muscle due to HFHS feeding was attenuated by SIRT3 overexpression in HFHS-fed SIRT3TG mice. SERCA1a and SERCA2a acetylation in mouse gastrocnemius was not altered by genotype or diet. These findings suggest SIRT3 overexpression improves SERCA function in mouse skeletal muscle.

Rho GTPases in Skeletal Muscle Development and Homeostasis.

Rho guanosine triphosphate hydrolases (GTPases) are molecular switches that cycle between an inactive guanosine diphosphate (GDP)-bound and an active guanosine triphosphate (GTP)-bound state during signal transduction. As such, they regulate a wide range of both cellular and physiological processes. In this review, we will summarize recent work on the role of Rho GTPase-regulated pathways in skeletal muscle development, regeneration, tissue mass homeostatic balance, and metabolism. In addition, we will present current evidence that links the dysregulation of these GTPases with diseases caused by skeletal muscle dysfunction. Overall, this information underscores the critical role of a number of members of the Rho GTPase subfamily in muscle development and the overall metabolic balance of mammalian species.

Acid Sphingomyelinase Controls Early Phases of Skeletal Muscle Regeneration by Shaping the Macrophage Phenotype.

Skeletal muscle regeneration is a complex process involving crosstalk between immune cells and myogenic precursor cells, i.e., satellite cells. In this scenario, macrophage recruitment in damaged muscles is a mandatory step for tissue repair since pro-inflammatory M1 macrophages promote the activation of satellite cells, stimulating their proliferation and then, after switching into anti-inflammatory M2 macrophages, they prompt satellite cells' differentiation into myotubes and resolve inflammation. Here, we show that acid sphingomyelinase (ASMase), a key enzyme in sphingolipid metabolism, is activated after skeletal muscle injury induced in vivo by the injection of cardiotoxin. ASMase ablation shortens the early phases of skeletal muscle regeneration without affecting satellite cell behavior. Of interest, ASMase regulates the balance between M1 and M2 macrophages in the injured muscles so that the absence of the enzyme reduces inflammation. The analysis of macrophage populations indicates that these events depend on the altered polarization of M1 macrophages towards an M2 phenotype. Our results unravel a novel role of ASMase in regulating immune response during muscle regeneration/repair and suggest ASMase as a supplemental therapeutic target in conditions of redundant inflammation that impairs muscle recovery.

Impaired Skeletal Muscle Development and Regeneration in Transglutaminase 2 Knockout Mice.

Skeletal muscle regeneration is triggered by local inflammation and is accompanied by phagocytosis of dead cells at the injury site. Efferocytosis regulates the inflammatory program in macrophages by initiating the conversion of their inflammatory phenotype into the healing one. While pro-inflammatory cytokines induce satellite cell proliferation and differentiation into myoblasts, growth factors, such as GDF3, released by healing macrophages drive myoblast fusion and myotube growth. Therefore, improper efferocytosis may lead to impaired muscle regeneration. Transglutaminase 2 (TG2) is a versatile enzyme participating in efferocytosis. Here, we show that TG2 ablation did not alter the skeletal muscle weights or sizes but led to the generation of small size myofibers and to decreased grip force in TG2 null mice. Following cardiotoxin-induced injury, the size of regenerating fibers was smaller, and the myoblast fusion was delayed in the tibialis anterior muscle of TG2 null mice. Loss of TG2 did not affect the efferocytic capacity of muscle macrophages but delayed their conversion to Ly6C-CD206+, GDF3 expressing cells. Finally, TG2 promoted myoblast fusion in differentiating C2C12 myoblasts. These results indicate that TG2 expressed by both macrophages and myoblasts contributes to proper myoblast fusion, and its ablation leads to impaired muscle development and regeneration in mice.

CAPN3: A muscle­specific calpain with an important role in the pathogenesis of diseases (Review).

Calpains are a family of Ca2+­dependent cysteine proteases that participate in various cellular processes. Calpain 3 (CAPN3) is a classical calpain with unique N­terminus and insertion sequence 1 and 2 domains that confer characteristics such as rapid autolysis, Ca2+­independent activation and Na+ activation of the protease. CAPN3 is the only muscle­specific calpain that has important roles in the promotion of calcium release from skeletal muscle fibers, calcium uptake of sarcoplasmic reticulum, muscle formation and muscle remodeling. Studies have indicated that recessive mutations in CAPN3 cause limb­girdle muscular dystrophy (MD) type 2A and other types of MD; eosinophilic myositis, melanoma and epilepsy are also closely related to CAPN3. In the present review, the characteristics of CAPN3, its biological functions and roles in the pathogenesis of a number of disorders are discussed.

Bioactive compounds from Artemisia dracunculus L. activate AMPK signaling in skeletal muscle.

An extract from Artemisia dracunculus L. (termed PMI-5011) improves glucose homeostasis by enhancing insulin action and reducing ectopic lipid accumulation, while increasing fat oxidation in skeletal muscle tissue in obese insulin resistant male mice. A chalcone, DMC-2, in PMI-5011 is the major bioactive that enhances insulin signaling and activation of AKT. However, the mechanism by which PMI-5011 improves lipid metabolism is unknown. AMPK is the cellular energy and metabolic sensor and a key regulator of lipid metabolism in muscle. This study examined PMI-5011 activation of AMPK signaling using murine C2C12 muscle cell culture and skeletal muscle tissue. Findings show that PMI-5011 increases Thr172-phosphorylation of AMPK in muscle cells and skeletal muscle tissue, while hepatic AMPK activation by PMI-5011 was not observed. Increased AMPK activity by PMI-5011 affects downstream signaling of AMPK, resulting in inhibition of ACC and increased SIRT1 protein levels. Selective deletion of DMC-2 from PMI-5011 demonstrates that compounds other than DMC-2 in a "DMC-2 knock out extract" (KOE) are responsible for AMPK activation and its downstream effects. Compared to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin, the phytochemical mixture characterizing the KOE appears to more efficiently activate AMPK in muscle cells. KOE-mediated AMPK activation was LKB-1 independent, suggesting KOE does not activate AMPK via LKB-1 stimulation. Through AMPK activation, compounds in PMI-5011 may regulate lipid metabolism in skeletal muscle. Thus, the AMPK-activating potential of the KOE adds therapeutic value to PMI-5011 and its constituents in treating insulin resistance or type 2 diabetes.

Assessing the Use of the sGC Stimulator BAY-747, as a Potential Treatment for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder, affecting one in 3500 to 5000 boys worldwide. The NO-sGC-cGMP pathway plays an important role in skeletal muscle function, primarily by improving blood flow and oxygen supply to the muscles during exercise. In fact, PDE5 inhibitors have previously been investigated as a potential therapy for DMD, however, a large-scale Phase III clinical trial did not meet its primary endpoint. Since the efficacy of PDE5i is dependent on sufficient endogenous NO production, which might be impaired in DMD, we investigated if NO-independent sGC stimulators, could have therapeutic benefits in a mouse model of DMD. Male mdx/mTRG2 mice aged six weeks were given food supplemented with the sGC stimulator, BAY-747 (150 mg/kg of food) or food alone (untreated) ad libitum for 16 weeks. Untreated C57BL6/J mice were used as wild type (WT) controls. Assessments of the four-limb hang, grip strength, running wheel and serum creatine kinase (CK) levels showed that mdx/mTRG2 mice had significantly reduced skeletal muscle function and severe muscle damage compared to WT mice. Treatment with BAY-747 improved grip strength and running speed, and these mice also had reduced CK levels compared to untreated mdx/mTRG2 mice. We also observed increased inflammation and fibrosis in the skeletal muscle of mdx/mTRG2 mice compared to WT. While gene expression of pro-inflammatory cytokines and some pro-fibrotic markers in the skeletal muscle was reduced following BAY-747 treatment, there was no reduction in infiltration of myeloid immune cells nor collagen deposition. In conclusion, treatment with BAY-747 significantly improves several functional and pathological parameters of the skeletal muscle in mdx/mTRG2 mice. However, the effect size was moderate and therefore, more studies are needed to fully understand the potential treatment benefit of sGC stimulators in DMD.

GSK2256294 Decreases sEH (Soluble Epoxide Hydrolase) Activity in Plasma, Muscle, and Adipose and Reduces F2-Isoprostanes but Does Not Alter Insulin Sensitivity in Humans.

[Figure: see text].

Metformin Increases Protein Phosphatase 2A Activity in Primary Human Skeletal Muscle Cells Derived from Lean Healthy Participants.

OBJECTIVE: To investigate if PP2A plays a role in metformin-induced insulin sensitivity improvement in human skeletal muscle cells. Participants. Eight lean insulin-sensitive nondiabetic participants (4 females and 4 males; age: 21.0 ± 1.0 years; BMI: 22.0 ± 0.7 kg/m2; 2-hour OGTT: 97.0 ± 6.0 mg/dl; HbA1c: 5.3 ± 0.1%; fasting plasma glucose: 87.0 ± 2.0 mg/dl; M value; 11.0 ± 1.0 mg/kgBW/min). DESIGN: A hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity in human subjects, and skeletal muscle biopsy samples were obtained. Primary human skeletal muscle cells (shown to retain metabolic characteristics of donors) were cultured from these muscle biopsies that included 8 lean insulin-sensitive participants. Cultured cells were expanded, differentiated into myotubes, and treated with 50 µM metformin for 24 hours before harvesting. PP2Ac activity was measured by a phosphatase activity assay kit (Millipore) according to the manufacturer's protocol. RESULTS: The results indicated that metformin significantly increased the activity of PP2A in the myotubes for all 8 lean insulin-sensitive nondiabetic participants, and the average fold increase is 1.54 ± 0.11 (P < 0.001). CONCLUSIONS: These results provided the first evidence that metformin can activate PP2A in human skeletal muscle cells derived from lean healthy insulin-sensitive participants and may help to understand metformin's action in skeletal muscle in humans.

A Systematic Review on the Role of SIRT1 in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a muscular disease characterized by progressive muscle degeneration. Life expectancy is between 30 and 50 years, and death is correlated with cardiac or respiratory complications. Currently, there is no cure, so there is a great interest in new pharmacological targets. Sirtuin1 (SIRT1) seems to be a potential target for DMD. In muscle tissue, SIRT1 exerts anti-inflammatory and antioxidant effects. The aim of this study is to summarize all the findings of in vivo and in vitro literature studies about the potential role of SIRT1 in DMD. A systematic literature search was performed according to PRISMA guidelines. Twenty-three articles satisfied the eligibility criteria. It emerged that SIRT1 inhibition led to muscle fragility, while conversely its activation improved muscle function. Additionally, resveratrol, a SIRT1 activator, has brought beneficial effects to the skeletal, cardiac and respiratory muscles by exerting anti-inflammatory activity that leads to reduced myofiber wasting.

Nrf2 activation induces mitophagy and reverses Parkin/Pink1 knock down-mediated neuronal and muscle degeneration phenotypes.

The balanced functionality of cellular proteostatic modules is central to both proteome stability and mitochondrial physiology; thus, the age-related decline of proteostasis also triggers mitochondrial dysfunction, which marks multiple degenerative disorders. Non-functional mitochondria are removed by mitophagy, including Parkin/Pink1-mediated mitophagy. A common feature of neuronal or muscle degenerative diseases, is the accumulation of damaged mitochondria due to disrupted mitophagy rates. Here, we exploit Drosophila as a model organism to investigate the functional role of Parkin/Pink1 in regulating mitophagy and proteostatic responses, as well as in suppressing degenerative phenotypes at the whole organism level. We found that Parkin or Pink1 knock down in young flies modulated proteostatic components in a tissue-dependent manner, increased cell oxidative load, and suppressed mitophagy in neuronal and muscle tissues, causing mitochondrial aggregation and neuromuscular degeneration. Concomitant to Parkin or Pink1 knock down cncC/Nrf2 overexpression, induced the proteostasis network, suppressed oxidative stress, restored mitochondrial function, and elevated mitophagy rates in flies' tissues; it also, largely rescued Parkin or Pink1 knock down-mediated neuromuscular degenerative phenotypes. Our in vivo findings highlight the critical role of the Parkin/Pink1 pathway in mitophagy, and support the therapeutic potency of Nrf2 (a druggable pathway) activation in age-related degenerative diseases.

Passive repetitive stretching is associated with greater muscle mass and cross-sectional area in the sarcopenic muscle.

Mechanical stimulation has benefits for muscle mass and function. Passive stretching is widely performed in clinical rehabilitation medicine. However, the hypertrophic effects of passive repetitive stretching on senescent skeletal muscles against muscle atrophy remain unknown. We used senescence-accelerated model SAM-P8 mice. The gastrocnemius muscle was passively repetitive stretched by manual ankle dorsiflexion for 15 min, 5 days a week for 2 weeks under deep anesthesia. We examined the effects of passive stretching on muscle mass, myofiber cross-sectional area, muscle fiber type composition, satellite cell and myonuclei content, signaling pathways involved in muscle protein synthesis, and myogenic regulatory factors. The gastrocnemius muscle weight and fiber cross-sectional area of the stretched side was found greater compared with that of the unstretched side. Passive repetitive stretching increased the mRNA expression level of Akt, p70S6K, 4E-BP1, Myf5, myogenin, MuRF1.The phosphorylation level of p70S6K significantly increased in the stretched muscles, whereas of Akt and 4E-BP1 remained unchanged, compared to the unstretched side. The Pax7+ cells and myonuclei content did not differ between the stretched and unstretched muscles. These findings suggest that the hypertrophic or suppressed atrophic observation in the stretched muscles are mainly attributable to the protein turnover provoked by stretching. These findings are applicable to clinical muscle strengthening and sarcopenia prevention.

Targeting HDAC8 to ameliorate skeletal muscle differentiation in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) causes progressive skeletal muscle degeneration and currently there are few therapeutic options. The identification of new drug targets and their validation in model systems of DMD could be a promising approach to make progress in finding new treatments for this lethal disease. Histone deacetylases (HDACs) play key roles in myogenesis and the therapeutic approach targeting HDACs in DMD is in an advanced phase of clinical trial. Here, we show that the expression of HDAC8, one of the members of the HDAC family, is increased in DMD patients and dystrophic zebrafish. The selective inhibition of HDAC8 with the PCI-34051 inhibitor rescues skeletal muscle defects, similarly to the treatment with the pan-HDAC inhibitor Givinostat. Through acetylation profile of zebrafish with HDAC8 dysregulation, we identified new HDAC8 targets involved in cytoskeleton organization such as tubulin that, when acetylated, is a marker of stable microtubules. Our work provides evidence of HDAC8 overexpression in DMD patients and zebrafish and supports its specific inhibition as a new valuable therapeutic approach in the treatment of this pathology.

FGGY carbohydrate kinase domain containing is expressed and alternatively spliced in skeletal muscle and attenuates MAP kinase and Akt signaling.

Skeletal muscle atrophy can result from a range of physiological conditions, including denervation, immobilization, hindlimb unweighting, and aging. To better characterize the molecular genetic events of atrophy, a microarray analysis revealed that FGGY carbohydrate kinase domain containing (Fggy) is expressed in skeletal muscle and is induced in response to denervation. Bioinformatic analysis of the Fggy gene locus revealed two validated isoforms with alternative transcription initiation sites that we have designated Fggy-L-552 and Fggy-S-387. Additionally, we cloned two novel alternative splice variants, designated Fggy-L-482 and Fggy-S-344, from cultured muscle cells suggesting that at least four Fggy splice variants are expressed in skeletal muscle. Quantitative RT-PCR was performed using RNA isolated from muscle cells and primers designed to distinguish the four alternative Fggy transcripts and found that the Fggy-L transcripts are more highly expressed during myoblast differentiation, while the Fggy-S transcripts show relatively stable expression in proliferating myoblasts and differentiated myotubes. Confocal fluorescent microscopy revealed that the Fggy-L variants appear to localize evenly throughout the cytoplasm, while the Fggy-S variants produce a more punctuate cytoplasmic localization pattern in proliferating muscle cells. Finally, ectopic expression of Fggy-L-552 and Fggy-S-387 resulted in inhibition of muscle cell differentiation and attenuation of the MAP kinase and Akt signaling pathways. The identification and characterization of novel genes such as Fggy helps to improve our understanding of the molecular and cellular events that lead to atrophy and may eventually result in the identification of new therapeutic targets for the treatment of muscle wasting.