Proteomic study of skeletal muscle in obesity and type 2 diabetes: progress and potential

ABSTRACT

Introduction: Skeletal muscle is the major site of insulin-stimulated glucose uptake and imparts the beneficial effects of exercise, and hence is an important site of insulin resistance in obesity and type 2 diabetes (T2D). Despite extensive molecular biology-oriented research the molecular mechanisms underlying insulin resistance in skeletal muscle remain to be established.

Areas covered: The proteomic capabilities have greatly improved over the last decades. This review summarizes the technical challenges in skeletal muscle proteomics studies as well as the results of quantitative proteomic studies of skeletal muscle in relation to obesity, T2D, and exercise.

Expert commentary: Current available proteomic studies contribute to the view that insulin resistance in obesity and T2D is associated with increased glycolysis and reduced mitochondrial oxidative metabolism in skeletal muscle, and that the latter can be improved by exercise. Future proteomics studies should be designed to markedly intensify the identification of abnormalities in metabolic and signaling pathways in skeletal muscle of insulin-resistant individuals to increase the understanding of the pathogenesis of T2D, but more importantly to identify multiple novel targets of treatment of which at least some can be safely targeted by novel drugs to treat and prevent T2D and reduce risk of cardiovascular disease.     

Lysophosphatidic acid-stimulated phosphorylation of PKD2 is mediated by PI3K p110β and PKCδ in myoblasts

Abstract

Context: G-protein coupled receptor (GPCR) signaling in skeletal muscle is incompletely understood; in particular, the signaling pathways that regulate GPCR-mediated signaling in skeletal muscle are only beginning to be established. Lysophosphatidic acid (LPA) is a GPCR agonist that has previously been shown to activate protein kinase D (PKD) in non-muscle cells; however, whether PKD is activated in response to LPA in skeletal muscle myoblasts, and the identities of signaling intermediates that regulate this activation, have not been defined. Objective: To determine whether PKD is activated in response to LPA administration in myoblasts, and to define the signaling pathways that mediate LPA-stimulated PKD phosphorylation. Methods: C2C12 myoblasts were treated with LPA and signaling pathways examined by means of Western immunoblotting and real-time PCR (RT-PCR). Pharmacological inhibition and RNA-interference were used to target specific molecules to determine their involvement in LPA-induced PKD phosphorylation. Results: Treatment of myoblasts with exogenous LPA revealed that PI3K p110β mediated PKD phosphorylation at Ser 748 and at Ser 916 through kinase-dependent and kinase-independent mechanisms. Loss of PKCδ, but not the loss of PKCα, prevented LPA-induced PKD phosphorylation. The PKD isoform responsive to LPA treatment was identified as PKD2. Conclusion: These results indicate that LPA-stimulated PKD2 phosphorylation requires PKCδ and non-catalytic actions of PI3K p110β, and provide new information with respect to GPCR-mediated signal transduction in myoblasts.     

Notch signaling genes: Myogenic DNA hypomethylation and 5-hydroxymethylcytosine

Notch intercellular signaling is critical for diverse developmental pathways and for homeostasis in various types of stem cells and progenitor cells. Because Notch gene products need to be precisely regulated spatially and temporally, epigenetics is likely to help control expression of Notch signaling genes. Reduced representation bisulfite sequencing (RRBS) indicated significant hypomethylation in myoblasts, myotubes, and skeletal muscle vs. many nonmuscle samples at intragenic or intergenic regions of the following Notch receptor or ligand genes: NOTCH1, NOTCH2, JAG2, and DLL1. An enzymatic assay of sites in or near these genes revealed unusually high enrichment of 5-hydroxymethylcytosine (up to 81%) in skeletal muscle, heart, and cerebellum. Epigenetics studies and gene expression profiles suggest that hypomethylation and/or hydroxymethylation help control expression of these genes in heart, brain, myoblasts, myotubes, and within skeletal muscle myofibers. Such regulation could promote cell renewal, cell maintenance, homeostasis, and a poised state for repair of tissue damage.     

Capsaicin mimics mechanical load-induced intracellular signaling events

Mechanical load-induced intracellular signaling events are important for subsequent skeletal muscle hypertrophy. We previously showed that load-induced activation of the cation channel TRPV1 caused an increase in intracellular calcium concentrations ([Ca²⁺]_i) and that this activated mammalian target of rapamycin (mTOR) and promoted muscle hypertrophy. However, the link between mechanical load-induced intracellular signaling events, and the TRPV1-mediated increases in [Ca²⁺]_i are not fully understood. Here we show that administration of the TRPV1 agonist, capsaicin, induces phosphorylation of mTOR, p70S6K, S6, Erk1/2 and p38 MAPK, but not Akt, AMPK or GSK3β. Furthermore, the TRPV1-induced phosphorylation patterns resembled those induced by mechanical load. Our results continue to highlight the importance of TRPV1-mediated calcium signaling in load-induced intracellular signaling pathways.     

Notch signaling genes

Notch intercellular signaling is critical for diverse developmental pathways and for homeostasis in various types of stem cells and progenitor cells. Because Notch gene products need to be precisely regulated spatially and temporally, epigenetics is likely to help control expression of Notch signaling genes. Reduced representation bisulfite sequencing (RRBS) indicated significant hypomethylation in myoblasts, myotubes, and skeletal muscle vs. many nonmuscle samples at intragenic or intergenic regions of the following Notch receptor or ligand genes: NOTCH1, NOTCH2, JAG2, and DLL1. An enzymatic assay of sites in or near these genes revealed unusually high enrichment of 5-hydroxymethylcytosine (up to 81%) in skeletal muscle, heart, and cerebellum. Epigenetics studies and gene expression profiles suggest that hypomethylation and/or hydroxymethylation help control expression of these genes in heart, brain, myoblasts, myotubes, and within skeletal muscle myofibers. Such regulation could promote cell renewal, cell maintenance, homeostasis, and a poised state for repair of tissue damage.     

Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy

Background

Chronic obstructive pulmonary disease (COPD) is accompanied by pulmonary inflammation and associated with extra-pulmonary manifestations, including skeletal muscle atrophy. Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of muscle protein- and myonuclear turnover; two crucial processes that determine muscle mass. In the present study we investigated the effect of the selective GSK-3 inhibitor SB216763 on muscle mass in a guinea pig model of lipopolysaccharide (LPS)-induced pulmonary inflammation-associated muscle atrophy.

Methods

Guinea pigs were pretreated with either intranasally instilled SB216763 or corresponding vehicle prior to each LPS/saline challenge twice weekly. Pulmonary inflammation was confirmed and indices of muscle mass were determined after 12 weeks. Additionally, cultured skeletal muscle cells were incubated with tumor necrosis factor α (TNF-α) or glucocorticoids (GCs) to model the systemic effects of pulmonary inflammation on myogenesis, in the presence or absence of GSK-3 inhibitors.

Results

Repeated LPS instillation induced muscle atrophy based on muscle weight and muscle fiber cross sectional area. Intriguingly, GSK-3 inhibition using SB216763 prevented the LPS-induced muscle mass decreases and myofiber atrophy. Indices of protein turnover signaling were unaltered in guinea pig muscle. Interestingly, inhibition of myogenesis of cultured muscle cells by TNF-α or synthetic GCs was prevented by GSK-3 inhibitors.

Conclusions

In a guinea pig model of LPS-induced pulmonary inflammation, GSK-3 inhibition prevents skeletal muscle atrophy without affecting pulmonary inflammation. Resistance to inflammation- or GC-induced impairment of myogenic differentiation, imposed by GSK-3 inhibition, suggests that sustained myogenesis may contribute to muscle mass maintenance despite persistent pulmonary inflammation. Collectively, these results warrant further exploration of GSK-3 as a potential novel drug target to prevent or reverse muscle wasting in COPD.     

Skeletal muscle reactive oxygen species: A target of good cop/bad cop for exercise and disease

Abstract

Metabolic stresses associated with disease, ageing, and exercise increase the levels of reactive oxygen species (ROS) in skeletal muscle. These ROS have been linked mechanistically to adaptations in skeletal muscle that can be favourable (i.e. in response to exercise) or detrimental (i.e. in response to disease). The magnitude, duration (acute versus chronic), and cellular origin of the ROS are important underlying factors in determining the metabolic perturbations associated with the ROS produced in skeletal muscle. In particular, insulin resistance has been linked to excess ROS production in skeletal muscle mitochondria. A chronic excess of mitochondrial ROS can impair normal insulin signalling pathways and glucose disposal in skeletal muscle. In contrast, ROS produced in skeletal muscle in response to exercise has been linked to beneficial metabolic adaptations including mitochondrial biogenesis and muscle hypertrophy. Moreover, unlike insulin resistance, exercise-induced ROS appears to be primarily of non-mitochondrial origin. The present review summarizes the diverse ROS-targeted metabolic outcomes associated with insulin resistance versus exercise in skeletal muscle, thus, presenting two contrasting perspectives of pathologically harmful versus physiologically beneficial ROS. Here, we discuss the key sites of ROS production during exercise and the effect of ROS in skeletal muscle of people with type 2 diabetes.     

Hedgehog信号通路与动物骨骼肌发育调控

Hedgehog信号通路在动物胚胎期及出生后骨骼肌的生长发育过程中发挥着重要作用。本文综述了Hedgehog信号通路对骨骼肌细胞增殖分化及肌纤维特性的调控作用及其在骨骼肌发育过程中与其它信号通路交互作用最新研究进展,为畜禽肉品质改良和肌肉相关疾病治疗提供理论基础。     

肥胖诱导的骨骼肌萎缩机制研究进展

骨骼肌是人体氨基酸和蛋白质的主要贮存、代谢库,其正常功能和代谢过程受到多种病理因素的影响。骨骼肌萎缩发生于骨骼肌稳态严重失衡状态下,对患者生活和社会医疗造成了沉重负担。近年来,由于世界肥胖人群数量激增,肥胖诱导的骨骼肌萎缩正日益成为公共卫生的严峻挑战之一。肥胖诱导的骨骼肌萎缩过程涉及多种信号分子或通路的改变,如泛素蛋白酶系统、自噬溶酶体系统、胰岛素/IGF1-PI3K-Akt、肌肉生长抑制素、白细胞介素-6、肿瘤坏死因子等;这些信号分子或通路在肥胖状态下被激活或抑制后,可共同影响蛋白质合成/分解平衡进而造成骨骼肌萎缩。基于上述信号分子或通路,系统总结并讨论了肥胖诱导的骨骼肌萎缩机制,以期为寻找缓解/治疗肥胖诱导的肌萎缩靶点和进一步开发利用天然植物化学物提供理论依据。     

BRCA1 is a novel regulator of metabolic function in skeletal muscle

Breast cancer type 1 (BRCA1) susceptibility protein is expressed across multiple tissues including skeletal muscle. The overall objective of this investigation was to define a functional role for BRCA1 in skeletal muscle using a translational approach. For the first time in both mice and humans, we identified the presence of multiple isoforms of BRCA1 in skeletal muscle. In response to an acute bout of exercise, we found increases in the interaction between the native forms of BRCA1 and the phosphorylated form of acetyl-CoA carboxylase. Decreasing BRCA1 content using a shRNA approach in cultured primary human myotubes resulted in decreased oxygen consumption by the mitochondria and increased reactive oxygen species production. The decreased BRCA1 content also resulted in increased storage of intracellular lipid and reduced insulin signaling. These results indicate that BRCA1 plays a critical role in the regulation of metabolic function in skeletal muscle. Collectively, these data reveal BRCA1 as a novel target to consider in our understanding of metabolic function and risk for development of metabolic-based diseases.     

Proteomic serum biomarkers for neuromuscular diseases

Introduction: The clinical evaluation of neuromuscular symptoms often includes the assessment of altered blood proteins or changed enzyme activities. However, the blood concentration of many muscle-derived serum markers is not specific for different neuromuscular disorders and also shows alterations in the course of these diseases. Thus, the establishment of more reliable biomarker signatures for improved muscle diagnostics is required.

Areas covered: To address the lack of muscle disease-specific marker molecules, mass spectrometry-based proteomics was applied to the systematic identification and biochemical characterization of new serum biomarker candidates. This article outlines serum proteomics in relation to neuromuscular disorders and reviews the bioanalytical results from recent proteomic profiling studies of representative neuromuscular disorders, including motor neuron disease, muscular dystrophies and sarcopenia of old age. Pathophysiological changes in the skeletal muscle proteome are reflected by serum alterations in a variety of sarcomeric proteins, metabolic enzymes and signaling proteins.

Expert commentary: Based on the proteomic identification of actively secreted or passively released skeletal muscle proteins following pathophysiological insults, new biomarker candidates can now be used to develop liquid biopsy procedures for superior diagnostic approaches, design novel prognostic tools and establish more reliable methods for the systematic evaluation of experimental therapies to treat neuromuscular disease.     

Recovery of muscle mass and muscle oxidative phenotype following disuse does not require GSK-3 inactivation

BackgroundPhysical inactivity contributes to muscle wasting and reductions in mitochondrial oxidative phenotype (OXPHEN), reducing physical performance and quality of life during aging and in chronic disease. Previously, it was shown that inactivation of glycogen synthase kinase (GSK)-3β stimulates muscle protein accretion, myogenesis, and mitochondrial biogenesis. Additionally, GSK-3β is inactivated during recovery of disuse-induced muscle atrophy.AimTherefore, we hypothesize that GSK-3 inhibition is required for reloading-induced recovery of skeletal muscle mass and OXPHEN.MethodsWild-type (WT) and whole-body constitutively active (C.A.) Ser^21/9 GSK-3α/β knock-in mice were subjected to a 14-day hind-limb suspension/14-day reloading protocol. Soleus muscle mass, fiber cross-sectional area (CSA), OXPHEN (abundance of sub-units of oxidative phosphorylation (OXPHOS) complexes and fiber-type composition), as well as expression levels of their main regulators (respectively protein synthesis/degradation, myogenesis and peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) signaling) were monitored.ResultsSubtle but consistent differences suggesting suppression of protein turnover signaling and decreased expression of several OXPHOS sub-units and PGC-1α signaling constituents were observed at baseline in C.A. GSK-3 versus WT mice. Although soleus mass recovery during reloading occurred more rapidly in C.A. GSK-3 mice, this was not accompanied by a parallel increased CSA. The OXPHEN response to reloading was not distinct between C.A. GSK-3 and WT mice. No consistent or significant differences in reloading-induced changes in the regulatory steps of protein turnover, myogenesis or muscle OXPHEN were observed in C.A. GSK-3 compared to WT muscle.ConclusionThis study indicates that GSK-3 inactivation is dispensable for reloading-induced recovery of muscle mass and OXPHEN.     

Ginsenoside Rb1 exerts antidiabetic action on C2C12 muscle cells by leptin receptor signaling pathway

Context: Ginsenoside Rb1 improves insulin sensitivity and glucose uptake in muscle cells via different signaling pathways; however, it is not clear that it has any effect on leptin signaling in skeletal muscle.

Objectives: The aim of this study was to investigate the effect of ginsenoside Rb1 on leptin receptors expression and main signaling pathways of leptin (STAT3, PI3 kinase and ERK kinase) in C2C12 skeletal muscle cells.

Materials and methods: C2C12 myotubes were incubated with various concentrations of Rb1 (0.1, 1 and 10?μM) for different incubation times (1–12?h). Leptin receptors expression and GLUT-4 translocation were analyzed using realtime PCR and western blot analyses, respectively. PI3 and ERK kinases were blocked using their specific inhibitors (wortmannin and PD98059) in the presence and absence of RB1 to determine the main signaling pathway related to leptin receptor activation in C2C12 cells.

Results: Rb1 could maximally stimulate both leptin receptors (OBRa and OBRb) mRNA and protein expression and phosphorylation of STAT3, PI3K and ERK2 in C2C12 myotubes at 10?μM for 3?h. Rb1 induced GLUT4 translocation was inhibited by the silencing of OBRb mRNA, demonstrated that glucose uptake was mediated via leptin receptor activation. GLUT4 recruitment to the cell surface induced by Rb1 was inhibited by wortmannin, an inhibitor of PI3K in combination with OBRb siRNA, but not by PD98059 an ERK2 kinase-1 inhibitor, indicating that GLUT4 translocation induced by Rb1 was associated with the leptin receptor upregulation and subsequent activation of PI3K.

Conclusions: Our results suggest that Rb1 promote translocation of GLUT4 by upregulation of leptin receptors and activation of PI3K.     

Mechanotransduction and the regulation of mTORC1 signaling in skeletal muscle

Mechanical stimuli play a major role in the regulation of skeletal muscle mass, and the maintenance of muscle mass contributes significantly to disease prevention and issues associated with the quality of life. Although the link between mechanical signals and the regulation of muscle mass has been recognized for decades, the mechanisms involved in converting mechanical information into the molecular events that control this process remain poorly defined. Nevertheless, our knowledge of these mechanisms is advancing and recent studies have revealed that signaling through a protein kinase called the mammalian target of rapamycin (mTOR) plays a central role in this event. In this review we will, (1) discuss the evidence which implicates mTOR in the mechanical regulation of skeletal muscle mass, (2) provide an overview of the mechanisms through which signaling by mTOR can be regulated, and (3) summarize our current knowledge of the potential mechanisms involved in the mechanical activation of mTOR signaling.     

HOX-1 and COX-2: Two differentially regulated key mediators of skeletal myoblast tolerance under oxidative stress

Abstract

The exact physiological role of oxidative stress as a primary cause for skeletal muscle pathological conditions involving muscle degeneration remains elusive. Therefore, the present study was performed so as to decipher the signalling pathways orchestrating the potential cytoprotective role of heme oxygenase 1 (HOX-1) as well as cyclooxygenase 2 (COX-2) in skeletal myoblasts exposed to H₂O₂. Cell treatment with H₂O₂ (0.5 mM) resulted in a time- and dose-dependent response of HOX-1 and COX-2 mRNA and protein levels, with ERK1/2, p38-MAPK and MSK1 found to mediate these effects. Furthermore, Src and JNKs blockade attenuated COX-2 response. Collectively, these novel findings highlight for the first time HOX-1 and COX-2 fundamental contribution to skeletal myoblast tolerance under oxidative stress, since their inhibition significantly attenuated viability of skeletal myoblasts. The data also delineate the various effectors regulating HOX-1 and COX-2 expression, probably alleviating muscle degeneration in related disorders.     

Skeletal muscle damage and impaired regeneration due to LPL-mediated lipotoxicity

According to the concept of lipotoxicity, ectopic accumulation of lipids in non-adipose tissue induces pathological changes. The most prominent effects are seen in fatty liver disease, lipid cardiomyopathy, non-insulin-dependent diabetes mellitus, insulin resistance and skeletal muscle myopathy. We used the MCK(m)-hLPL mouse distinguished by skeletal and cardiac muscle-specific human lipoprotein lipase (hLPL) overexpression to investigate effects of lipid overload in skeletal muscle. We were intrigued to find that ectopic lipid accumulation induced proteasomal activity, apoptosis and skeletal muscle damage. In line with these findings we observed reduced Musculus gastrocnemius and Musculus quadriceps mass in transgenic animals, accompanied by severely impaired physical endurance. We suggest that muscle loss was aggravated by impaired muscle regeneration as evidenced by reduced cross-sectional area of regenerating myofibers after cardiotoxin-induced injury in MCK(m)-hLPL mice. Similarly, an almost complete loss of myogenic potential was observed in C2C12 murine myoblasts upon overexpression of LPL. Our findings directly link lipid overload to muscle damage, impaired regeneration and loss of performance. These findings support the concept of lipotoxicity and are a further step to explain pathological effects seen in muscle of obese patients, patients with the metabolic syndrome and patients with cancer-associated cachexia.