Interleukin-6 mimics insulin-dependent cellular distribution of some cytoskeletal proteins and Glut4 transporter without effect on glucose uptake in 3T3-L1 adipocytes.

Interleukin (IL)-6, a known proinflammatory cytokine, is released in both visceral adipose tissue and contracting skeletal muscle. In this study, we used microRNA profiling as a screening method to identify miRNA species modified by IL-6 treatment in mouse 3T3-L1 adipocytes. miRNA microarray analysis and qRT-PCR revealed increased expression of miR-146b-3p in adipocytes exposed to IL-6 (1 ng/ml) during 8-day differentiation. On the basis of ontological analysis of potential targets, selected proteins associated with cytoskeleton and transport were examined in the context of adipocyte response to insulin, using immunofluorescence and confocal microscopy. We concluded that IL-6: (i) does not affect insulin action on actin cellular distribution; (ii) modulates the effect of insulin on myosin light chain kinase (Mylk) distribution by preventing its shift toward cytoplasm; (iii) mimics the effect of insulin on dynein distribution by increasing its near-nuclear accumulation; (iv) mimics the effect of insulin on glucose transporter Glut4 distribution, especially by increasing its near-nuclear accumulation; (v) supports insulin action on early endosome marker Rab4A near-nuclear accumulation. Moreover, as IL-6 did not disturb insulin-dependent glucose uptake, our results do not confirm the IL-6-induced impairment of insulin action observed in some in vitro studies, suggesting that the effect of IL-6 is dose dependent.

Interleukin-6 affects pacsin3, ephrinA4 expression and cytoskeletal proteins in differentiating primary skeletal myoblasts through transcriptional and post-transcriptional mechanisms.

Interleukin (IL)-6 is a proinflammatory cytokine released in injured and contracting skeletal muscles. In this study, we examined cellular expression of proteins associated with cytoskeleton organization and cell migration, chosen on the basis of microRNA profiling, in rat primary skeletal muscle cells (RSkMC) treated with IL-6 (1 ng/ml) for 11 days. MiRNA microarray analysis and qRT-PCR revealed increased expression of miR-154-3p and miR-338-3p in muscle cells treated with IL-6. Pacsin3 was downregulated post-transcriptionally by IL-6, but not by IGF-I. Ephrin4A protein was increased both in IL-6- and IGF-I-treated myocytes. IL-6, but not IGF-I, stimulated migratory ability of RSkMC, examined in wound healing assay. Alpha-actinin protein was slightly augmented in RSKMC treated with IL-6, similarly to IGF-I. IL-6, but not IGF-I, upregulated desmin in differentiating RSkMC. IL-6 supplementation caused accumulation of alpha-actinin and desmin in near-nuclear area of muscle cells, which was manifested by increased ratio: mean near-nuclear fluorescence/mean peripheral cytoplasm fluorescence of these proteins. We concluded that IL-6, a known proinflammatory cytokine and a physical activity-associated myokine, acting during differentiation of primary skeletal muscle cells, alters expression of nonmuscle-specific miRNAs. This cytokine causes differential effects on pacsin-3 and ephrinA4, through post-transcriptional inhibition and stimulation, respectively. IL-6-exerted modifications of cytoskeletal proteins in muscle cells include both transcriptional (desmin and dynein heavy chain 5) and post-transcriptional activation (alpha-actinin). Moreover, IL-6 augments near-nuclear distribution of cytoskeletal proteins, alpha-actinin and desmin and promotes migration of myocytes. Such effects suggest that IL-6 plays a role during skeletal muscle regeneration, acting through mechanisms independent of regulation of myogenic program.

Interleukin-8 enhances myocilin expression, Akt-FoxO3 signaling and myogenic differentiation in rat skeletal muscle cells.

Interleukin (IL)-8 is released both in visceral adipose tissue and in contracting skeletal muscles. In this study, we examined cellular pathways associated with muscle hypertrophy, chosen on the basis of microRNA profiling, in differentiating rat primary skeletal muscle cells (RSkMC) treated with IL-8 (1 ng/ml) for 11 days. IL-8 increased myocilin expression, Akt phosphorylation, FoxO3 dispersion throughout the cytoplasm, and reduced FoxO3 level. IL-8 decreased the expression of atrogin and MuRF1 and increased myotube length and diameter. We concluded that IL-8 present in extracellular environment of myoblasts induced to differentiation stimulates expression of myocilin, a protein important for skeletal muscle hypertrophy. This phenomenon was associated with: (a) activation of myogenic transcription, (b) increased phosphorylation and activation of PKB/Akt, leading to (c) cytoplasm distribution and degradation of a transcription factor FoxO3, (d) decreased expression of gene markers of proteolysis, atrogin and Murf1, and (e) increased myotube length and diameter. In this regard, IL-8 affects skeletal muscle cells similarly to IGF-I and can be considered as a potent anticatabolic factor for skeletal muscle.

Transcriptomic Profiling During Myogenesis.

Microarray-based transcriptomic profiling enables simultaneous measurement of expression of multiple genes from one biological sample. Here we describe a detailed protocol, which serves to examine global gene expression using whole genome oligonucleotide microarrays. We also provide examples of bioinformatics tools, which are helpful in analyses and interpretation of microarray data, and propose further biological assays, to warrant conclusions drawn from transcriptomic signature.

Insulin-dependent cytoplasmic distribution of Rab4a in mouse adipocytes is inhibited by interleukin-6, -8, and -15.

The purpose of the study was to examine the effect of interleukins, IL-6, IL-8, and IL-15, on insulin-mediated redistribution of Rab4a, an early endosome marker, in mouse 3T3-L1 adipocytes. The interleukins did not affect cell viability; however, cell number was slightly but significantly higher in cultures exposed to IL-8 and IL-15. IL-8 and IL-15 decreased lipid storage in adipocytes, whereas IL-6 had no effect. Rab4A showed cytoplasmic localization, and in control unstimulated adipocytes it was found primarily nearby nucleus, that was supported by cellular fluorescence distribution profile, and by calculated indices, that is, high percentage of near-nuclear area fluorescence and a low mean peripheral cytoplasmic fluorescence/mean near-nuclear fluorescence ratio. Insulin stimulation (100 nmol/l, 30 min) altered the cytoplasmic localization of Rab4a in control adipocytes, which was manifested by its redistribution towards plasma membrane. This effect of insulin was prevented in adipocytes exposed to IL-6, IL-8, or IL-15. We concluded that insulin-dependent Rab4a redistribution, probably reflecting stimulation of vesicle-mediated transport, is inhibited in adipocytes subjected to differentiation in the presence of IL-6, IL-8, or IL-15. Such alterations may be involved in the mechanisms contributing to development of insulin resistance associated with inflammation; however, further studies in this field are required.

Palmitate exerts opposite effects on proliferation and differentiation of skeletal myoblasts.

The purpose of the study was to examine mechanisms controlling cell cycle progression/arrest and differentiation of mouse C2C12 myoblasts exposed to long-chain saturated fatty acid salt, palmitate. Treatment of proliferating myoblasts with palmitate (0.1 mmol/l) markedly decreased myoblast number. Cyclin A and cyclin D1 levels decreased, whereas total p21 and p21 complexed with cyclin-dependent kinase-4 (cdk4) increased in myoblasts treated with palmitate. In cells induced to differentiation addition of palmitate augmented the level of cyclin D3, the early (myogenin) and late (α-actinin, myosin heavy chain) markers of myogenesis, and caused an increase of myotube diameter. In conclusion, exposure to palmitate inhibits proliferation of myoblasts through a decrease in cyclin A and cyclin D1 levels and an increase of p21-cdk4 complex formation; however, it promotes cell cycle exit, myogenic differentiation and myotube growth.

Insulin-Like Growth Factor-I Increases Laminin, Integrin Subunits and Metalloprotease ADAM12 in Mouse Myoblasts.

The extracellular matrix (ECM) is considered a part of the myogenesis signaling mechanism. we hypothesized that insulin-like growth factor-I (IGF-I) modifies ECM during differentiation of mouse C2C12 cells. The myogenic effect of IGF-I (30 nmol/l) was manifested by increased myogenin and myosin heavy chain (MyHC) levels as well as fusion index (2.6 times over control) on the 3rd day of differentiation. IGF-I markedly augmented laminin, but not fibronectin. Cellular contents of integrin α3, α5 and ß1 during 3-day differentiation increased in the presence of IGF-I. Treatment with IGF-I increased the expression of the long form of metalloprotease ADAM12 (100 kDa) in myocytes. In conclusion: i) IGF-I caused an increase of laminin, integrin α3 and ß1 in C2C12 myogenic cells that can be secondary to stimulation of myogenesis; ii) IGF-I augmented integrin α5 and ADAM12 levels, suggesting a role of this growth factor in determination of the pool of reserve cells during myogenesis.

Transcriptional regulation of important cellular processes in skeletal myogenesis through interferon-γ.

The purpose of the present study was to investigate the effect of interferon (IFN)-γ on the transcriptomic profile of differentiating mouse C2C12 myogenic cells. Global gene expression was evaluated using whole mouse genome oligonucleotide microarrays, and the results were validated through real-time PCR. IFN-γ (1 ng/mL) increased myoblast proliferation but decreased cell respiration and myosin heavy chain content and slightly decreased the fusion index in differentiating C2C12 cell cultures. The genes upregulated through IFN-γ were involved in cell cycle; regulation of cell proliferation; programmed cell death; chemotaxis; and cytokine, growth factor, and peptidase activity, whereas the genes downregulated through IFN-γ primarily contributed to the regulation of transcription, cell-cell signaling, nitrogen compound biosynthesis, ser/thr protein kinase signaling, and regulation of the Wnt pathway. In conclusion, IFN-γ affects the expression of numerous genes associated with the regulation of several processes in myogenesis. The effects of IFN-γ on cellular transcription include (1) alteration of cytokine/growth factor expression, promoting cell proliferation and migration but inhibiting differentiation, (2) impairment of pro-myogenic transcription, (3) disruption of cell adhesion and sarcolemma/cytoskeleton organization, and (4) increased peptidase activity leading to enhanced proteolysis and apoptosis.

[Developmental programming of metabolic diseases--a review of studies on experimental animal models]. / Programowanie rozwojowe chorób metabolicznych--przeglad wyników badan na zwierzecych modelach doswiadczalnych.

Growth and development in utero is a complex and dynamic process that requires interaction between the mother organism and the fetus. The delivery of macro--and micronutrients, oxygen and endocrine signals has crucial importance for providing a high level of proliferation, growth and differentiation of cells, and a disruption in food intake not only has an influence on the growth of the fetus, but also has negative consequences for the offspring's health in the future. Diseases that traditionally are linked to inappropriate life style of adults, such as type 2 diabetes, obesity, and arterial hypertension, can be "programmed" in the early stage of life and the disturbed growth of the fetus leads to the symptoms of the metabolic syndrome. The structural changes of some organs, such as the brain, pancreas and kidney, modifications of the signaling and metabolic pathways in skeletal muscles and in fatty tissue, epigenetic mechanisms and mitochondrial dysfunction are the basis of the metabolic disruptions. The programming of the metabolic disturbances is connected with the disruption in the intrauterine environment experienced in the early and late gestation period. It causes the changes in deposition of triglycerides, activation of the hormonal "stress axis" and disturbances in the offspring's glucose tolerance. The present review summarizes experimental results that led to the identification of the above-mentioned links and it underlines the role of animal models in the studies of this important concept.

[Interactions of proliferation and differentiation signaling pathways in myogenesis]. / Interakcje szlaków sygnalowych proliferacji i róznicowania w miogenezie

The commitment of myogenic cells in skeletal muscle differentiation requires earlier irreversible interruption of the cell cycle. At the molecular level, several key regulators of the cell cycle have been identified: cyclin-dependent kinases and their cyclins stimulate the cell cycle progress and its arrest is determined by the activity of cdk inhibitors (Cip/Kip and INK protein families) and pocket protein family: Rb, p107 and p130. The biological activity of cyclin/cdk complexes allows the successive phases of the cell cycle to occur. Myoblast specialization, differentiation and fusion require the activity of myogenic regulatory factors, which include MyoD, myogenin, Myf5 and MRF4. MyoD and Myf5 play a role in muscle cell specialization, myogenin controls the differentiation process, whereas MRF4 is involved in myotube maturation. The deregulation of the cell cycle leads to uncontrolled proliferation, which antagonizes the functions of myogenic factors and it explains the lack of differentiation-specific gene expression in dividing cells. Conversely, the myogenic factor MyoD seems to cooperate with cell cycle inhibitors leading to inhibition of cell cycle progress and commitment to the differentiation process. The hypophosphorylated form of Rb and cdk inhibitors play an important role in permanent arrest of the cell cycle in differentiated myotubes. Furthermore, cyclin/cdk complexes not only regulate cell division by phosphorylation of several substrates, but may also control other cellular processes such as signal transduction, differentiation and apoptosis. Beyond regulating the cell cycle, Cip/Kip proteins play an important role in cell death, transcription regulation, cell fate determination, cell migration and cytoskeletal dynamics. The article summarizes current knowledge concerning the interactions of intracellular signaling pathways controlling crucial stages of fetal and regenerative myogenesis.

[Systemic and local mechanisms leading to cachexia in cancer]. / Ogólnoustrojowe i miejscowe mechanizmy prowadzace do kacheksji w chorobach nowotworowych.

Cachexia is a multifactorial syndrome of atrophy of skeletal muscle and adipose tissue, resulting in progressive loss of body weight associated with low quality of life and poor prognosis in cancer. Studies on experimental animal models and observations on patients have shown that the soluble factors secreted by tumor cells and tissues of the patient can participate in regulation of the wasting process. Cachexia is often accompanied by anorexia, which is caused by predominance of signals inhibiting appetite in the hypothalamus, such as release of proopiomelanocortin and anorexigenic action of proinflammatory cytokines (IL-1α, IL-1ß, IL-6, TNF-α). Cachexia is also accompanied by extensive metabolic changes consisting of increase of resting energy expenditure and disturbance of carbohydrate, protein and lipid metabolism. Increased expression of protein uncoupling phosphorylation leads to increased thermogenesis in skeletal muscle. Tumor tissue hypoxia caused by its growth beyond blood vessels activates the transcription factor HIF-1, which results in increase in glycolysis, and leads to lactic acid accumulation and activation of the energy inefficient Cori cycle. Loss of fat tissue is caused by increase of lipolysis induced by lipid-mobilizing factor (LMF) and proinflammatory cytokines. Skeletal muscle wasting in cachexia is caused by a reduction of protein synthesis at the stage of initiation and elongation of translation and the simultaneous increase of protein degradation via ubiquitin-dependent and lysosomal pathways. The main mediators of skeletal muscle wasting in cancer are proteolysis-inducing factor (PIF), proinflammatory cytokines, and angiotensin II acting through increased levels of reactive oxygen species (ROS) and nuclear factor NF-κB activation, as well as glucocorticoid activated FOXO transcription factors and myostatin. Understanding of the complexity of the interaction of factors produced by the tumor and the patient's body may form the basis for the development of effective treatments for cachexia in cancer and other pathological conditions.

[Maternal obesity and the development of skeletal muscle in offspring--fetal origin of metabolic disorders]. / Matczyna otylosc a rozwój miesni szkieletowych u potomstwa--plodowe pochodzenie zaburzen metabolicznych.

Suboptimal fetal environments due to inadequate maternal nutrition, obesity, inflammation or gestational diabetes expose the fetus to humoral cues that alter metabolism and growth parameters leading to metabolic disturbances later in life. The fetal stage is crucial for the development of skeletal muscle, a tissue playing an important role in metabolism. Maternal obesity induces inflammation in the fetus causing modifications in the development of fetal skeletal muscle. Changes in the normal course of myogenesis may arise through several mechanisms: changes in WNT/ß-catenin signaling pathway, decreased AMPK activity evoked by TNF-α, increased activity of NF-κB in response to inflammation, which leads to a decrease in myogenic factor MyoD, and increased expression of TGF ß1. Modification in fetal development associated with maternal obesity is attributed to epigenetic changes. Polyunsaturated fatty acids supplied in the diet did affect the development of insulin-sensitive tissues during both the fetal and postnatal period. The specific phenotype of skeletal muscle fibers may play a role in the development of obesity, i.e. fiber phenotype I (slow, oxidative) may protect against obesity and insulin resistance. Exploring the mechanisms of direct impact of maternal obesity on the development of tissues in the offspring may help to reduce the occurrence of metabolic diseases in later life.

Treatment with TNF-alpha and IFN-gamma alters the activation of SER/THR protein kinases and the metabolic response to IGF-I in mouse c2c12 myogenic cells.

UNLABELLED: The aim of this study was to compare the effects of TNF-alpha, IL-1beta and IFN-gamma on the activation of protein kinase B (PKB), p70(S6k), mitogen-activated protein kinase (MAPK) and p90( rsk ), and on IGF-I-stimulated glucose uptake and protein synthesis in mouse C2C12 myotubes. 100 nmol/l IGF-I stimulated glucose uptake in C2C12 myotubes by 198.1% and 10 ng/ml TNF-alpha abolished this effect. Glucose uptake in cells differentiated in the presence of 10 ng/ml IFN-gamma increased by 167.2% but did not undergo significant further modification upon the addition of IGF-I. IGF-I increased the rate of protein synthesis by 249.8%. Neither TNF-alpha nor IFN-gamma influenced basal protein synthesis, but both cytokines prevented the IGF-I effect. 10 ng/ml IL-1beta did not modify either the basal or IGF-I-dependent glucose uptake and protein synthesis. With the exception of TNF-alpha causing an 18% decrease in the level of PKB protein, the cellular levels of PKB, p70(S6k), p42(MAPK), p44(MAPK) and p90( rsk ) were not affected by the cytokines. IGF-I caused the phosphorylation of PKB (an approximate 8-fold increase above the basal value after 40 min of IGF-I treatment), p42(MAPK) (a 2.81-fold increase after 50 min), and the activation of p70(S6k) and p90( rsk ), manifesting as gel mobility retardation. In cells differentiated in the presence of TNF-alpha or IFN-gamma, this IGF-I-mediated PKB and p70(S6k) phosphorylation was significantly diminished, and the increase in p42(MAPK) and p90( rsk ) phosphorylation was prevented. The basal p42(MAPK) phosphorylation in C2C12 cells treated with IFN-gamma was high and comparable with the activation of this kinase by IGF-I. Pretreatment of myogenic cells with IL-1beta did not modify the IGF-I-stimulated phosphorylation of PKB, p70(S6k), p42(MAPK) and p90( rsk ). IN CONCLUSION: i) TNF-alpha and IFN-gamma, but not IL-1beta, if present in the extracellular environment during C2C12 myoblast differentiation, prevent the stimulatory action of IGF-I on protein synthesis. ii) TNF-alpha- and IFN-gamma-induced IGF-I resistance of protein synthesis could be associated with the decreased phosphorylation of PKB and p70(S6k). iii) The activation of glucose uptake in C2C12 myogenic cells treated with IFN-gamma is PKB independent. iv) The similar effects of TNF-alpha and IFN-gamma on the signalling and action of IGF-I on protein synthesis in myogenic cells could suggest the involvement of both of these cytokines in protein loss in skeletal muscle.

Treatment with IFN-gamma prevents insulin-dependent PKB, p70S6k phosphorylation and protein synthesis in mouse C2C12 myogenic cells.

The purpose of the present study was to examine the potential effect of IFN-gamma (interferon-gamma) on the cellular content and phosphorylation of PKB (protein kinase B), p70S6k (p70 S6 kinase) and MAPK (mitogen-activated protein kinase), and on the ability of insulin to stimulate the glucose uptake and protein synthesis in mouse C2C12 myotubes. Insulin (100 nmol/l) stimulated glucose uptake in C2C12 myotubes by 203.4%. Glucose uptake in cells differentiated in the presence of IFN-gamma (10 ng/ml) was increased by 165.8% and was not further significantly modified by the addition of insulin (183.4% of control value). Insulin increased the rate of protein synthesis by 198.8%. The basal rate of protein synthesis was not affected by IFN-gamma; however, this cytokine abolished the insulin effect. Cellular levels of PKB, p70S6k, p42MAPK and p44MAPK were not modified by IFN-gamma. Insulin caused the phosphorylation of PKB and the activation of p70S6k, but not p42MAPK and p44MAPK. In cells differentiated in the presence of IFN-gamma, the insulin-mediated PKB phosphorylation was significantly diminished, whereas the phosphorylation of p70S6k was completely prevented. Pretreatment of C2C12 myogenic cells with IFN-gamma led to the marked increase in p42MAPK phosphorylation. Exposure of C2C12 myoblasts to IFN-gamma impaired MyoD and myogenin expression and decreased the fusion index on the fifth day of differentiation. In conclusion, (i) IFN-gamma present in the extracellular environment during C2C12 myoblast differentiation prevents the stimulatory action of insulin on protein synthesis; (ii) IFN-gamma-induced insulin resistance of protein synthesis in myogenic cells can be associated with the decreased phosphorylation of PKB and p70S6k, as well as with the augmented basal phosphorylation of p42MAPK; (iii) this cytokine effect can be partly explained by alterations in the differentiation process.