Tumor necrosis factor-α alters integrins and metalloprotease ADAM12 levels and signaling in differentiating myoblasts.

The extracellular matrix (ECM) is important in the regulation of myogenesis. We hypothesized that tumor necrosis factor-α (TNF-α) modifies ECM during differentiation of mouse C2C12 myoblasts. Exogenous TNF-α (1 ng/ml) stimulated myoblast fusion on the 3rd day (by 160% vs control) but not on the 5th day of myogenesis. The level of integrin α5 was significantly augmented by TNF-α during 5 day-differentiation; however, integrin ß1 was higher than control only on the 3rd day of cytokine treatment. Both the abundance of integrin α5 bound to actin and the level of integrin ß1 complexed with integrin α5 increased in the presence of TNF-α, especially on the 3rd day of differentiation. Similarly, the stimulatory effects of TNF-α on integrin α3, metalloprotease ADAM12 and kinases related to integrins, FAK and ILK, were limited to the 3rd day of differentiation. We concluded that TNF-α-induced changes in ECM components in differentiating myogenic cells, i.e. i) increased expression of integrin α5, ß1, α3, and metalloprotease ADAM12, ii) enhanced formation of α5ß1 integrin receptors and interaction of integrin α5-cytoskeleton, and iii) increased expression of kinases associated with integrin signaling, FAK and ILK, were temporarily associated with the onset of myocyte fusion.

The effect of palmitate supplementation on gene expression profile in proliferating myoblasts.

High-fat diet, exposure to saturated fatty acids, or the presence of adipocytes in myoblast microenvironment affects skeletal muscle growth and function. The aim of the present study was to investigate the effect of palmitate supplementation on transcriptomic profile of mouse C2C12 myoblasts. Global gene expression was evaluated using whole mouse genome oligonucleotide microarrays, and the results were validated through qPCR. A total of 4047 genes were identified as differentially expressed, including 3492 downregulated and 555 upregulated genes, during a 48-h exposure to palmitate (0.1 mmol/l). Functional classification showed the involvement of these genes in several processes which regulate cell growth. In conclusion, the addition of palmitate modifies the expression of genes associated with (1) myoblast responsiveness to hormones and growth factors, (2) cytokine and growth factor expression, and (3) regulation of cell-cell and cell-matrix communication. Such alterations can affect myoblast growth and differentiation; however, further studies in this field are required.

The influence of high glucose and high insulin on mechanisms controlling cell cycle progression and arrest in mouse C2C12 myoblasts: the comparison with IGF-I effect.

BACKGROUND: Myogenesis is susceptible to the availability of nutrients and humoral factors and suboptimal fetal environments affect the number of myofibers and muscle mass. AIM: We examined the mechanisms regulating cell cycle progression and arrest in skeletal myoblasts. MATERIALS AND METHODS: Mouse C2C12 myoblasts were subjected to proliferation or induction of differentiation in the presence of high glucose and high insulin (HGHI glucose 15 mmol/l, insulin 50 nmol/l), and these effects were compared with the influence of anabolic factor for skeletal muscle, insulin-like growth factor-I (IGF-I 30 nmol/l). RESULTS: High glucose and high insulin, similarly to IGF-I, increased the intracellular level of cyclin A, cyclin B1 and cyclin D1 during myoblast proliferation. In HGHI-treated myoblasts, these cyclins were localized mostly in the nuclei, and the level of cdk4-bound cyclin D1 was augmented. HGHI significantly stimulated the expression of cyclin D3, total level of p21 and cdk-bound fraction of p21 in differentiating cells. The cellular level of MyoD was augmented by HGHI both in proliferating and differentiating myogenic cells. CONCLUSIONS: High glucose and insulin modify the mechanisms controlling cell cycle progression and the onset of myogenesis by: (1) increase of cyclin A, cyclin B1 and cyclin D1 in myoblast nuclei, and stimulation of cyclin D1-cdk4 binding; (2) increase in cyclin D3 and MyoD levels, and the p21-cdk4 complexes after induction of differentiation. Hyperglycemia/hyperinsulinemia during fetal or postnatal life could exert effects similar to IGF-I and can be, therefore, favourable for skeletal muscle growth and regeneration.

Interleukin-1beta stimulates early myogenesis of mouse C2C12 myoblasts: the impact on myogenic regulatory factors, extracellular matrix components, IGF binding proteins and protein kinases.

The purpose of the study was to examine the mechanisms important for early myogenesis in mouse C2C12 myogenic cells exposed to interleukin-1beta. Cyclin A and cyclin B1 were increased by interleukin-1beta (1 ng/ml), but the level of cyclin D1 and total DNA content was unaffected. Fusion index and the rate of protein synthesis was increased in the presence of IL-1beta, but these effects were limited to 3-day-treatment. IL-1beta increased the level of MyoD, myogenin and MHC on the 3rd day of differentiation, without altering the content of the active form of myostatin, as well as it augmented the level of fibronectin, integrin beta1 and full length 100 kDa form of ADAM12. IL-1beta caused a decrease in IGFBP-4 and IGFBP-6 levels and a marked increase in IGFBP-5. The phosphorylation of PKB and ERK1/2 and the cellular content of p38 were elevated by IL-1beta. We conclude that the myogenic effect of IL-1beta was limited to the onset of myoblast fusion and was associated with: i) increase in the level of myogenic transcription factors i.e. MyoD and myogenin expression, ii) modification of extracellular matrix assembly and signaling, manifested by an increase in fibronectin, integrin-beta1 and ADAM12 content, iii) drop in IGFBP-4 and IGFBP-6, and an increase in IGFBP-5, that could alter the local IGF-1 bioavailability, and iv) increase in phosphorylation of PKB and ERK1/2, and the expression of p38 kinase, leading to activation of intracellular pathways essential for myogenic differentiation.

High glucose-mediated alterations of mechanisms important in myogenesis of mouse C2C12 myoblasts.

We have examined the progression and regulation of myogenesis, cellular levels of IGFBP-4, -5, -6, and several extracellular matrix (ECM) proteins (fibronectin, integrin α5, ß1 subunits and a disintegrin metalloprotease ADAM12) in murine C2C12 myoblasts during 3-day differentiation under high glucose alone or combined with high insulin, factors characteristic for type 1 and 2 diabetes. High ambient glucose inhibited myogenesis of C2C12 myoblasts, an effect manifested by a twofold decrease in myoblast fusion, a drop in intracellular MyoD, myogenin and MHC levels, and increased cellular content of active myostatin isoform. Reduction in myogenesis by high glucose is accompanied by increase in cellular levels of IGFBP-4 and -6 and decrease in IGFBP-5. High glucose could modify ECM components assembly, by the increase in fibronectin levels and the decrease in metalloprotease ADAM12, without the effect on integrin α5 and ß1 subunits. In contrast, high glucose and high insulin activate myoblast differentiation, manifested by an increase in fusion index and myogenin, as well as a drop in myostatin levels. The presence of high insulin prevented high-glucose-dependent changes in IGFBPs and ECM proteins. The data indicate the potential mechanisms of the influence of extracellular environment associated with maternal diabetes and insulin resistance on foetal myogenesis.

Growth factor and cytokine interactions in myogenesis. Part I. The effect of TNF-alpha and IFN-gamma on IGF-I-dependent differentiation in mouse C2C12 myogenic cells.

UNLABELLED: The aim of this study was to examine the potential interactions of IGF-I with TNF-alpha and IFN-gamma with regard to regulation of the myogenesis and proliferative potential of mouse C2C12 myoblasts. The stimulation of myogenesis by IGF-I (30 nmol/l) was manifested by an enhanced myoblast fusion and expression of myosin heavy chain (MHC) during the first 3 days of differentiation. IGF-I-dependent fusion and MHC expression was reduced by TNF-alpha and IFN-gamma. Both cytokines prevented the stimulatory effect of IGF-I on MyoD expression with minor modification of the myogenin level. Both TNF-alpha and IFN-gamma activated the expression of cyclin A in myoblasts restimulated to proliferation; however, when used in combination with IGF-I these cytokines prevented the rise in cyclin A induced by growth factor. IN CONCLUSION: i) TNF-alpha and IFN-gamma reduce IGF-I-dependent myogenesis which was manifested by the reduction of myoblast fusion and MHC cellular levels, ii) Molecular mechanisms of inhibitory action of TNF-alpha and IFN-gamma on IGF-I-mediated differentiation involve a decrease in MyoD whereas myogenin level plays a minor role, iii) TNF-alpha and IFN-gamma increase the proliferative potential of myoblasts; however, they reduced the mitogenic effect of IGF-I, manifested by a decrease of IGF-I-stimulated cyclin A expression in myoblasts reinduced to proliferation. Interactions among IGF-I and proinflammatory cytokines are therefore important to establish a number of myoblasts and the onset of myogenesis during muscle regeneration.

Growth factor and cytokine interactions in myogenesis. Part II. Expression of IGF binding proteins and protein kinases essential for myogenesis in mouse C2C12 myogenic cells exposed to TNF-alpha and IFN-gamma.

The aim of the study was to examine potential interactions among IGF-I and proinflammatory cytokines, TNF-alpha and IFN-gamma, in the regulation of local IGF-I bioavailability and cellular proteins mediating myogenic signals. We investigated levels of IGFBP-4, -5, -6, protein kinase Czeta (PKC zeta), p38 and extracellular signal-regulated kinase (ERK1/2) in differentiating mouse C2C12 myoblasts. IGF-I significantly stimulated expression of IGFBP-5. TNF-alpha and IFN-gamma attenuated the expression of IGFBP-4 and -6 under basal conditions and in the presence of IGF-I, and inhibited IGF-I-induced IGFBP-5 expression during 5-day myogenesis. TNF-alpha and IFN-gamma markedly attenuated p38 expression in the presence of IGF-I on the 5th day of myogenesis. When combined with IGF-I the cytokines exerted opposite effects on the PKC zeta level, i.e. TNF-alpha caused an increase, whereas IFN-gamma reduced the cellular content of this kinase. Exposition of C2C12 myoblasts to IGF-I or cytokines led to the stimulation of ERK1/2 phosphorylation; however, both TNF-alpha and IFN-gamma exerted an inhibitory effect on the activation of ERK1/2 in myoblasts cultured in the presence of IGF-I. We concluded as follows: i) TNF-alpha and IFN-gamma present in the extracellular environment of differentiating C2C12 myoblasts can alter the local bioavailability of IGF-I by inhibiting the expression of IGFBP-4, -5, and -6, ii) the decrease in p38 expression and ERK1/2 phosphorylation in C2C12 myoblasts exposed to cytokines can lead to disturbances in IGF-I-regulated myogenesis.

Transcriptional dysregulation of skeletal muscle protein metabolism in streptozotocin-diabetic mice.

The purpose of the study was to evaluate potential changes in expression of genes involved in protein metabolism and myogenic differentiation markers in skeletal muscle of streptozotocin-diabetic mice. Microarray analysis revealed alterations in the expression of 84 gene transcripts in gastrocnemius muscle of diabetic mice. Regarding protein metabolism a marked downregulation in gene transcripts for: general transcription factor IIA1 (-1.88, P=0.016309), TATA box binding protein (-2.17, P=0.037373), eukaryotic translation initiation factor 4E nuclear import factor 1 (-1.61, P=0.037373), eukaryotic translation elongation factor Ibeta2 (-1.95, P=0.010406), ubiquitin-like 5 (-1.67, P=0.024975) and ubiquitin conjugating enzyme 7 interacting protein 1 (-1.68, P=0.016309) was observed. STZ-diabetes caused a drop in the expression of myogenin, whereas myostatin level was significantly elevated. In conclusion, 1) STZ-diabetes attenuates expression of gene transcripts involved in the process of transcription and translation, which may affect skeletal muscle protein synthesis and lead to nitrogen imbalance, 2) impaired expression of gene transcripts involved in the regulation and activity of the ubiquitin-proteasome pathway may contribute to attenuation of mechanisms eliminating damaged proteins in STZ-diabetes, 3) changes in the expression of key myogenic factors, manifested by a decrease in myogenin level and enhancement of myostatin expression may be one of the mechanisms limiting skeletal muscle growth and regeneration associated with diabetes.

Exposure to TNF-alpha but not IL-1beta impairs insulin-dependent phosphorylation of protein kinase B and p70S6k in mouse C2C12 myogenic cells.

UNLABELLED: Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine considered to play an important role in muscle catabolism, but little is known about the mechanisms of its action. The aim of the present study was therefore to examine the effect of TNF-alpha pretreatment on glucose uptake and protein synthesis as well as the cellular content and phosphorylation of protein kinase B (PKB), p70S6k, Mitogen Activated Protein (MAP) kinase and p90rsk in mouse C2C12 myotubes stimulated with insulin. To determine whether interleukin (IL)-1beta might be involved in the catabolic action of TNF-alpha, the effects of IL-1beta were also tested. Experiments were performed on mouse C2C12 myoblasts subjected to differentiation in the presence of increasing concentrations of TNF-alpha (0.1-100 ng/ml) or IL-1 (5-50 ng/ml) for 5 or 6 days. Insulin (100 nmol/l) markedly stimulated glucose uptake in C2C12 myotubes (202.6% of control). This effect was profoundly attenuated by pretreatment with TNF-alpha at a concentration of 1 ng/ml (122.2% of control) and completely abolished by higher cytokine concentrations. Pretreatment of cells with TNF-alpha at a concentration of 1 ng/ml was also effective in diminishing the effect of insulin on protein synthesis, whereas higher cytokine concentrations prevented hormonal stimulation of protein synthesis in C2C12 myotubes. Pretreatment with TNF-alpha caused a significant decrease in PKB protein content. Insulin-mediated activation of protein kinase B was significantly diminished in cells differentiated in the presence of TNF-alpha. Treatment of C2C12 cells with insulin led to the gel mobility retardation of p70S6k indicating its phosphorylation and activation. In cells differentiated in the presence of TNF-alpha an approximately 2-fold decrease of insulin-mediated p70S6k phosphorylation was noted. Six-day differentiation of myogenic cells in the presence of TNF-alpha did not affect the protein content of p42MAPK, p44MAPK, p90rsk and phosphorylation of p42MAPK. Neither glucose uptake nor protein synthesis stimulated by insulin were affected significantly by pretreatment with IL-beta. Preincubation of myogenic cells with IL-1beta did not modify either the protein content of PKB and p70S6k or the insulin-stimulated phosphorylation of these kinases. IN CONCLUSION: i) high concentrations of TNF-alpha, but not IL-beta, present in the extracellular environment during myoblast differentiation prevent the stimulatory action of insulin on glucose uptake and protein synthesis; ii) insulin resistance induced by TNF-alpha in C2C12 myogenic cells could be associated with the decreased insulin-mediated phosphorylation of PKB and p70s6k, but not with the basal phosphorylation of p42MAPK.

Changes of insulin-mediated protein kinases phosphorylation and the expression of IGFBP-3 in skeletal muscle of streptozotocin-diabetic mice.

UNLABELLED: The purpose of the present study was to investigate potential changes in expression and activation of Ser/Thr protein kinases as well as in the level of insulin-like growth factor-binding proteins (IGFBPs) in skeletal muscle of streptozotocin (STZ)-diabetic mice. We have examined the basal and insulin-mediated phosphorylation of protein kinase B (PKB), protein kinase Czeta (PKCzeta), p70(S6k), mitogen-activated protein kinase (MAPK)/p90(rsk) pathway and the expression of IGFBP-3, -4, and -5 in mice selected for body weight gain (line C) and reduction (line L). Apart from IGFBP-3 level, which was higher in C line, the diabetes-associated changes in signaling components examined in present work were similar in both lines of mice. The expression of PKB in skeletal muscle was similar in control and diabetic mice. Insulin increased the Ser473 phosphorylation of PKB in both experimental groups however, in diabetic mice the insulin-dependent PKB phosphorylation was more evident in comparison to control group. Neither protein level nor insulin-stimulated p70(S6k) activation were modified by STZ-diabetes. Basal PKC phosphorylation was augmented in muscle of diabetic mice and it was not increased following insulin injection. No apparent differences in levels of p42(MAPK), p44(MAPK) and p90(rsk) protein in gastrocnemius muscles between control and STZ-treated mice were observed. Basal phosphorylation of p90(rsk) in diabetic mice was markedly elevated in comparison to the control. In muscle of C-line mice, insulin stimulated the p90(rsk) activity to the same extent in both experimental groups (+22% over appropriate basal value). Insulin-mediated stimulation of p90(rsk) in muscle of L-line mice amounted to +26% and +14%, for control and diabetic mice, respectively. Protein level of IGFBP-3 in muscle of diabetic C-line mice was augmented by approx. 28% when compared to the control, whereas the expression of IGFBP-4 and -5 was not modified by STZ-diabetes. IN CONCLUSION: diabetes-associated changes in the insulin signaling in skeletal muscle involve: 1) enhanced insulin-dependent phosphorylation of PKB; 2) increased basal phosphorylation of PKC and its resistance to stimulatory action of insulin; 3) increased basal phopshorylation of p90(rsk), and 4) augmented IGFBP-3 protein level, which can potentially contribute to disruption of anabolic signals in this tissue.

The impairment of IGF-I-stimulated protein synthesis and activation of protein kinase B, p70(S6k), MAP kinase, and p90(rsk) in mouse C2C12 myogenic cells exposed to high glucose and high insulin.

The aim of the present study was to examine the effect of high glucose alone and in combination with high insulin on IGF-I-stimulated protein synthesis and the activation of IGF-I signaling pathways in mouse C2C12 myogenic cells. Experiments were performed on mouse C2C12 myoblasts subjected to differentiation under normal glucose (5 mmol/I), high glucose alone (15 mmol/l), or in combination with high insulin (50 nmol/l). Six-day differentiation under high glucose alone or in combination with high insulin resulted in IGF-I resistance, which was manifested by the abolition of the stimulatory effect on protein synthesis. IGF-I caused the activation of protein kinase B (PKB) in control C2C12 myogenic cells. Pretreatment with high glucose did not affect PKB phosphorylation whereas in cells differentiated under high glucose and high insulin PKB activation by IGF-I was markedly decreased as compared with control (differentiation under normal glucose). Neither the p70(S6k) protein content nor the pattern of IGF-I-mediated kinase activation was affected by pretreatment with high glucose, however high glucose and high insulin in combination caused an impairment of the p70(S6k) phosphorylation, in relation to the control. An increase in p42(MAPK) phosphorylation occurred under normal glucose conditions after the stimulation with IGF-I. The MAP kinase was not phosphorylated in response to IGF-I in cells preincubated with high glucose alone or in combination with high insulin. The pattern of p90(rsk) activation by IGF-I was not modified by pretreatment with high glucose, however no activation of p90(rsk) was found in cells pretreated with high glucose and high insulin in combination. In conclusions: 1) high glucose abolishes the stimulatory action of IGF-I on protein synthesis and it does not affect the activation of PKB, p70(S6k), and p90(rsk) in mouse C2C12 myogenic cells, 2) high glucose with high insulin in combination also abolish the stimulatory effect of IGF-I, but this phenomenon is accompanied by attenuated PKB and p70(S6k) activation and the lack of activation of p90(rsk), 3) apart from PKB, p70(S6k) and p90(rsk), other kinases are probably involved in the regulation of IGF-I-mediated protein synthesis in myogenic cells.