Proposed mechanism of LPS inhibition of myogenesis in differentiating myoblasts.

<p>Proposed mechanism of LPS inhibition of myogenesis in differentiating myoblasts.</p

Effect of a TLR2-neutralizing antibody on NF-κB activity in LPS-treated myoblasts.

<p>(A) C2C12 myoblasts were cultured for 48 h in DM alone, LPS (1 μg/mL), or LPS (1 μg/mL) plus TLR2-neutralizing antibody (10 μg/mL). Data are the mean ± SEM of 7 independent experiments performed in duplicate. (B) C2C12 myoblasts were cultured for 48 h in DM with LPS (1 μg/mL), LPS (1 μg/mL) plus TAK-242 (1 μM), or LPS (1 μg/mL) plus TLR2-neutralizing antibody (10 μg/mL). NF-κB activity was measured by ELISA. Data are the mean ± SEM of 7–10 independent experiments performed in duplicate. **p < 0.01, *p < 0.05 by one-way ANOVA followed by Tukey’s honest significant difference test.</p

Effect of TAK-242 on LPS-induced perturbation of muscle regulatory factors and NF-κB activity.

<p>(A) TAK-242 partially reverses the LPS-induced downregulation of myogenin, but not of MyoD. C2C12 myoblasts were cultured for 48 h in DM alone, with LPS (1 μg/mL or 0.1 μg/mL), or with a combination of LPS (1 μg/mL) and TAK-242 (1 μM). A representative western blot probed with antibodies to myogenin, MyoD, or β-tubulin (internal standard) is shown. (B and C) Quantification of the data presented in (A). Data are the mean ± SEM of 5–7 independent experiments. (D) TAK-242 partially reverses the LPS-induced upregulation of myostatin expression. C2C12 myoblasts were cultured for 144 h as described in (A). A representative western blot probed with antibodies to myostatin or β-tubulin (internal standard) is shown. (E) Quantification of the data presented in (D). Data are the mean ± SEM of 14–16 independent experiments. (F) TAK-242 inhibits NF-κB activity in LPS-treated differentiating myoblasts. Cells were treated as described in (A), and NF-κB activity was analyzed using a TransAM ELISA kit. Data are the mean ± SEM of 5 independent experiments performed in duplicate. ***p < 0.001, *p < 0.05, #p < 0.05 by one-way ANOVA followed by Tukey’s honest significant difference test.</p

Lipopolysaccharide inhibits myogenic differentiation of C2C12 myoblasts through the Toll-like receptor 4-nuclear factor-κB signaling pathway and myoblast-derived tumor necrosis factor-α

<div><p>Background</p><p>Circulating lipopolysaccharide (LPS) concentrations are often elevated in patients with sepsis or with various endogenous diseases that are associated with metabolic endotoxemia. Involuntary loss of skeletal muscle, termed muscle wasting, is commonly observed in these conditions, suggesting that circulating LPS might play an essential role in its development. Although impairment of muscle regeneration is an important determinant of skeletal muscle wasting, it is unclear whether LPS affects this process and, if so, by what mechanism. Here, we used the C2C12 myoblast cell line to investigate the effects of LPS on myogenesis.</p><p>Methods</p><p>C2C12 myoblasts were grown to 80% confluence and induced to differentiate in the absence or presence of LPS (0.1 or 1 μg/mL); TAK-242 (1 μM), a specific inhibitor of Toll-like receptor 4 (TLR4) signaling; and a tumor necrosis factor (TNF)-α neutralizing antibody (5 μg/mL). Expression of a skeletal muscle differentiation marker (myosin heavy chain II), two essential myogenic regulatory factors (myogenin and MyoD), and a muscle negative regulatory factor (myostatin) was analyzed by western blotting. Nuclear factor-κB (NF-κB) DNA-binding activity was measured using an enzyme-linked immunosorbent assay.</p><p>Results</p><p>LPS dose-dependently and significantly decreased the formation of multinucleated myotubes and the expression of myosin heavy chain II, myogenin, and MyoD, and increased NF-κB DNA-binding activity and myostatin expression. The inhibitory effect of LPS on myogenic differentiation was reversible, suggesting that it was not caused by nonspecific toxicity. Both TAK-242 and anti-TNF-α reduced the LPS-induced increase in NF-κB DNA-binding activity, downregulation of myogenic regulatory factors, and upregulation of myostatin, thereby partially rescuing the impairment of myogenesis.</p><p>Conclusions</p><p>Our data suggest that LPS inhibits myogenic differentiation via a TLR4–NF-κB-dependent pathway and an autocrine/paracrine TNF-α-induced pathway. These pathways may be involved in the development of muscle wasting caused by sepsis or metabolic endotoxemia.</p></div

Effect of LPS on C2C12 myogenesis.

<p>(A) LPS inhibits the formation of myotubes. Myoblasts were cultured for 144 h in DM alone (control), DM plus LPS 0.1 μg/mL, or DM plus LPS 1 μg/mL, then fixed and subjected to May–Grünwald–Giemsa staining. Representative images are shown. Scale bar = 100 μm. Arrows indicate differentiated myotubes. (B) LPS decreases the myogenic index. Cells were treated as described in (A). Data are the mean ± SEM of 10–12 independent experiments, each examining 5 randomly selected fields (total 50–60 fields per treatment group). (C and D) LPS induces myotube atrophy. Cells were treated as described in (A), and the distribution of myotube widths (C) and mean myotube widths (D) were calculated. Data are the mean ± SEM of 10–12 independent experiments, each examining 5 randomly selected fields (total 175–296 myotubes from 50–60 fields per treatment group). (E) Representative western blot probed with antibodies to MyHC II or β-tubulin (internal standard). Cells were treated as described in (A). (F) Quantification of the data presented in (E). Data are the mean ± SEM of 13–14 independent experiments. ***p < 0.001, **p < 0.01, ###p < 0.001, ##p < 0.01 by one-way ANOVA followed by Tukey’s honest significant difference test.</p

Effect of TAK-242 on LPS-induced inhibition of myogenesis.

<p>(A) May–Grünwald–Giemsa staining of C2C12 cells cultured for 144 h in DM alone, DM plus LPS (1 μg/mL), or DM plus LPS (1 μg/mL) and TAK-242 (1 μM). Representative images are shown. Scale bar = 100 μm. Arrows indicate differentiated myotubes. (B) TAK-242 partially reversed the LPS-induced decrease in myogenic index. Cells were treated as described in (A). Data are the mean ± SEM of 10 independent experiments per treatment group, each examining 5 randomly selected fields (total 50 fields). (C and D) TAK-242 partially ameliorated the LPS-induced myotube atrophy. Cells were treated as described in (A), and the distribution of myotube widths (C) and mean myotube widths (D) were calculated for each treatment group. Data are the mean ± SEM of 10–12 independent experiments, each examining 5 randomly selected fields (total 194–296 myotubes from 50–60 fields per treatment group). (E) Representative western blot probed with antibodies to MyHC II or β-tubulin (internal standard). Cells were treated as described in (A). (F) Quantification of the data presented in (E). Data are the mean ± SEM of 12–13 independent experiments. ***p < 0.001, ###p < 0.001, #p < 0.05 by one-way ANOVA followed by Tukey’s honest significant difference test.</p

TLR4 mRNA expression in differentiating C2C12 myoblasts.

<p>(A–C) TLR4 mRNA levels in C2C12 myoblasts cultured in DM for 2, 48, 96, or 144 h with or without LPS. (A) Agarose gel of TLR4 and GAPDH (internal standard) mRNA levels during differentiation. (B) Real-time RT-PCR quantification of TLR4 mRNA levels. Data were normalized to CK2 mRNA levels and the ratio in cells cultured in DM for 2 h was set at 1.0. Data are the mean ± SEM of 3–4 independent experiments, each performed in duplicate. (C) Quantification of TLR4 mRNA by real-time RT-PCR. C2C12 cells were cultured with the indicated concentrations of LPS for 144 h. Data were normalized to CK2 mRNA levels and the ratio in untreated cells was set at 1.0. Data are the mean ± SEM of 3 independent experiments performed in duplicate. In (B) and (C), P > 0.05 for all comparisons.</p

Effect of a TNF-α-neutralizing antibody on LPS-induced perturbation of muscle differentiation.

<p>(A) C2C12 myoblasts were cultured for 48 h in DM alone, LPS (1 μg/mL), or LPS (1 μg/mL) plus TNF-α-neutralizing antibody (5 μg/mL). A representative western blot probed with antibodies to myogenin, MyoD, or β-tubulin (internal standard) is shown. (B and C) Quantification of the data presented in (A). Data are the mean ± SEM of 7–15 independent experiments. (D) C2C12 myoblasts were cultured for 144 h as described in (A). A representative western blot probed with antibodies to MyHC II, myostatin, or β-tubulin (internal standard) is shown. (E and F) Quantification of the data presented in (D). Data are the mean ± SEM of 5–21 independent experiments. (G) Cells were treated as described in (A), and NF-κB activity was analyzed by ELISA. Data are the mean ± SEM of 7 independent experiments performed in duplicate. ***p < 0.001, **p < 0.01, *p < 0.05, ###p < 0.001, ##p < 0.01, #p < 0.05 by one-way ANOVA followed by Tukey’s honest significant difference test.</p

Effect of LPS on muscle regulatory factors and NF-κB activity in differentiating myoblasts.

<p>(A) LPS downregulates expression of the positive myogenic regulatory factors myogenin and MyoD in a dose-dependent manner. C2C12 myoblasts were incubated in DM with or without LPS (0.1 or 1 μg/mL) for 48 h. A representative western blot probed with antibodies to myogenin, MyoD, or β-tubulin (internal standard) is shown. (B and C) Quantification of the data presented in (A). Data are the mean ± SEM of 6–7 independent experiments. (D) LPS upregulates expression of the negative myogenic regulatory factor myostatin in a dose-dependent manner. C2C12 myoblasts were treated for 144 h as described in (A). A representative western blot probed with antibodies to myostatin or β-tubulin (internal standard) is shown. (E) Quantification of the data presented in (D). Data are the mean ± SEM of 14 independent experiments. (F) LPS increases NF-κB DNA-binding activity in a dose-dependent manner. C2C12 myoblasts were incubated in DM with or without LPS (0.1 or 1 μg/mL) for 48 h, and NF-κB activity was analyzed using a TransAM ELISA kit. Data are the mean ± SEM of 5 independent experiments performed in duplicate. ***p < 0.001, **p < 0.01, *p < 0.05, #p < 0.05 by one-way ANOVA followed by Tukey’s honest significant difference test.</p

Molecular Mass and Molecular-Mass Distribution of TEMPO-Oxidized Celluloses and TEMPO-Oxidized Cellulose Nanofibrils

Native wood cellulose was oxidized by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation, and the fibrous TEMPO-oxidized celluloses (TOCs) thus obtained were disintegrated in water to prepare TOC nanofibrils (TOCNs). The carboxyl groups of TOCs and TOCNs were methyl-esterified, and the methylated samples were dissolved in 8% LiCl/<i>N</i>,<i>N</i>-dimethylacetamide for size-exclusion chromatography/multiangle laser-light scattering (SEC-MALLS) analysis to obtain their molecular-mass (MM) values and MM distributions (MMDs). The results showed that remarkable depolymerization occurred in TOCs and TOCNs and depended on the oxidation and sonication conditions. Because single peaks without bimodal patterns were observed in the MMDs for all of the TOC and TOCN samples, depolymerization may have randomly occurred on whole cellulose molecules and oxidized cellulose molecules in the microfibrils during these treatments. Compared with the MM values obtained by SEC-MALLS, the intrinsic viscosities of TOCs dissolved in 0.5 M copper ethylenediamine solution provided lower MM values owing to depolymerization during the dissolution and postreduction processes