TLR2 deficiency attenuates skeletal muscle atrophy in mice.

Oxidative stress and inflammation are associated with skeletal muscle atrophy. Because the activation of toll-like receptor (TLR) 2 induces oxidative stress and inflammation, TLR2 may be directly linked to skeletal muscle atrophy. This study examined the role of TLR2 in skeletal muscle atrophy in wild-type (WT) and TLR2 knockout (KO) mice. Immobilization for 2 weeks increased the expression of cytokine genes and the levels of carbonylated proteins and nitrotyrosine in the skeletal muscle, but these increases were lower in the TLR2 KO mice. Muscle weight loss and a reduction in treadmill running times induced by immobilization were also attenuated in TLR2 KO mice. Furthermore, immobilization increased the protein levels of forkhead box O 1/3, atrogin-1 and muscle ring finger 1 in the WT mice, which was attenuated in TLR2 KO mice. In addition, immobilization-associated increases in ubiquitinated protein levels were lower in the TLR2 KO mice. Immobilization increased the phosphorylation of Akt and p70S6K similarly in WT and KO mice. Furthermore, cardiotoxin injection into the skeletal muscle increased the protein levels of atrogin-1, interleukin-6, and nitrotyrosine and increased the levels of ubiquitinated proteins, although these levels were increased to a lesser extent in TLR2 KO mice. These results suggest that TLR2 is involved in skeletal muscle atrophy, and the inhibition of TLR2 offers a potential target for preventing skeletal muscle atrophy.

AMPK activator, AICAR, inhibits palmitate-induced apoptosis in osteoblast.

Osteoblast apoptosis reduces bone mineral density. Apoptosis can be induced in a variety of cells by palmitate, which is one of the most common saturated fatty acids in dietary fat. The AMPK activator, AICAR, has been shown to inhibit palmitate-induced apoptosis. However, the role of palmitate in osteoblast apoptosis is currently unknown. This study examined whether palmitate could induce apoptosis in osteoblasts, and if so, whether AICAR could alleviate palmitate-induced apoptosis. Palmitate reduced cell survival and induced apoptosis in a dose- and time-dependent manner in human fetal osteoblasts (hFOB) 1.19. While the long-chain acyl-CoA synthetase inhibitor, triacsin C, inhibited palmitate-induced apoptosis, anti-oxidants and ceramide synthesis inhibitors did not attenuate the apoptosis. AICAR prevented palmitate-induced apoptosis and the inhibition of AICAR-mediated increase in fatty acid oxidation by etomoxir did not affect the prevention of apoptosis by AICAR. Constitutively-active AMPK also inhibited palmitate-induced apoptosis. Treatment with an AMPK inhibitor (compound C) and a dominant-negative AMPK adenovirus suppressed the inhibitory effect of AICAR on apoptosis. Palmitate impaired the activation of ERK by fetal bovine serum, which was blocked by AICAR. Moreover, AICAR increased ERK activation, and ERK inhibitors, PD98059 and U0126, as well as a dominant-negative MEK1, abolished the inhibitory effect of AICAR on palmitate-induced apoptosis. AICAR also inhibited palmitate-induced apoptosis in osteoblastic differentiated cells from human bone marrow, which was accompanied by recovered ERK activity. These results suggest that palmitate induces apoptosis in osteoblasts through the impaired activation of ERK, and the activation of AMPK inhibits palmitate-induced apoptosis by activating ERK.

Treatment of thoracolumbar fracture.

The most common fractures of the spine are associated with the thoracolumbar junction. The goals of treatment of thoracolumbar fracture are leading to early mobilization and rehabilitation by restoring mechanical stability of fracture and inducing neurologic recovery, thereby enabling patients to return to the workplace. However, it is still debatable about the treatment methods. Neurologic injury should be identified by thorough physical examination for motor and sensory nerve system in order to determine the appropriate treatment. The mechanical stability of fracture also should be evaluated by plain radiographs and computed tomography. In some cases, magnetic resonance imaging is required to evaluate soft tissue injury involving neurologic structure or posterior ligament complex. Based on these physical examinations and imaging studies, fracture stability is evaluated and it is determined whether to use the conservative or operative treatment. The development of instruments have led to more interests on the operative treatment which saves mobile segments without fusion and on instrumentation through minimal invasive approach in recent years. It is still controversial for the use of these treatments because there have not been verified evidences yet. However, the morbidity of patients can be decreased and good clinical and radiologic outcomes can be achieved if the recent operative treatments are used carefully considering the fracture pattern and the injury severity.

Hemin, heme oxygenase-1 inducer, attenuates immobilization-induced skeletal muscle atrophy in mice.

AIMS: The present study examined the effect of the heme oxygenase (HO)-1 inducer hemin on skeletal muscle atrophy induced by single limb immobilization in mice. MAIN METHODS: Immobilization was conducted in the left hindlimb of C57BL/6 mice for 1 week and the right hindlimb was used as a control. Hemin (30 mg/kg) was administered intraperitoneally once a day during the immobilization period. Gastrocnemius muscles were used for analysis. Muscle weight was measured to quantify degree of atrophy, and exhaustion treadmill test was performed to assess muscle function. KEY FINDINGS: Immobilization increased HO-1 protein levels in skeletal muscle, which was further increased by hemin treatment. Immobilization induced weight loss and a functional reduction in skeletal muscle, which were attenuated by hemin treatment. Gene expression and protein levels of MuRF1 and atrogin-1 were increased by immobilization and hemin treatment attenuated the increment. The phosphorylation of mTOR and p70S6k was decreased by immobilization in skeletal muscle and hemin had no effect on mTOR and p70S6k phosphorylation. Gene expression of the antioxidants superoxide dismutase and glutathione peroxidase 1 in skeletal muscle was reduced by immobilization and hemin treatment recovered the reduction. Immobilization increased levels of carbonylated protein and nitrotyrosine in skeletal muscle, which was reversed by hemin treatment. Gene expression of inflammatory cytokines was increased by immobilization and was normalized as a result of hemin treatment. SIGNIFICANCE: These results suggest that hemin attenuates immobilization-induced skeletal muscle atrophy through the suppression of protein degradation via its anti-oxidant and anti-inflammatory properties.

Deficiency of inducible nitric oxide synthase attenuates immobilization-induced skeletal muscle atrophy in mice.

The present study examined the effects of inducible nitric oxide synthase (iNOS) deficiency on skeletal muscle atrophy in single leg-immobilized iNOS knockout (KO) and wild-type (WT) mice. The left leg was immobilized for 1 wk, and the right leg was used as the control. Muscle weight and contraction-stimulated glucose uptake were reduced by immobilization in WT mice, which was accompanied with increased iNOS expression in skeletal muscle. Deficiency of iNOS attenuated muscle weight loss and the reduction in contraction-stimulated glucose uptake by immobilization. Phosphorylation of Akt, mTOR, and p70S6K was reduced to a similar extent by immobilization in both WT and iNOS KO mice. Immobilization decreased FoxO1 phosphorylation and increased mRNA and protein levels of MuRF1 and atrogin-1 in WT mice, which were attenuated in iNOS KO mice. Aconitase and superoxide dismutase activities were reduced by immobilization in WT mice, and deficiency of iNOS normalized these enzyme activities. Increased nitrotyrosine and carbonylated protein levels by immobilization in WT mice were reversed in iNOS KO mice. Phosphorylation of ERK and p38 was increased by immobilization in WT mice, which was reduced in iNOS KO mice. Immobilization-induced muscle atrophy was also attenuated by an iNOS-specific inhibitor N(6)-(1-iminoethyl)-l-lysine, and this finding was accompanied by increased FoxO1 phosphorylation and reduced MuRF1 and atrogin-1 levels. These results suggest that deficiency of iNOS attenuates immobilization-induced skeletal muscle atrophy through reduced oxidative stress, and iNOS-induced oxidative stress may be required for immobilization-induced skeletal muscle atrophy.

Inhibition of Inducible Nitric Oxide Synthase Attenuates Monosodium Urate-induced Inflammation in Mice.

The present study elucidated the effect of the selective inducible nitric oxide synthase (iNOS) inhibitor N(6)-(1-iminoethyl)-L-lysine (L-NIL) on monosodium urate (MSU) crystal-induced inflammation and edema in mice feet. L-NIL (5 or 10 mg/kg/day) was administered intraperitoneally 4 h before injection of MSU (4 mg) into the soles of mice hindlimb feet. Twenty-four hours after MSU injection, foot thickness was increased by 160% and L-NIL pretreatment reduced food pad swelling in a dose dependent manner. Pretreatment of 10 mg/kg/day L-NIL significantly suppressed the foot pad swelling by MSU. Plasma level of nitric oxide (NO) metabolites and gene expression and protein level of iNOS in feet were increased by MSU, which was suppressed by L-NIL pretreatment. Similar pattern of change was observed in nitrotyrosine level. MSU increased the gene expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß and L-NIL pretreatment suppressed MSU-induced cytokines expression. The mRNA levels of superoxide dismutase and glutathione peroxidase1 were increased by MSU and L-NIL pretreatment normalized the gene expression. Phosphorylation of extracellular signal-regulated kinase 1/2 and p38 was increased by MSU, which was suppressed by L-NIL pretreatment. The mRNA levels of iNOS, TNF-α, and IL-1ß were increased by MSU in human dermal fibroblasts, C2C12 myoblasts, and human fetal osteoblasts in vitro, which was attenuated by L-NIL in a dose dependent manner. This study shows that L-NIL inhibits MSU-induced inflammation and edema in mice feet suggesting that iNOS might be involved in MSU-induced inflammation.

Deficiency of iNOS Does Not Prevent Isoproterenol-induced Cardiac Hypertrophy in Mice.

We investigated whether deficiency of inducible nitric oxide synthase (iNOS) could prevent isoproterenol-induced cardiac hypertrophy in iNOS knockout (KO) mice. Isoproterenol was continuously infused subcutaneously (15 mg/kg/day) using an osmotic minipump. Isoproterenol reduced body weight and fat mass in both iNOS KO and wild-type mice compared with saline-infused wild-type mice. Isoproterenol increased the heart weight in both iNOS KO and wild-type mice but there was no difference between iNOS KO and wild-type mice. Posterior wall thickness of left ventricle showed the same tendency with heart weight. Protein level of iNOS in the left ventricle was increased in isoproterenol-infused wild-type mice. The gene expression of interleukin-6 (IL-6) and transforming growth factor-beta (TGF-beta) in isoproterenol-infused wild-type was measured at 2, 4, 24, and 48-hour and isoproterenol increased both IL-6 (2, 4, 24, and 48-hour) and TGF-beta (4 and 24-hour). Isoproterenol infusion for 7 days increased the mRNA level of IL-6 and TGF-beta in iNOS KO mice, whereas the gene expression in wild-type mice was not increased. Phosphorylated form of extracellular signal-regulated kinases (pERK) was also increased by isoproterenol at 2 and 4-hour but was not increased at 7 days after infusion in wild-type mice. However, the increased pERK level in iNOS KO mice was maintained even at 7 days after isoproterenol infusion. These results suggest that deficiency of iNOS does not prevent isoproterenol-induced cardiac hypertrophy and may have potentially harmful effects on cardiac hypertrophy.

Inhibition of lipid infusion-induced skeletal muscle insulin resistance by cotreatment with tempol and glutathione in mice.

In the present study, we examined whether lipid infusion-induced insulin resistance in skeletal muscle could be reversed by the antioxidants tempol, glutathione (GSH), or tempol with GSH in male C57BL/6J mice via hyperinsulinemic-euglycemic clamp. Lipid infusion increased the mRNA level of mitochondrial type superoxide dismutase (Mn-SOD), glutathione peroxidase 1, tumor necrosis factor-alpha, and interleukin-6. Lipid infusion decreased GSH and GSH/glutathione disulfide (GSSG) ratio and increased the activities of JNK and p38 in skeletal muscle. Lipid infusion induced insulin resistance in whole body and skeletal muscle. Treatment with the SOD mimetic tempol did not prevent oxidative stress, the inflammatory response, and insulin resistance induced by lipid infusion. Tempol alone increased oxidative stress and aggravated the lipid-induced inflammatory response. GSH at 100 and 200 micromol. kg(-1) . h(-1) did not prevent insulin resistance and the inflammatory response by lipid infusion. On the contrary, GSH at 100 micromol. kg(-1) . h(-1) with tempol prevented insulin resistance in the whole body and skeletal muscle, and it completely reversed oxidative stress and the inflammatory response. These results suggest that lipid infusion-induced insulin resistance in skeletal muscle is produced by oxidative stress and cotreatment with tempol and GSH inhibits lipid-induced insulin resistance.