Arsenic induces apoptosis in myoblasts through a reactive oxygen species-induced endoplasmic reticulum stress and mitochondrial dysfunction pathway.

A pool of myoblasts available for myogenesis is important for skeletal muscle size. The decreased number of skeletal muscle fibers could be due to the decreased myoblast proliferation or cytotoxicity. Identification of toxicants that regulate myoblast apoptosis is important in skeletal muscle development or regeneration. Here, we investigate the cytotoxic effect and its possible mechanisms of arsenic trioxide (As(2)O(3)) on myoblasts. C2C12 myoblasts underwent apoptosis in response to As(2)O(3) (1-10 µM), accompanied by increased Bax/Bcl-2 ratio, decreased mitochondria membrane potential, increased cytochrome c release, increased caspase-3/-9 activity, and increased poly (ADP-ribose) polymerase (PARP) cleavage. Moreover, As(2)O(3) triggered the endoplasmic reticulum (ER) stress indentified through several key molecules of the unfolded protein response, including glucose-regulated protein (GRP)-78, GRP-94, PERK, eIF2α, ATF6, and caspase-12. Pretreatment with antioxidant N-acetylcysteine (NAC, 0.5 mM) dramatically suppressed the increases in reactive oxygen species (ROS), lipid peroxidation, ER stress, caspase cascade activity, and apoptosis in As(2)O(3) (10 µM)-treated myoblasts. Furthermore, As(2)O(3) (10 µM) effectively decreased the phosphorylation of Akt, which could be reversed by NAC. Over-expression of constitutive activation of Akt (c.a. Akt) also significantly attenuated As(2)O(3)-induced myoblast apoptosis. Taken together, these results suggest that As(2)O(3) may exert its cytotoxicity on myoblasts by inducing apoptosis through a ROS-induced mitochondrial dysfunction, ER stress, and Akt inactivation signaling pathway.

Islet-like clusters derived from skeletal muscle-derived stem/progenitor cells for autologous transplantation to control type 1 diabetes in mice.

A population of muscle-derived stem/progenitor cells (MDSPCs) contained in skeletal muscle is responsible for muscle regeneration. MDSPCs from mouse muscle have been shown to be capable of differentiating into pancreatic islet-like cells. However, the potency of MDSPCs to differentiate into functional islet-like cluster remains to be confirmed. The therapeutic potential of autologous MDSPCs transplantation on type 1 diabetes still remains unclear. Here, we investigated a four-stage method to induce the differentiation of MDSPCs into insulin-producing clusters in vitro, and tested the autologous transplantation to control type 1 diabetes in mice. MDSPCs isolated from the skeletal muscles of mice possessed the ability to form islet-like clusters through several stages of differentiation. The expressions of pancreatic progenitor-related genes, insulin, and islet-related genes were significantly upregulated in islet-like clusters determined by the quantitative reverse transcription polymerase chain reaction. The autologous islet-like clusters transplantation effectively improved hyperglycaemia and glucose intolerance and increased the survival rate in streptozotocin-induced diabetic mice without the use of immunosuppressants. Taken together, these results provide evidence that MDSPCs from murine muscle tissues are capable of differentiating into insulin-producing clusters, which possess insulin-producing ability in vitro and in vivo, and have the potential for autologous transplantation to control type 1 diabetes.

Low-Concentration Arsenic Trioxide Inhibits Skeletal Myoblast Cell Proliferation via a Reactive Oxygen Species-Independent Pathway.

Myoblast proliferation and differentiation are essential for skeletal muscle regeneration. Myoblast proliferation is a critical step in the growth and maintenance of skeletal muscle. The precise action of inorganic arsenic on myoblast growth has not been investigated. Here, we investigated the in vitro effect of inorganic arsenic trioxide (As2O3) on the growth of C2C12 myoblasts. As2O3 decreased myoblast growth at submicromolar concentrations (0.25-1 µM) after 72 h of treatment. Submicromolar concentrations of As2O3 did not induce the myoblast apoptosis. Low-concentration As2O3 (0.5 and 1 µM) significantly suppressed the myoblast cell proliferative activity, which was accompanied by a small proportion of bromodeoxyuridine (BrdU) incorporation and decreased proliferating cell nuclear antigen (PCNA) protein expression. As2O3 (0.5 and 1 µM) increased the intracellular arsenic content but did not affect the reactive oxygen species (ROS) levels in the myoblasts. Cell cycle analysis indicated that low-concentrations of As2O3 inhibited cell proliferation via cell cycle arrest in the G1 and G2/M phases. As2O3 also decreased the protein expressions of cyclin D1, cyclin E, cyclin B1, cyclin-dependent kinase (CDK) 2, and CDK4, but did not affect the protein expressions of p21 and p27. Furthermore, As2O3 inhibited the phosphorylation of Akt. Insulin-like growth factor-1 significantly reversed the inhibitory effect of As2O3 on Akt phosphorylation and cell proliferation in the myoblasts. These results suggest that submicromolar concentrations of As2O3 alter cell cycle progression and reduce myoblast proliferation, at least in part, through a ROS-independent Akt inhibition pathway.

Low-dose benzo(a)pyrene and its epoxide metabolite inhibit myogenic differentiation in human skeletal muscle-derived progenitor cells.

The risk of low birth weights is elevated in prenatal exposure to polycyclic aromatic hydrocarbons (PAHs), which are ubiquitous environmental pollutants generated from combustion of organic compounds, including cigarette smoke. We hypothesized that benzo(a)pyrene (BaP), a member of PAHs existing in cigarette smoke, may affect the myogenesis to cause low birth weights. We investigated the effects of BaP and its main metabolite, benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), on the myogenic differentiation of human skeletal muscle-derived progenitor cells (HSMPCs). HSMPCs were isolated by a modified preplate technique and cultured in myogenic differentiation media with or without BaP and BPDE (0.25 and 0.5 µM) for 4 days. The multinucleated myotube formation was morphologically analyzed by hematoxylin and eosin staining. The expressions of myogenic differentiation markers and related signaling proteins were determined by Western blotting. Both BaP and BPDE at the submicromolar concentrations (0.25 and 0.5 µM) dose-dependently repressed HSMPCs myogenic differentiation without obvious cell toxicity. Both BaP and BPDE inhibited the muscle-specific protein expressions (myogenin and myosin heavy chain) and phosphorylation of Akt (a known modulator in myogenesis), which could be significantly reversed by the inhibitors for aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and nuclear factor (NF)-κB. BaP- and BPDE-activated NF-κB-p65 protein phosphorylation could also be attenuated by both AhR and ER inhibitors. The inhibitory effects of BaP and BPDE on myogenesis were reversed after withdrawing BaP exposure, but not after BPDE withdrawal. These results suggest that both BaP and BPDE are capable of inhibiting myogenesis via an AhR- or/and ER-regulated NF-κB/Akt signaling pathway.

Antidiabetic effects of pterosin A, a small-molecular-weight natural product, on diabetic mouse models.

The therapeutic effect of pterosin A, a small-molecular-weight natural product, on diabetes was investigated. Pterosin A, administered orally for 4 weeks, effectively improved hyperglycemia and glucose intolerance in streptozotocin, high-fat diet-fed, and db/db diabetic mice. There were no adverse effects in normal or diabetic mice treated with pterosin A for 4 weeks. Pterosin A significantly reversed the increased serum insulin and insulin resistance (IR) in dexamethasone-IR mice and in db/db mice. Pterosin A significantly reversed the reduced muscle GLUT-4 translocation and the increased liver phosphoenolpyruvate carboxyl kinase (PEPCK) expression in diabetic mice. Pterosin A also significantly reversed the decreased phosphorylations of AMP-activated protein kinase (AMPK) and Akt in muscles of diabetic mice. The decreased AMPK phosphorylation and increased p38 phosphorylation in livers of db/db mice were effectively reversed by pterosin A. Pterosin A enhanced glucose uptake and AMPK phosphorylation in cultured human muscle cells. In cultured liver cells, pterosin A inhibited inducer-enhanced PEPCK expression, triggered the phosphorylations of AMPK, acetyl CoA carboxylase, and glycogen synthase kinase-3, decreased glycogen synthase phosphorylation, and increased the intracellular glycogen level. These findings indicate that pterosin A may be a potential therapeutic option for diabetes.

Caffeine enhances osteoclast differentiation from bone marrow hematopoietic cells and reduces bone mineral density in growing rats.

Caffeine-containing beverage consumption has been associated with low bone mass and increased fracture risk in some, but not most, observational studies. The effects of caffeine on bone metabolism are still controversial. We investigated the effects of caffeine on the differentiation of bone progenitor cells and bone mineral density (BMD) by in vitro and in vivo experiments. Low-concentration caffeine (0.005-0.1 mM) did not affect the bone marrow cell viability and alkaline phosphatase activity during osteoblast differentiation from bone marrow stromal cells, but it effectively enhanced the osteoclastogenesis from bone marrow hematopoietic cells and the bone resorption activity by pit formation assay. Moreover, caffeine effectively enhanced the receptor activator of NF-κB ligand (RANKL), but reduced the osteoprotegerin protein expressions in osteoblast MC3T3-E1 cells. Caffeine could also increase the cyclooxygenase-2 (COX-2) protein expression and prostaglandin (PG)E(2) production in cultured neonatal mouse calvariae. In animal study, BMD in lumbar vertebra, femur, or tibia was significantly lowered in growing rats supplemented with 0.2% caffeine in diets for 20 weeks compared with the control group. The calcium contents in tibia and femur of caffeine-treated rats were also lower than that in the control group. The osteoclastogenesis of bone marrow cells isolated from caffeine-treated rats was markedly enhanced as compared with the control group. Taken together, these results suggest that caffeine may reduce BMD in growing rats through the enhancement in osteoclastogenesis. Caffeine may possess the ability to enhance a COX-2/PGE(2) -regulated RANKL-mediated osteoclastogenesis.

Arsenic inhibits myogenic differentiation and muscle regeneration.

BACKGROUND: The incidence of low birth weights is increased in offspring of women who are exposed to high concentrations of arsenic in drinking water compared with other women. We hypothesized that effects of arsenic on birth weight may be related to effects on myogenic differentiation. OBJECTIVE: We investigated the effects of arsenic trioxide (As2O3) on the myogenic differentiation of myoblasts in vitro and muscle regeneration in vivo. METHODS: C2C12 myoblasts and primary mouse and human myoblasts were cultured in differentiation media with or without As2O3 (0.1-0.5 microM) for 4 days. Myogenic differentiation was assessed by myogenin and myosin heavy chain expression and multinucleated myotube formation in vitro; skeletal muscle regeneration was tested using an in vivo mouse model with experimental glycerol myopathy. RESULTS: A submicromolar concentration of As2O3 dose-dependently inhibited myogenic differentiation without apparent effects on cell viability. As2O3 significantly and dose-dependently decreased phosphorylation of Akt and p70s6k proteins during myogenic differentiation. As2O3-induced inhibition in myotube formation and muscle-specific protein expression was reversed by transfection with the constitutively active form of Akt. Sections of soleus muscles stained with hematoxylin and eosin showed typical changes of injury and regeneration after local glycerol injection in mice. Regeneration of glycerol-injured soleus muscles, myogenin expression, and Akt phosphorylation were suppressed in muscles isolated from As2O3-treated mice compared with untreated mice. CONCLUSION: Our results suggest that As2O3 inhibits myogenic differentiation by inhibiting Akt-regulated signaling.