Antidiabetic effect of Chrysophyllum albidum is mediated by enzyme inhibition and enhancement of glucose uptake via 3T3-L1 adipocytes and C2C12 myotubes

Objective: To investigate the in vivo and in vitro antidiabetic potential of Chrysophyllum albidum. Methods: The effects of oral treatment with hydro-ethanolic extract (125, 250 and 500 mg/kg) of the stem bark of Chrysophyllum albidum and glibenclamide for 21 d on glucose level, serum enzyme markers for liver function, lipid profile, total protein, serum urea, serum creatinine, and body weight were evaluated in experimental diabetic rats administered with 45 mg/kg of streptozotocin. In vitro assays including glucose uptake in C2C12 cells and 3T3-L1 adipose tissues, α-glucosidase and α-amylase inhibition were employed to evaluate the possible mechanism of hypoglycemic action of the extract. DPPH and nitric oxide radical antioxidant activity of the extract was also measured. Results: The increased levels of blood glucose, triglycerides, low-density lipoprotein, total cholesterol, serum aspartate, and alanine transaminases, creatinine, and urea in the diabetic animals were reduced significantly (P<0.01) after treatment with Chrysophyllum albidum extract. The decreased total protein and high-density lipoprotein concentrations were normalized after treatment. In addition, the extract significantly (P<0.01) increased the transport of glucose in 3T3-L1 cells and C2C12 myotubes and exhibited considerable potential to inhibit α-amylase and α-glucosidase. It also demonstrated potent antioxidant action by scavenging considerably DPPH and nitric oxide radicals. Conclusions: Chrysophyllum albidum stem bark extract exhibits considerable antidiabetic effect by stimulating glucose uptake and utilization in C2C12 myotubes and 3T3-L1 adipocytes as well as inhibiting the activities of α-amylase and α-glucosidase.

Antidiabetic effect of Chrysophyllum albidum is mediated by enzyme inhibition and enhancement of glucose uptake via 3T3-L1 adipocytes and C2C12 myotubes

Objective: To investigate the in vivo and in vitro antidiabetic potential of Chrysophyllum albidum. Methods: The effects of oral treatment with hydro-ethanolic extract (125, 250 and 500 mg/kg) of the stem bark of Chrysophyllum albidum and glibenclamide for 21 d on glucose level, serum enzyme markers for liver function, lipid profile, total protein, serum urea, serum creatinine, and body weight were evaluated in experimental diabetic rats administered with 45 mg/kg of streptozotocin. In vitro assays including glucose uptake in C2C12 cells and 3T3-L1 adipose tissues, α-glucosidase and α-amylase inhibition were employed to evaluate the possible mechanism of hypoglycemic action of the extract. DPPH and nitric oxide radical antioxidant activity of the extract was also measured. Results: The increased levels of blood glucose, triglycerides, low-density lipoprotein, total cholesterol, serum aspartate, and alanine transaminases, creatinine, and urea in the diabetic animals were reduced significantly (P<0.01) after treatment with Chrysophyllum albidum extract. The decreased total protein and high-density lipoprotein concentrations were normalized after treatment. In addition, the extract significantly (P<0.01) increased the transport of glucose in 3T3-L1 cells and C2C12 myotubes and exhibited considerable potential to inhibit α-amylase and α-glucosidase. It also demonstrated potent antioxidant action by scavenging considerably DPPH and nitric oxide radicals. Conclusions: Chrysophyllum albidum stem bark extract exhibits considerable antidiabetic effect by stimulating glucose uptake and utilization in C2C12 myotubes and 3T3-L1 adipocytes as well as inhibiting the activities of α-amylase and α-glucosidase.

Modes of muscle satellite cells fusing myotubes of spontaneous contraction in vitro / 中华地方病学杂志

Objective To investigate the cell-fusion modes and contractive characteristics of myotubes formed by muscle satellite cells in vitro. Methods The muscle satellite cells in quadriceps femoris of six 2-day-old SD rats were isolated, cultured and identified by immunofluorescence; the cell-fusion modes and contractive characteristics of myotubes were observed and recorded under microscope. Results The positve cells expressing marker Pax7 were obtained by immunofluorescence, and the purity was 90.3%. Myotubes were fused by several cells, cell and myotube or myotube and myotube. There were myotubes of spontaneous contraction on the conditions of no acetylcholine, no electric stimulation or no contiguous cells. Conclusion The myotubes of spontaneous contraction are fused by muscle satellite cells through three cell-fusion modes in vitro.

Methanolic extract of Momordica cymbalaria enhances glucose uptake in L6 myotubes in vitro by up-regulating PPAR-γ and GLUT-4 / 中国天然药物

The present study was undertaken to evaluate the influence of the methanolic fruit extract of Momordica cymbalaria (MFMC) on PPARγ (Peroxisome Proliferator Activated Receptor gamma) and GLUT-4 (Glucose transporter-4) with respect to glucose transport. Various concentrations of MFMC ranging from 62.5 to 500 μg·mL(-1) were evaluated for glucose uptake activity in vitro using L6 myotubes, rosiglitazone was used as a reference standard. The MFMC showed significant and dose-dependent increase in glucose uptake at the tested concentrations, further, the glucose uptake activity of MFMC (500 μg·mL(-1)) was comparable with rosigilitazone. Furthermore, MFMC has shown up-regulation of GLUT-4 and PPARγ gene expressions in L6 myotubes. In addition, the MFMC when incubated along with cycloheximide (CHX), which is a protein synthesis inhibitor, has shown complete blockade of glucose uptake. This indicates that new protein synthesis is required for increased GLUT-4 translocation. In conclusion, these findings suggest that MFMC is enhancing the glucose uptake significantly and dose dependently through the enhanced expression of PPARγ and GLUT-4 in vitro.

Mechanism of inhibition effect of HGF on the expression of CTGF in myotubes induced by TGF-β1 / 实用医学杂志

Objective To study the influence and mechanism of hepatocyte growth factor (HGF) on myotube phenotype by myotube transdifferentiation induced by transforming growth factor-β1 (TGF-β1). Methods C2C12 cells were cultured in differentiation medium to induce myotubes formation. The cells were randomly devided into 3 groups. The control group without growth factor interruption. The induction group was supplemented with TGF-β1 (5 ng/mL) while the inhibition group was supplenmented with both TGF-β1 (5 ng/mL) and HGF (30 ng/mL). After 12 hours, the expressions of connective tissue growth factor (CTGF) protein in myotubes were detected by Western blot, the levels of CTGF mRNA were measured by RT-PCR. Results Compared to the control group, the protein and mRNA levels of CTGF significantly increased in TGF-β1 treated group , whereas the protein and mRNA levels of CTGF were significantly lower in inhibition group than those in induction group (P < 0.05). Conclusion HGF can inhibit the effect of TGF-β1 on the expression of CTGF in myotubes , which provides the evidences on the study of skeletal muscle cell transdifferentiation.

In vitro glucose uptake activity of Tinospora crispa in skeletal muscle cells

Background: In South Asian countries, stems of the plant, Tinnospora crispa, Linn (TC) are often used as a folk medicine in the treatment of type 2 diabetes mellitus. Though TC’s antiglycemic activity has been demonstrated in diabetic rats, the mechanism for its action has not yet been elucidated. Objective: To investigate the effect of TC’s aqueous extract (TCA) on glucose transport activity in skeletal muscle cell line. Materials and methods: A skeletal muscle cell line, L6 myoblasts, was used for this study. The myoblasts grown to the stage of fused myotubes were pre-incubated with and without TCA for 24 hours. Then, a 2-[³H]-deoxy-D-glucose (2-DG) uptake test was made in a 24-well plate for 10 minutes. In the downstream transport regulation studies, the TCA pre-incubated cells was either treated or untreated with specific inhibitors of the PI3-Kinase (wortmannin) and p38 MAP-Kinase (SB203580) pathways prior to the uptake test. All studies were carried out in triplicate with a minimum of three independent experiments. Results were expressed as mean±SE and compared with student’s t test for a level of significance at p \< 0.05. Results: TCA at 4 mg/mL significantly enhances glucose uptake of L6 myotubes in dose and time dependent manner with the half maximum effects at 24 hours (196.60±11.09%, p \< 0.05). The effect was completely abolished by a cytoskeletal blockade (10 μM of cytochalasin B), supportive of active glucose transport activity. Both wortmannin and SB203580 have no effect on the TCA-stimulated glucose uptake. Conclusion: Tinospora crispa enhances glucose transport of L6 myotubes in an insulin-independent pathway in a time- and dose-dependent manner.

EFFECTS OF TUMOR NECROSIS FACTOR ON DEGRADATION OF LONG-LIVED PROTEIN IN CULTURED MYOTUBES / 解放军医学杂志

Objective To study the effects of tumor necrosis factord (TNF ?)on degradation of long lived protein in cultured myotubes, myoblasts were proliferated in tissue block culture and fused into myotubes. Methods Then the protein in myotubes was radiolabelled with L [3,5 3 H] tyrosine. Myotubes were either cultured with TNF ? 2000U/mL or without TNF ?, and 12h, 24h, 36h, 48h later, the amounts of L [3,5 3 H] tyrosine in culture medium and cells were determined, and the degradation rates of long lived protein were calculated. Other myotubes were cultured either with 50?mol/mL proteasome inhibitor MG132 or 50?mol/mL MG132 and TNF ? 2000U/mL, and long lived proteolytic rates were calculated by the same method after 24h culture. Results The long lived proteolytic rates in myotubes cultured with TNF ? were increased significantly at all time points compared with control group ( P

Immunohistochemical Localization of NCAM at Neuromuscular Contacts in Rat Muscle-Rat Nerve Cocultures / 대한해부학회지

Neuromuscular junction formation is one of the hot research area for understanding synapse formation, and the contact and adhesion between muscle and neurons during this procedure is regarded as one of important steps for synaptogenesis. The changes of neuronal cell adhesion molecules during nerve-muscle contats has not been revealed yet. In this study, we isolated skeletal muscle cells and ventral spinal cord neurons from Sprague-Dawley rats and observed the contact areas with a transmission electron microscpe and studied the presence of NCAM at the contact sites by immunohistochemistry. The ventral spinal cord neuronal processes contact intimately with skeletal muscle cells, some of which were submerged into the muscle surface and had synaptic vesicles. NCAM was expressed on neuronal processes, only sialylated form were associated with acetylcholine receptor aggregates. These results confirmed the significance of adhesion in neuromuscular junction formation and NCAM may participate in this process by preventing the separation of 2 cells at the contact site.

Cytoskeletons of Cultured Rat Fetal Muscle Cells / 대한해부학회지

Myoblasts fuse together to form multinucleated myotubes. However, only a few studies have been reported on the cytoskeletal changes during the fusion process. To understand the change of cytoskeleton during the fusion process, isolated myoblasts from embryonic day 21 Sprague-Dawley rats were cultured on formvar-, carbon-, and gelatin-coated gold grids for electron microscopy. The cells were fixed and plasma membrane and cytoplasm were extracted with triton X-100 and observed directly with Hitachi H-600 transmission electron microscope without staining. Fusiform myoblasts have complex cytoskeletal networks at the center of the cells, which were too dense to be resolved, however the margins of myoblasts and myotubes have bundles of cytoskeletons running in the longitudinal direction with reticulated cytoskeletal networks in between. Lamellipodial ruffles at both ends of myoblasts were characterized by a cytoskeletal lattice at the base and a few radiating strands into the filopodia-like processes. Radiating cytoskeletons originated either from the longitudinally oriented cytoskeletal bundles or reticular lattice continuous to them. The fusion areas were characterized by thin filaments connecting adjacent cells and the connection of longitudinal filament bundles from the fusing cells. These results suggest the modification of cytoskeletons during myoblast fusion.