The Effects of Tetrapeptides Designed to Fit the Androgen Binding Site of ZIP9 on Myogenic and Osteogenic Cells

ZIP9 is a recently identified membrane-bound androgen receptor of physiological significance that may mediate certain physiological responses to androgens. Using in silico methods, six tetrapeptides with the best docking properties at the testosterone binding site of ZIP9 were synthesized and further investigated. All tetrapeptides displaced T-BSA-FITC, a membrane-impermeable testosterone analog, from the surface of mouse myogenic L6 cells that express ZIP9 but not the classical androgen receptor (AR). Silencing the expression of ZIP9 with siRNA prevented this labeling. All tetrapeptides were found to be pro-androgenic; in L6 cells they stimulated the expression of myogenin, triggered activation of focal adhesion kinase, and prompted the fusion of L6 myocytes to syncytial myotubes. In human osteoblastic SAOS-2 cells that express AR and ZIP9, they reduced the expression of alkaline phosphatase and stimulated mineralization. These latter effects were prevented by silencing ZIP9 expression, indicating that the osteoblast/osteocyte conversion is exclusively mediated through ZIP9. Our results demonstrate that the synthetic tetrapeptides, by acting as ZIP9-specific androgens, have the potential to replace testosterone or testosterone analogs in the treatment of bone- or muscle-related disorders by circumventing the undesirable effects mediated through the classical AR.

Identification of C21 Steroidal Glycosides from Gymnema sylvestre (Retz.) and Evaluation of Their Glucose Uptake Activities

Gymnema sylvestre (Retz.) Schult is a multi-purpose traditional medicine that has long been used for the treatment of various diseases. To discover the potential bioactive composition of G. sylvestre, a chemical investigation was thus performed. In this research, four new C21 steroidal glycosides sylvepregosides A-D (1–4) were isolated along with four known compounds, gymnepregoside H (5), deacetylkidjoladinin (6), gymnepregoside G (7) and gymnepregoside I (8), from the ethyl acetate fraction of G. sylvestre. The structures of the new compounds were established by extensive 1D and 2D nuclear magnetic resonance (NMR) spectra with mass spectroscopy data. Compounds 1–6 promoted glucose uptake by the range of 1.10- to 2.37-fold, respectively. Compound 1 showed the most potent glucose uptake, with 1.37-fold enhancement. Further study showed that compounds 1 and 5 could promote GLUT-4 fusion with the plasma membrane in L6 cells. The result attained in this study indicated that the separation and characterization of these compounds play an important role in the research and development of new anti-diabetic drugs and pharmaceutical industry.

Anthocyanins from Aristotelia chilensis Prevent Olanzapine-Induced Hepatic-Lipid Accumulation but Not Insulin Resistance in Skeletal Muscle Cells

Type 2 diabetes and obesity are major problems worldwide and dietary polyphenols have shown efficacy to ameliorate signs of these diseases. Anthocyanins from berries display potent antioxidants and protect against weight gain and insulin resistance in different models of diet-induced metabolic syndrome. Olanzapine is known to induce an accelerated form of metabolic syndrome. Due to the aforementioned, we evaluated whether delphinidin-3,5-O-diglucoside (DG) and delphinidin-3-O-sambubioside-5-O-glucoside (DS), two potent antidiabetic anthocyanins isolated from Aristotelia chilensis fruit, could prevent olanzapine-induced steatosis and insulin resistance in liver and skeletal muscle cells, respectively. HepG2 liver cells and L6 skeletal muscle cells were co-incubated with DG 50 μg/mL or DS 50 μg/mL plus olanzapine 50 μg/mL. Lipid accumulation was determined in HepG2 cells while the expression of p-Akt as a key regulator of the insulin-activated signaling pathways, mitochondrial function, and glucose uptake was assessed in L6 cells. DS and DG prevented olanzapine-induced lipid accumulation in liver cells. However, insulin signaling impairment induced by olanzapine in L6 cells was not rescued by DS and DG. Thus, anthocyanins modulate lipid metabolism, which is a relevant factor in hepatic tissue, but do not significantly influence skeletal muscle, where a potent antioxidant effect of olanzapine was found.

Cytotoxicity, Anti-Obesity and Anti-Diabetic Activities of Heteromorpha arborescens (Spreng.) Cham Leaves

This study investigated the cytotoxicity, anti-obesity and anti-diabetic potentials of blanched, aqueous and ethanol extracts of Heteromorpha arborescens (Spreng.) Cham leaves. The results revealed that both ethanol and aqueous extracts exhibited considerable inhibition against α-glucosidase (IC50 of 627.29 ± 4.62 µg/mL and 576.46 ± 3.21 µg/mL respectively), while the blanched extract showed weak α-glucosidase inhibition (IC50; 855.38 ± 4.29 µg/mL) and the aqueous extract showed the best α-amylase inhibition (IC50; 583.74 ± 5.87 µg/mL). However, weak α-amylase inhibition was observed in the ethanol (IC50; 724.60 ± 4.33 µg/mL) and blanched extracts (IC50; 791.63 ± 3.76 µg/mL). The toxicity of the extracts is indicated by LC50 values as 154.75 µg/mL, 125 µg/mL and 90.58 µg/mL for ethanol, aqueous and blanched extracts respectively, indicating the blanched extract to be the most toxic. Moderate glucose utilization in both C3A and L6 cells was also observed for the aqueous and ethanol extracts which may be attributed to the relatively lower toxicity levels present. However, glucose utilization was very weak for the blanched extract, which may be due to higher level of cytotoxicity it possessed. Relatively weaker lipase inhibition was observed for the ethanol (IC50; 699.3 ± 1.33 µg/mL), aqueous (IC50; 811.52 ± 3.52 µg/mL) and blanched extracts (IC50; 1152.7 ± 4.61 µg/mL) compared to orlistat (IC50; 56.88 ± 0.11 µg/mL). However, there was no reasonable reduction in lipid accumulation observed in all the extract treated cells. These observations suggest that ethanol and aqueous extracts of H. arborescens leaf are promising as new agents for the treatment of diabetes and its acclaimed anti-obesity potentials are likely due to its lipase, α-amylase and α-glucosidase inhibition.

Androgen receptor regulates the proliferation of myoblasts under appropriate or excessive stretch through IGF-1 receptor mediated p38 and ERK1/2 pathways

Background Androgen receptor (AR) exerts important roles in exercise-induced alterations of muscle mass, in which the proliferation and differentiation of satellite cells or myoblasts are crucial. Our previous study in C2C12 myoblasts demonstrated that 15% (mimic appropriate exercise) and 20% (mimic excessive exercise) stretches promoted and inhibited the proliferation respectively; and AR played a crucial role in 15% stretch-induced pro-proliferation through IGF-1-modulated PI3K/Akt, p38 and ERK1/2 pathways, but AR’s role in stretches-modulated proliferation of general myoblasts, especially 20% stretch, remains unclear, and the mechanisms need to be further clarified. Methods Firstly, the discrepancy in proliferation and the above indicators between L6 (without AR) and C2C12 (with AR) myoblasts were compared under 15% or 20% stretch. Then the influences of transfection AR or exogenous IGF-1 treatment on proliferation and these indicators were detected in stretched L6 myoblasts. Results (1) Under un-stretched state, the proliferation of L6 was slower than C2C12 cells. Furthermore, AR knockdown in C2C12 myoblasts repressed, while AR overexpression in L6 myoblasts promoted the proliferation. (2) 15% stretch-induced increases in the proliferation and activities of p38 and ERK1/2 were lower in L6 than C2C12 cells; AR overexpression enhanced the proliferation of 15% stretched L6 cells accompanied with the increases of p38 and ERK1/2 activities. (3) 20% stretch-induced anti-proliferation and inhibition of p38 activity were severer in L6 than C2C12 myoblasts; AR overexpression reversed the anti-proliferation of 20% stretch and enhanced p38 activity in L6 myoblasts. (4) In stretched L6 myoblasts, AR overexpression increased IGF-1R level despite no detectable IGF-1; and recombinant IGF-1 increased the proliferation, the level of IGF-1R, and the activities of p38 and ERK1/2 in 15% stretched L6 myoblasts. Conclusions The study demonstrated AR's crucial roles in stretches-regulated proliferation of myoblasts, and increased AR fulfilled 15% stretch's pro-proliferation via activating IGF-1R- p38 and ERK1/2 pathways while decreased AR achieved 20% stretch's anti-proliferation via inhibiting IGF-1R- p38 pathway, which is useful to understand in depth the role and mechanisms of AR in appropriate exercise increasing while excessive exercise decreasing muscle mass.

Aloperine Relieves Type 2 Diabetes Mellitus via Enhancing GLUT4 Expression and Translocation

Aloperine (ALO), a quinolizidine alkaloid isolated from Sophora alopecuroides L. used in the traditional Uygur medicine, induced a significant increase in cellular glucose uptake of L6 cells, suggesting it has the potential to relieve hyperglycemia. Therefore, we investigated the effects of ALO on type 2 diabetes mellitus (T2DM) through in vitro and in vivo studies. The translocation of glucose transporter 4 (GLUT4) and changes in intracellular Ca2+ levels were real-time monitored in L6 cells using a laser scanning confocal microscope and related protein kinase inhibitors were used to explore the mechanism of action of ALO. Furthermore, high fat diet combined with low-dose streptozotocin (STZ) was used to induce T2DM in rats, and ALO was given to the stomach of T2DM rats for 4 weeks. In vitro results showed that ALO-induced enhancement of GLUT4 expression and translocation were mediated by G protein-PLC-PKC and PI3K/Akt pathways and ALO-enhanced intracellular Ca2+ was involved in activating PKC via G protein-PLC-IP3R-Ca2+ pathway, resulting in promoted GLUT4 plasma membrane fusion and subsequent glucose uptake. ALO treatment effectively ameliorated hyperglycemia, glucose intolerance, insulin resistance and dyslipidemia, alleviated hepatic steatosis, protected pancreatic islet function and activated GLUT4 expression in insulin target tissues of T2DM rats. These findings demonstrated that ALO deserves attention as a potential hypoglycemic agent.

TMT-Based L6 Quantitative Proteomics Analysis Reveals the Effect of Bovine Derived MFG-E8 against Oxidative Stress on Rat L6 Cells

Milk fat globule-EGF factor 8 (MFG-E8) is a secreted matrix glycoprotein exert a crucial role in regulating tissue homeostasis and protecting against skeletal muscle injury. To explore the molecular mechanism...

Androgen Receptor Regulates the Proliferation of Stretched Myoblasts and Its Mechanisms: IGF-1R-mediated p38 and ERK1/2 Pathways

BackgroundOur previous study has indicated that in C2C12 myoblasts androgen receptor (AR) plays a crucial role in 15% stretch-induced proliferation through insulin-like growth factor (IGF-1)-modulated PI3K/Akt, p38 and ERK1/2 pathways. In this study we further explored AR's effects on 15% and 20% stretches-modulated myoblasts proliferation and involved pathways in L6 (no detectable AR) and C2C12 (abundant AR) myoblasts.MethodsThe proliferation and the above indicators were compared between stretched L6 and C2C12 myoblasts by CCK8, ELISA and Western blot, then were detected again in stretched L6 myoblasts after transfection with AR overexpression plasmid utilizing LipoPlusTM 2000 reagent or treatment with different concentrations (200, 500, 1000 ng/ml) IGF-1 recombinant polypeptide.ResultsWe found that ①Under un-stretched states, the proliferation rate of L6 cells was slower than that of C2C12 cells, and the proliferation of C2C12 cells was repressed by AR knockdown using siRNA interference, while L6 myoblasts proliferation was promoted by overexpression AR. ②The proliferation rate as well as the activities of p38 and ERK1/2 increased by 15% stretch were much lower in L6 than that in C2C12 cells; AR overexpression enhanced the proliferation of 15% stretched L6 cells accompanied with increased activities of p38 and ERK1/2; ③20% stretch decreased the proliferation and activity of p38 in L6 more than that in C2C12 myoblasts; overexpression of AR totally reversed the anti-proliferation of 20% stretch and significantly enhanced p38 activity in L6 myoblasts;④In L6 myoblasts, overexpression AR increased IGF-1R expression despite no detectable IGF-1 was secreted; and recombinant IGF-1 increased not only the proliferation, but also the protein level of IGF-1R and activities of p38 and ERK1/2 in a dose-dependent manner in 15% stretched L6 myoblasts. ConclusionsAR fulfilled 15% stretch-induced pro-proliferation of myoblasts by activating IGF-1R-p38 and ERK1/2 pathways, while 20% stretch-induced anti-proliferation was mediated by inhibiting AR-IGF-1R-p38 pathway. This study is beneficial to understand in depth the role and mechanisms of AR on appropriate exercise increases while excessive exercise decreases muscle mass.