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TGR5 plays an important role in many physiological processes. However, the functions of TGR5 in the regulation of the glucose metabolism and insulin sensitivity in the skeletal muscles have not been fully elucidated. We synthesized MN6 as a potent and selective TGR5 agonist. Here, the effect of MN6 on insulin resistance in skeletal muscles was evaluated in diet-induced obese (DIO) mice and C2C12 myotubes, and the underlying mechanisms were explored. The activation of MN6 on human and mouse TGR5 was evaluated by a cAMP assay in HEK293 cell lines stable expressing hTGR5/CRE or mTGR5/CRE cells. GLP-1 secretion was measured in NCI-H716 cells and CD1 mice. The acute and chronic effects of MN6 on regulating metabolic abnormalities were observed in ob/ob and DIO mice. 2-deoxyglucose uptake was examined in isolated skeletal muscles. Akt phosphorylation, glucose uptake and glycogen synthesis were examined to assess the effects of MN6 on palmitate-induced insulin resistance in C2C12 myotubes. MN6 potently activated human and mouse TGR5 with EC 50 values of 15.9 and 17.9 nmol/L, respectively, and stimulated GLP-1 secretion in NCI-H716 cells and CD1 mice. A single oral dose of MN6 significantly decreased the blood glucose levels in ob/ob mice. Treatment with MN6 for 15 days reduced the fasting blood glucose and HbA1c levels in ob/ob mice. MN6 improved glucose and insulin tolerance and enhanced the insulin-stimulated glucose uptake of skeletal muscles in DIO mice. The palmitate-induced impairment of insulin-stimulated Akt phosphorylation, glucose uptake and glycogen synthesis in C2C12 myotubes could be prevented by MN6. The effect of MN6 on palmitate-impaired insulin-stimulated Akt phosphorylation was abolished by siRNA-mediated knockdown of TGR5 or by the inhibition of adenylate cyclase or protein kinase A, suggesting that this effect is dependent on the activation of TGR5 and the cAMP/PKA pathway. Our study identified that a TGR5 agonist could ameliorate insulin resistance by the cAMP/PKA pathway in skeletal muscles; this uncovered a new effect of the TGR5 agonist on regulating the glucose metabolism and insulin sensitivity in skeletal muscles and further strengthened its potential value for the treatment of type 2 diabetes. • MN6 potently activated TGR5 and stimulated GLP-1 secretion. • MN6 improved glucose homeostasis in ob/ob and DIO mice. • MN6 enhanced insulin-stimulated glucose uptake in skeletal muscles of DIO mice. • MN6 prevented palmitate-induced insulin resistance in C2C12 myotubes. • MN6 ameliorated palmitate-induced insulin resistance by TGR5/cAMP/PKA pathway. [ABSTRACT FROM AUTHOR]

Under the known pharmacological activation mechanisms, activators allosterically or directly open potassium channel gates. However, herein, molecular dynamics simulations on TREK-1, a member of the channel class gated at the filter, suggested that negatively charged activators act with a gate-independent mechanism where compounds increase currents by promoting ions passing through the central cavity. Then, based on studies of KCNQ2, we uncovered that this noncanonical activation mechanism is shared by the other channel class gated at the helix-bundle crossing. Rational drug design found a novel KCNQ2 agonist, CLE030, which stably binds to the central cavity. Functional analysis, molecular dynamics simulations, and calculations of the potential of mean force revealed that the carbonyl oxygen of CLE030 influences permeant ions in the central cavity to contribute to its activation effects. Together, this study discovered a ligand-to-ion activation mechanism for channels that bypasses their gates and thus is conserved across subfamilies with different gates.

Background: Due to chemotherapy resistance in osteosarcoma subgroups, the prognosis of these patients is still poor, and the development of new agents is of utmost importance. The aim of our study was to test the antitumor effects of two novel alpha-aryloxy-alpha-methylhydrocinnamic acid derivatives as peroxisome proliferator-activated receptor (PPAR) gamma agonists, together with rosiglitazone, a well-known thiazolidinedione (TZD) acting on several osteosarcoma cell lines., Methods/results: The MTT assay revealed that cell viability was inhibited in a dose-dependent manner with IC(50) 6.2-15.8 microM for the two novel compounds and rosiglitazone (48.4-83.5 microM). Exposure to DG8 and DH9 at low micromolar concentrations induced a dose-dependent block of DNA synthesis and colony formation. In these antitumor assays, DG8 and DH9 were more effective than rosiglitazone, although the PPARgamma agonistic activity of rosiglitazone is much higher. The SiRNA approach to downregulate specifically PPARgamma in U-2OS cells did not affect the cytotoxic efficiency of either the two novel compounds or rosiglitazone., Conclusion: These observations suggest that non-TZDs with less PPARgamma agonistic activity might show more potent antitumor efficacy independent of PPARgamma in human osteosarcoma cells, which supports the possibility that they could be beneficial in the treatment of osteosarcoma patients., (Copyright 2009 S. Karger AG, Basel.)