The steroid hormone 20-hydroxyecdysone (20HE) is an essential signaling molecule that modulates molting response in insects and may function as a putative anabolic factor in vertebrate animals, although no mammalian 20HE receptor has been identified. Here we show that in H4IIE cell culture, 20HE treatment decreased expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), reduced glucose production, and induced Akt2 phosphorylation sensitive to the phosphoinositide-3 kinase pathway-specific inhibitor LY-294002. Daily oral administration of 20HE (10 mg/kg for 13 wk) ameliorated obesity and insulin resistance in C57BL/6J mice fed a high-fat diet and produced a significant decrease of body weight gain and body fat mass compared with nontreated animals as demonstrated by dual-energy X-ray absorptiometry analysis. In addition, plasma insulin levels and glucose tolerance were significantly lowered by 20HE treatment. These changes were accompanied by the reduced hepatic expression of PEPCK and G6Pase and increased adiponectin production by visceral fat tissue. These studies demonstrate the anti-obesity and anti-diabetic effects of 20HE and begin to elucidate its putative cellular targets both in vitro and in vivo.

The ionotropic glutamate receptor GluR6 exhibits strongly and rapidly desensitizing current responses. Treatment of heterologically expressed GluR6 with the lectin concanavalin A (ConA) in Xenopus oocytes as well as in human embryonic kidney-293 cells results in a considerable increase of the steady-state current, presumably by inhibiting receptor desensitization. In the present study, we investigated the molecular basis of this effect using a systematic mutagenesis approach. We found that although N-glycosylation is an absolute prerequisite for the lectin-mediated inhibition of desensitization, no single one of the nine extracellular consensus sites for N-glycosylation of GluR6 is required. Rather, each of the nine N-linked carbohydrate side chains is independently capable of modulatory interaction with the lectin. Moreover, even artificially introduced N-glycosylation sites can substitute for native sites. Thus, the specific site of the lectin binding does not appear to be important for its desensitization-inhibiting action. Furthermore, we show that the extent of the receptor's ConA sensitivity depends on its state of activation, because the desensitized GluR6 exhibits significantly lower lectin sensitivity than the nondesensitized receptor. We conclude that binding of ConA "locks" the receptor in the activatable state, thereby inhibiting conformational changes required to shift the receptor to the desensitized state.

We report here the design, development and performance of a novel formulation of liposome- encapsulated glucocorticoids (GCs). A highly efficient (>90%) and stable GC encapsulation was obtained based on a transmembrane calcium acetate gradient driving the active accumulation of an amphipathic weak acid GC pro-drug into the intraliposome aqueous compartment, where it forms a GC-calcium precipitate. We demonstrate fabrication principles that derive from the physicochemical properties of the GC and the liposomal lipids, which play a crucial role in GC release rate and kinetics. These principles allow fabrication of formulations that exhibit either a fast, second-order (t1/2 ∼1 h), or a slow, zero-order release rate (t1/2 ∼ 50 h) kinetics. A high therapeutic efficacy was found in murine models of experimental autoimmune encephalomyelitis (EAE) and hematological malignancies.

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The present study shows the advantages of liposome-based nano-drugs as a novel strategy of delivering active pharmaceutical ingredients for treatment of neurodegenerative diseases that involve neuroinflammation. We used the most common animal model for multiple sclerosis (MS), mice experimental autoimmune encephalomyelitis (EAE). The main challenges to overcome are the drugs’ unfavorable pharmacokinetics and biodistribution, which result in inadequate therapeutic efficacy and in drug toxicity (due to high and repeated dosage). We designed two different liposomal nano-drugs, i.e., nano sterically stabilized liposomes (NSSL), remote loaded with: (a) a “water-soluble” amphipathic weak acid glucocorticosteroid prodrug, methylprednisolone hemisuccinate (MPS) or (b) the amphipathic weak base nitroxide, Tempamine (TMN). For the NSSL-MPS we also compared the effect of passive targeting alone and of active targeting based on short peptide fragments of ApoE or of β-amyloid. Our results clearly show that for NSSL-MPS, active targeting is not superior to passive targeting. For the NSSL-MPS and the NSSL-TMN it was demonstrated that these nano-drugs ameliorate the clinical signs and the pathology of EAE. We have further investigated the MPS nano-drug’s therapeutic efficacy and its mechanism of action in both the acute and the adoptive transfer EAE models, as well as optimizing the perfomance of the TMN nano-drug. The highly efficacious anti-inflammatory therapeutic feature of these two nano-drugs meets the criteria of disease-modifying drugs and supports further development and evaluation of these nano-drugs as potential therapeutic agents for diseases with an inflammatory component.