The involvement of Ca(2+) in the insulin-mediated signaling cascade, resulting in glucose uptake in skeletal muscle, is uncertain. Here, we test the hypothesis that Ca(2+) influx through canonical transient receptor potential 3 (TRPC3) channels modulates insulin-mediated glucose uptake in adult skeletal muscle. Experiments were performed on adult skeletal muscle cells of wild-type (WT) and obese, insulin-resistant ob/ob mice. Application of the diacylglycerol analog 1-oleyl-2-acetyl-sn-glycerol (OAG) induced a nonselective cation current, which was inhibited by the addition of anti-TRPC3 antibody in the patch pipette and smaller in ob/ob than in WT cells. Knockdown of TRPC3, using a novel technique based on small interfering RNA (siRNA) coupled to functionalized carbon nanotubes, resulted in pronounced (approximately 70%) decreases in OAG-induced Ca(2+) influx and insulin-mediated glucose uptake. TRPC3 and the insulin-sensitive glucose transporter 4 (GLUT4) coimmunoprecipitated, and immunofluorescence staining showed that they were colocalized in the proximity of the transverse tubular system, which is the predominant site of insulin-mediated glucose transport in skeletal muscle. In conclusion, our results indicate that TRPC3 interacts functionally and physically with GLUT4, and Ca(2+) influx through TRPC3 modulates insulin-mediated glucose uptake. Thus, TRPC3 is a potential target for treatment of insulin-resistant conditions.

Hemolin is the most abundant bacteria-induced proteins in Hyalophora cecropia hemolymph. Its structural features, both at the protein and gene level, ascribe this molecule to the immunoglobulin gene superfamily (IgSF) with particular homology to neural cell adhesion molecules. An increasing number of evidence suggest a role in immune recognition and in cell adhesion events. Hemolin is also developmentally regulated as suggested by changes in its concentration during larval and pupal ecdysis (Trenczek, T., 1998. Endogenous defense mechanisms of insects. Zoology 101, 298-315; Lanz-Mendoza, H., Faye, I., 1999. Physiological aspects of the immunoglobulin superfamily in invertebrates. Dev. Comp. Immunol. 23, 359-374). In the present study the expression of hemolin was investigated in oogenesis and in early embryogenesis. Our results reveal that hemolin is expressed in follicles and in epidermal and neural tissues of embryos.

Development and innate immune defence are two vital processes that have been demonstrated to use the same or similar molecules and signalling pathways in insects. Hemolin is a moth haemolymph protein belonging to the immunoglobulin superfamily. It is strongly induced upon bacterial infection. However, recent studies indicate a developmental regulation of hemolin. We show that the steroid hormone 20-hydroxyecdysone (20E) can activate the expression of Hyalophora cecropia Hemolin (HcHemolin) in the fat body of diapausing pupae. Using the protein synthesis inhibitor cycloheximide we demonstrate that Hemolin up-regulation by 20E requires ongoing protein synthesis. Moreover, 20E enhances transcription of the Hemolin gene in response to bacteria. Comparing the upstream regions of Manduca sexta Hemolin (MsHemolin) and HcHemolin, we identified four putative regulatory sites. Two are putative hormone response elements (HREs), one with an imperfect inverted repeat (HRE-IR) and one with a monomeric site (HRE-M). An additional monomeric hormone receptor site (MRE) is present only in HcHemolin. The third conserved motif is similar to the interferon (IFN) regulatory factor binding element (IRF-E) and IFN-stimulated response element (ISRE). The fourth conserved element is a kappaB motif situated between the Cap-site and the TATA-box. Finally, by electrophoresis mobility shift assay we demonstrate that the HRE-IR forms specific complexes with nuclear extract proteins of normal pupae that increase after 20E stimulation.

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The increased IFN-γ-producing cell responses induced by the combination of Ad35-CS/BCG-CS and sustained type 1 antibody profile together with high levels of LLPCs may be essential for the development of long-term protective immunity against liver-stage parasites.