After stimulus, cells were treated as was previously described23. well as the involvement of LRP1 in this process in the human Mller glial-derived cell line (MIO-M1). We found that IGF-1 produced GLUT1 translocation to the PM, in a time-dependent manner involving the intracellular signaling activation of MAPK/ERK and PI3K/Akt pathways, and generated a significant glucose uptake. Moreover, we found a molecular association between LRP1 and GLUT1, which was significantly reduced by IGF-1. Finally, cells treated with specific siRNA for LRP1 showed an impaired GLUT1 expression on PM and decreased glucose uptake induced by IGF-1. We conclude that IGF-1 regulates glucose homeostasis in MGCs involving the expression of LRP1. experiments will lithospermic acid be needed to evaluate if this mechanism may control other retinal cells that are metabolically dependent of MGCs, such as neurons and endothelial cells, which could have lithospermic acid clinical and therapeutic connotations in neurodegenerative diseases of the retina. Open in a separate window Figure 6 Schematic model of LRP1 mediation in GLUT1 translocation to cell surface and glucose uptake induced by IGF-1. (A) Representative image in which, in non-stimulated MIO-M1 cells, LRP1 and GLUT1 are stored in same, but uncharacterized vesicles, since they are molecularly associated through a possible direct interaction or mediated by adaptor proteins. This molecular association would be necessary to retain GLUT1 inside the cells. (B) IGF-1 induces MAPK/ERK and PI3K/Akt signaling activation through its cognate receptor (IGF-1R). (C) This IGF-1-induced activation promptly leads to the molecular dissociation of LRP1 and GLUT1, promoting the intracellular traffic of both membrane proteins to the PM and glucose uptake. However, if both intracellular signaling pathways have different downstream focuses on within the GLUT1 traffic are still unfamiliar. (D) The LRP1 knockdown fully abrogates the IGF-1R intracellular signaling, the GLUT1 translocation and glucose uptake processes. Taken account these considerations, we propose that the LRP1 mediation in the IGF-1-induced glucose control in MIO-M1 cells may be focused at two levels: (1) by regulating the intracellular traffic of GLUT1, and (2) by acting like a scaffold protein for the IGF-1R activation. Ganglion cell coating (GCL), inner plexiform coating (IPL), inner nuclear coating (INL), outer plexiform coating (OPL), outer nuclear coating (ONL), outer section layer (OS). Methods Cell ethnicities and reagents MIO-M1 cells, a spontaneously immortalized human being Mller cell collection (Moorfields/Institute of Ophthalmology-Mller 1), was provided by Dr G. Astrid Limb (University or college College London, Institute of Ophthalmology and Moorfields Attention Hospital, London, UK) and managed in DMEM-high glucose (4.5?mg/ml) stabilized with 2?mM l-glutamine (GlutaMAX; Invitrogen, Buenos Aires, Argentina) and supplemented with 110?mg/ml sodium pyruvate, 10% (v/v) fetal bovine serum (FBS) (Invitrogen, Buenos Aires, Argentina) and 100 U/ml penicillin/streptomycin (Invitrogen, Buenos Aires, PIK3CD Argentina) at 37?C with 5% CO222,45. Human being IGF-1, Wortmannin and PD98059 were from Sigma-Aldrich (St. Louis, MO). Rabbit anti-pIGF-1R (#28897, T1316), rabbit anti-Akt (#9272), rabbit anti-pAkt (#9275S, T308), rabbit anti-ERK1/2 (#4695) and rabbit anti-pERK (#9101, Thr202/Tyr204) antibodies were from Cell Signaling Technology (Beverly, MA). Mouse monoclonal anti–actin (#A2228) antibody was from SigmaCAldrich (St. Louis, MO). Rabbit anti-LRP1 (#ab92544), rabbit anti-IGF-1R (#ab182408) and mouse monoclonal anti-GLUT1 (#ab40084), rabbit anti-GLUT2 (#ab95256) and rabbit anti-GLUT4 (#ab654) were from Abcam (Cambridge, MA). Mouse monoclonal anti-APT1A1 (#M7-PB-E9) was from ThermoFisher Scientific (Rockford, IL). Several Alexa Fluor conjugated secondary antibodies (goat anti-rabbit IgG- and anti-mouse IgG-Alexa Fluor 594 or 488; Invitrogen, Buenos Aires, Argentina) were utilized for immunofluorescence assays (antibody dilution: 1/800)22,29. Transfection procedure for the silencing LRP1 MIO-M1 cells (4??105 cells/well) were cultured in 6-well plates and transiently transfected with 5?pmol/well of siRNA-LRP1 (#s8280; Ambion, Austin, TX) for 48?h, using Lipofectamine RNAiMAX reagent (Invitrogen) and Opti-MEM 1 (Gibco, Thermo Fischer Scientific, Buenos Aires, Argentina). As control, the silence select bad control siRNA (#4390846; Ambion) was used22. Western blot assays MIO-M1 cell protein extracts were prepared using RIPA buffer (50?mM TrisCHCl pH 8.0, 150?mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, lithospermic acid 0.1% lithospermic acid SDS, 1?mM PMSF, 10?mM sodium ortho-vanadate, and protease inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA)). Forty micrograms of cell protein extracts were diluted in sample buffer 5 with DTT (dithiothreitol) and then heated for 5?min at 95?C. Electrophoresis on 10% SDSCpolyacrylamide gels46 was applied and proteins were electrotransferred to nitrocellulose membrane47 (GE Healthcare Life Technology, Amsterdam, The Netherlands). Nonspecific binding was clogged with 5% non-fat dry milk inside a TrisCHCl saline buffer comprising 0.01% Tween 20 (TBS-T) for 60?min at room temperature. The membranes were incubated over night at 4?C with diluted main antibodies and secondary antibodies raised in goat anti-mouse IgG IRDye 680CW and goat anti-rabbit IgG IRDye 800CW (LI-COR Biosciences, Lincoln, NE) diluted 1/10,000 for 1?h at room temperature. The specific bands were exposed using Odyssey CLx near-infrared fluorescence imaging system (LI-COR Biosciences). Biotin-labeling.