y-conglutin, the lupin seed-glucose lowering glycoprotein, interferes on insulin pathway in HepG2 cells

Gamma-conglutin (\u3b3-C), a Lupinus albus, seed glycoprotein, has been shown to lower blood glucose in hyperglycaemic rats and increase glucose consumption in HepG2 cells. Aim of the present work was to assess the modalities of interaction of this protein with the target cells, so as to contribute unveiling its mechanism of action. \u3b3-Conglutin internalization: HepG2 cells, pretreated for 30\u2019 w/wo inhibitors of caveolae mediated endocytosis [filipin (5 \u3bcg/ml) and genistein (200 \u3bcM)], clathrin-mediated pathway [chlorpromazine (25 \u3bcM), methyl-\u3b2-cyclodextrin (5 mM)], and macropinocytosis [amiloride (5 mM)], were incubated with FICT-\u3b3-conglutin (50 \u3bcg/ml) for 4 h at 37\ub0C, followed by FACS analysis. The macropinocitosis pathway was the mechanism of \u3b3-conglutin internalization (69% inhibition). 2D-electrophoresis of cell lysates and antibody reaction of the blotted maps showed that \u3b3-conglutin is internalized in intact form. Moreover, the protein is modified inside the cell by multiple phosphorylation. Insulin pathway: reduced effect on AMPK activity (-50% vs controls) and increased expression of phosphorylated AKT form (+ 25% vs controls) were recorded in HepG2 cells, following western-blot analysis, exposed to 10 mM \u3b3-conglutin and/or 100 nM insulin, or metformin 10 mM