Internalization of intact γ‐conglutin, the lupin seed glucoselowering glycoprotein, by Hep G2 cells: biochemical and microscopy studies

Abstract

Gamma‐conglutin (γ‐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 monitor the localization of this protein in HepG2 cells in parallel to the glucose uptake. HepG2 cells treated with γ‐C were processed to confocal microscopy and transmission electron microscopy (TEM): the protein was detected with specific antibodies. After 3 h treatment, γ‐C was accumulated inside the cells and at 6 h it was diffused as protein aggregates into the cytoplasm. At the same time, glucose uptake increases by 64% vs the control cells. At 24 h the intracellular staining was reduced and protein aggregates stacked onto the cell membranes were visible. TEM showed that microvilli were involved in trapping the protein at the beginning of the process. Microscopy observations were confirmed by 2D electrophoresis showing that the protein inside the cells consisted of its intact subunits. These findings, while confirming the remarkable glucose uptake capacity by HepG2 cells, showed that γ‐C is internalized by the cells, thus opening the way to new strategies for the understanding of the described lupin γ‐conglutin insulin‐mimetic activity.This work was partially supported by a grant of MIUR from Italy