Decreased Mrp2 expression by fructose in the primary culture model in sandwich-cultured rat hepatocytes (SCRHs)

BAROSSO, ISMAEL R.; MEDEOT, ANABELA C.; SCHUCK, VIRGINIA S.; FAZZI, AGUSTINA; HILLOTE, GERALDINE L; SALAS, GIMENA; SANCHEZ POZZI, ENRIQUE J.

Buenos Aires

Congreso; Reunión Anual SAFIS 2023; 2023

Sociedad Argentina de Fisiología

The increase in fructose consumption in the global diet contributes to an increase in total caloric intake and it is related to an increase in the incidence of Metabolic Syndrome (MS). Previously, we demonstrated that administration of 10% fructose in drinking water for 8 weeks to normal rats, a model of MS, reduced bile flow and decreased the expression of canalicular transporters Mrp2 (multidrug resistance-associated protein 2). and Bsep (export of bile salts bomb). Aim: To develop a model to study the signaling pathways involved in the actions of fructose, we evaluated whether incubation of a primary culture of rat hepatocytes in collagen sandwich configuration (SCRHs) with fructose can mimic the effect on Mrp2 observed in vivo. Methods: Once the hepatocytes were polarized, they were treated with 22 mM fructose for 24 hours. LDH (lactate dehydrogenase)release in the media was measured by a detention kit (Wienner Lab). Mrp2 activity was evaluated by secretion of fluorescent glutathione methylfluorescein (GMF) using the index BEI (biliary excretion index). For that, treatments were performed duplicated, in one the experiment was performed with standard buffer whereas in the other a Ca2+/Mg2+ free buffer was used. The BEI of GMF was calculated as: BEI = (fluorescenceCa2+/Mg2+ - fluorescenceCa2+/Mg2+ -free) / fluorescence Ca2+/Mg2+ x100%. Mrp2 expression was evaluated by SDS-polyacrylamide gel electrophoresis and transfer to PVDF membranes of cell homogenates from both groups using primary antibodies directed against Mrp2. Results: (Average ± SEM). Fructose 24 mM did not affect LDH (C: 100±5%; F: 105±3%; n=3) and produced a decrease in BEI by 60% with respect to the Control (C: 24±4; F: 9±4; n=4; *p