CONICET | Buscador de Institutos y Recursos Humanos

In eukaryotic cells, lipids are stored in lipid droplets (LDs), organellescomposed of a hydrophobic core covered by a monolayer ofsurface phospholipids. MTP locates in the lumen of the endoplasmicreticulum (ER) and participates in the secretion of lipids fromthe liver as very low density lipoproteins (VLDL). There is evidencethat MTP could be associated with cytosolic LDs. However, no studieswere performed in hepatic LDs. The objective of this work wasto study the subcellular distribution of MTP and its interaction withLDs in hepatocytes. First, we obtained hepatic LDs from mice andevaluated the associated proteins by mass spectrometry followed byimmunoblotting. Upon checking fraction purity with the appropriateantibodies, we found that amongst the 325 proteins associated tothe LDs, several were from the ER, such as calnexin, carboxylesteraseCes3/Tgh, and, interestingly, also MTP. Then, we worked withprimary rat hepatocytes stimulated with 0,4mM oleic acid complexedwith bovine serum albumin (OA/BSA) to produce LDs. Using confocalmicroscopy, we observed that MTP localized in the ER underbasal conditions, but located around the LDs after incubationwith OA/BSA. Then, we analyzed the distribution of MTP in microsomaland LDs fractions by subcellular fractionation followed byimmunoblotting. We found that MTP protein expression increased inLD fractions in a time-dependent manner upon OA/BSA incubation,with a concomitant decrease in the microsomal fraction. Finally, wegenerated a stable clone expressing the MTP-FLAG construct usingHuh7 cells. So far, we found that the stable cell line is functional,increasing VLDL secretion compared to control cells. Conclusion:our results demonstrated the role of MTP as a multifunctional protein.We believe that through its interaction with cytosolic LDs, MTPwould be participating not only in the secretion of VLDL, but also inLDs metabolism.