Functional analysis of fatty acid binding proteins expressed in enterocyte

RODRIGUEZ SAWICKI, L; FALOMIR LOCKHART LJ; BOTASSO N; STORCH J; CÓRSICO B

Buenos Aires

Conferencia; The Weber Conference on Advanced Fluorescence Microscopy Techniques; 2011

UBA - Fluorescence Foundation

Long chain fatty acids (LCFA) are needed as a source of metabolic energy, membrane biogenesis, second messengers, modulation of gene expression; and are associated with cell growth, differentiation, apoptosis and inflammatory processes. Lipid hydrolysis in the intestinal lumen releases great quantities of LCFA. Once inside the enterocyte, LCFA can be reversibly bound to two homologous proteins: Intestinal and Liver Fatty Acid Binding Proteins (I- and LFABP) (1,2,3). Classical functions proposed include cytosolic buffers and transporters of hydrophobic ligands, but new studies position them as key components for regulatory systems. The present work was conducted to evaluate FABP´s specific roles in the lipid metabolism and inflammatory response of the enterocyte. For this purpose, we obtained a cellular model of Caco-2 cells with ablated expression of LFABP and analyzed the assimilation and metabolism of LCFA. Knock-down clones showed marked variations in LCFA assimilation rates and differences in oleate distribution among complex lipid species. Cell proliferation and differentiation were also slowed in the clones. In addition, the effect of LCFA in the induction of an inflammatory response in Caco-2 cells was studied, showing increased levels of cytokines’ mRNA after LCFA exposure. These results indicate that LFABP plays a specific role in the enterocyte lipid metabolism. We plan to expand this line of research including the análisis of FABP-protein interactions with candidate proteins using Fluorescene Resonance Transfer Energy (FRET). Although some interacting partners have been reported for several members of this family (4, 5, 6), no one is yet known for IFABP. Proteins participating in lipid assimilation, its metabolism and transcription factors are strong candidates to interact with FABPs. At the moment, LFABP, IFABP and candidate proteins’ cDNAs are being ligated to different visible fluorescent proteins. Pairs of proteins will be transfected in Caco-2 cells and analyzed using confocal microscopy for FRET signal. The information obtained combining lipid metabolism, immunological processes and protein-protein interactions will contribute to asses intestinal FABPs specific functions and their importance in the enterocyte cell biology.