title: The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics.
abstract: BACKGROUND
  Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization.
  
  
  RESULTS
  Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-β-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has been proposed to play many functions in vivo, including the formation of lipoproteins and the control of their size. The association of ApoA-IV with CLD was confirmed by confocal and immunoelectron microscopy and validated in vivo in the jejunum of mice fed with a high-fat diet.
  
  
  CONCLUSIONS
  We report for the first time the protein endowment of Caco-2/TC7 enterocyte CLDs. Our results suggest that their formation and mobilization may participate in the control of enterocyte TRL secretion in a cell-specific manner.
authors.affiliation: Université Pierre et Marie Curie-Paris 6, UMR S 872, Les Cordeliers, Paris 75006, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Bouchoux
authors.forename: Frauke
authors.initials: F
authors.lastname: Beilstein
authors.forename: Thomas
authors.initials: T
authors.lastname: Pauquai
authors.forename: I Chiara
authors.initials: IC
authors.lastname: Guerrera
authors.forename: Danielle
authors.initials: D
authors.lastname: Chateau
authors.forename: Nathalie
authors.initials: N
authors.lastname: Ly
authors.forename: Malik
authors.initials: M
authors.lastname: Alqub
authors.forename: Christophe
authors.initials: C
authors.lastname: Klein
authors.forename: Jean
authors.initials: J
authors.lastname: Chambaz
authors.forename: Monique
authors.initials: M
authors.lastname: Rousset
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Lacorte
authors.forename: Etienne
authors.initials: E
authors.lastname: Morel
authors.forename: Sylvie
authors.initials: S
authors.lastname: Demignot
mesh_terms: D000818:Animals; D018938:Caco-2 Cells; D002454:Cell Differentiation; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism*; D020895:Enterocytes / Q000166:cytology* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D050356:Lipid Metabolism; D008055:Lipids / Q000302:isolation & purification*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009928:Organ Specificity; D020543:Proteome / Q000378:metabolism*
chemical_list: D008055:Lipids; D020543:Proteome