The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics

Abstract

Background information. 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.

The Retinol Dehydrogenase Rdh10 Localizes to Lipid Droplets during Acyl Ester Biosynthesis

“…17␤HSD11) have been identified in the proteomes of lipid droplets (51,53,54). Dhrs3 reduces retinal in the eye and metabolizes retinal derived from ␤-carotene cleavage (55,56).…”

“…The proteome of the lipid droplet includes multiple varieties of proteins, in addition to perilipins, lipases, and acyltransferases, likely partially typical of specific cell types (51)(52)(53). These include SDR that catalyze steroid metabolism, such as isoforms of 3␤-and 17␤-hydroxysteroid dehydrogenases, indicating that the metabolism of lipids in addition to acyl esters occurs on the surfaces of lipid droplets.…”

Intrahepatic Role of Exchangeable Apolipoproteins in Lipoprotein Assembly and Secretion

“…29 It has been shown that apoA-IV was associated with cytosolic lipid droplets in intestinal enterocytes. 30 The mechanism by which apoA-IV can exist as a cytosolic protein has not been defined, nor is it clear whether the cytosolic lipid dropletbound apoA-IV exerts an impact on chylomicron assembly.…”

Dictyostelium Lipid Droplets Host Novel Proteins

“…Our selection finally yielded 72 candidate proteins (Table 1), of which the majority were also identified in recent proteomic studies on yeast (38) and three mammalian cell types (39)(40)(41). We grouped our candidates into enzymes of lipid metabolism (15 enzymes), small GTPases (31 members), constituents of the (rough) endoplasmic reticulum (11 proteins), or cytoskeletal proteins (6 proteins).…”