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      Exposure to dietary lipid leads to rapid production of cytosolic lipid droplets near the brush border membrane

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          Abstract

          Background

          Intestinal absorption of dietary lipids involves their hydrolysis in the lumen of proximal intestine as well as uptake, intracellular transport and re-assembly of hydrolyzed lipids in enterocytes, leading to the formation and secretion of the lipoproteins chylomicrons and HDL. In this study, we examined the potential involvement of cytosolic lipid droplets (CLD) whose function in the process of lipid absorption is poorly understood.

          Methods

          Intestinal lipid absorption was studied in mouse after gavage. Three populations of CLD were purified by density ultracentrifugations, as well as the brush border membranes, which were analyzed by western-blots. Immunofluorescent localization of membranes transporters or metabolic enzymes, as well as kinetics of CLD production, were also studied in intestine or Caco-2 cells.

          Results

          We isolated three populations of CLD (ranging from 15 to 1000 nm) which showed differential expression of the major lipid transporters scavenger receptor BI (SR-BI), cluster of differentiation 36 (CD-36), Niemann Pick C-like 1 (NPC1L1), and the ATP-binding cassette transporters ABCG5/G8 but also caveolin 2 and fatty acid binding proteins. The enzyme monoacylglycerol acyltransferase 2 (MGAT2) was identified in the brush border membrane (BBM) in addition to the endoplasmic reticulum, suggesting local synthesis of triglycerides and CLD at both places.

          Conclusions

          We show a very fast production of CLD by enterocytes associated with a transfer of apical constituents as lipid transporters. Our findings suggest that following their uptake by enterocytes, lipids can be partially metabolized at the BBM and packaged into CLD for their transportation to the ER.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12986-016-0107-9) contains supplementary material, which is available to authorized users.

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          Most cited references59

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          Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.

          Lipid droplets (LDs) store metabolic energy and membrane lipid precursors. With excess metabolic energy, cells synthesize triacylglycerol (TG) and form LDs that grow dramatically. It is unclear how TG synthesis relates to LD formation and growth. Here, we identify two LD subpopulations: smaller LDs of relatively constant size, and LDs that grow larger. The latter population contains isoenzymes for each step of TG synthesis. Glycerol-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the first and rate-limiting step, relocalizes from the endoplasmic reticulum (ER) to a subset of forming LDs, where it becomes stably associated. ER-to-LD targeting of GPAT4 and other LD-localized TG synthesis isozymes is required for LD growth. Key features of GPAT4 ER-to-LD targeting and function in LD growth are conserved between Drosophila and mammalian cells. Our results explain how TG synthesis is coupled with LD growth and identify two distinct LD subpopulations based on their capacity for localized TG synthesis. Copyright © 2013 Elsevier Inc. All rights reserved.
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            Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes.

            Adipocytes hold the body's major energy reserve as triacylglycerols packaged in large lipid droplets. Perilipins, the most abundant proteins on these lipid droplets, play a critical role in facilitating both triacylglycerol storage and hydrolysis. The stimulation of lipolysis by beta-adrenergic agonists triggers rapid phosphorylation of perilipin and translocation of hormone-sensitive lipase to the surfaces of lipid droplets and more gradual fragmentation and dispersion of micro-lipid droplets. Because few lipid droplet-associated proteins have been identified in adipocytes, we isolated lipid droplets from basal and lipolytically stimulated 3T3-L1 adipocytes and identified the component proteins by mass spectrometry. Structural proteins identified in both preparations include perilipin, S3-12, vimentin, and TIP47; in contrast, adipophilin, caveolin-1, and tubulin selectively localized to droplets in lipolytically stimulated cells. Lipid metabolic enzymes identified in both preparations include hormone-sensitive lipase, lanosterol synthase, NAD(P)-dependent steroid dehydrogenase-like protein, acyl-CoA synthetase, long chain family member (ACSL) 1, and CGI-58. 17-beta-Hydroxysteroid dehydrogenase, type 7, was identified only in basal preparations, whereas ACSL3 and 4 and two short-chain reductase/dehydrogenases were identified on droplets from lipolytically stimulated cells. Additionally, both preparations contained FSP27, ribophorin I, EHD2, diaphorase I, and ancient ubiquitous protein. Basal preparations contained CGI-49, whereas lipid droplets from lipolytically stimulated cells contained several Rab GTPases and tumor protein D54. A close association of mitochondria with lipid droplets was suggested by the identification of pyruvate carboxylase, prohibitin, and a subunit of ATP synthase in the preparations. Thus, adipocyte lipid droplets contain specific structural proteins as well as lipid metabolic enzymes; the structural reorganization of lipid droplets in response to the hormonal stimulation of lipolysis is accompanied by increases in the relative mass of several proteins and the recruitment of additional proteins.
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              Biogenesis of the multifunctional lipid droplet: Lipids, proteins, and sites

              Lipid droplets (LDs) are ubiquitous dynamic organelles that store and supply lipids in all eukaryotic and some prokaryotic cells for energy metabolism, membrane synthesis, and production of essential lipid-derived molecules. Interest in the organelle’s cell biology has exponentially increased over the last decade due to the link between LDs and prevalent human diseases and the discovery of new and unexpected functions of LDs. As a result, there has been significant recent progress toward understanding where and how LDs are formed, and the specific lipid pathways that coordinate LD biogenesis.
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                Author and article information

                Contributors
                (33)582066329 , Christine.Comera@toulouse.inra.fr
                Journal
                Nutr Metab (Lond)
                Nutr Metab (Lond)
                Nutrition & Metabolism
                BioMed Central (London )
                1743-7075
                28 July 2016
                28 July 2016
                2016
                : 13
                : 48
                Affiliations
                [1 ]Institut des Maladies Métaboliques et Cardiovasculaires - I2MC, UMR 1048, Institut National de la Santé et de la Recherche Médicale, Université Toulouse III Paul Sabatier, Toulouse, F-31000 France
                [2 ]Leibniz-Institut für Arterioskleroseforschung, Universität Münster, Münster, Germany
                [3 ]UMR 1331 Toxalim, INRA, Université de Toulouse, ENVT, INP-Purpan, 180 chemin de Tournefeuille, BP 93173, 31027 Toulouse, cedex 3, France
                [4 ]INSERM UMR 1043 (INSERM/UPS/CNRS/USC Inra), CHU Purpan, Toulouse, France
                [5 ]CMEAB, Faculté de Médecine Rangueil, Toulouse, France
                [6 ]INSERM/UPS - US006/CREFRE, Service d’Histopathologie, CHU Purpan, Toulouse, France
                [7 ]Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, WI USA
                Article
                107
                10.1186/s12986-016-0107-9
                4965885
                27478484
                6ae56197-ffaa-425a-9dfe-461608f30d5f
                © The Author(s). 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 19 April 2016
                : 21 July 2016
                Funding
                Funded by: ANR/PNRA Absinthe
                Award ID: 5.35
                Award Recipient :
                Funded by: Ministère de la Recherche et de l'Enseignement Français.
                Funded by: US National Institutes of Health
                Award ID: DK088210
                Award Recipient :
                Funded by: ANR (France)
                Award ID: SVSE 1-2012 project SENSOFAT2
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2016

                Nutrition & Dietetics
                lipid droplets,diet and dietary lipids,intestine,fatty acid/metabolism,fatty acid/transport

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