Characterization of the Proteome of Cytoplasmic Lipid Droplets in Mouse Enterocytes after a Dietary Fat Challenge

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Abstract

Dietary fat absorption by the small intestine is a multistep process that regulates the uptake and delivery of essential nutrients and energy. One step of this process is the temporary storage of dietary fat in cytoplasmic lipid droplets (CLDs). The storage and mobilization of dietary fat is thought to be regulated by proteins that associate with the CLD; however, mechanistic details of this process are currently unknown. In this study we analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following a dietary fat challenge. In this analysis we identified 181 proteins associated with the CLD fraction, of which 37 are associated with known lipid related metabolic pathways. We confirmed the localization of several of these proteins on or around the CLD through confocal and electron microscopy, including perilipin 3, apolipoprotein A-IV, and acyl-CoA synthetase long-chain family member 5. The identification of the enterocyte CLD proteome provides new insight into potential regulators of CLD metabolism and the process of dietary fat absorption.

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emmanuel boutet, Claire O'Donovan (2014)

The mission of the Universal Protein Resource (UniProt) (http://www.uniprot.org) is to provide the scientific community with a comprehensive, high-quality and freely accessible resource of protein sequences and functional annotation. It integrates, interprets and standardizes data from literature and numerous resources to achieve the most comprehensive catalog possible of protein information. The central activities are the biocuration of the UniProt Knowledgebase and the dissemination of these data through our Web site and web services. UniProt is produced by the UniProt Consortium, which consists of groups from the European Bioinformatics Institute (EBI), the SIB Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR). UniProt is updated and distributed every 4 weeks and can be accessed online for searches or downloads.

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

Florian Wilfling, Huajin Wang, Joel T. Haas … (2013)

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.

Dawn L Brasaemle, Georgia Dolios, Lawrence Shapiro … (2004)

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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Author and article information

Contributors

François Blachier: Role: Academic Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 18 May 2015

Publication date Collection: 2015

Volume: 10

Issue: 5

Electronic Location Identifier: e0126823

Affiliations

[1 ]Department of Nutrition Science, Purdue University, West Lafayette, Indiana, United States of America

[2 ]Department of Biological Sciences, Purdue University, West Lafayette, Indiana, United States of America

[3 ]Bindley Bioscience Center, Purdue University, West Lafayette, Indiana, United States of America

[4 ]Department of Entomology, Purdue University, West Lafayette, Indiana, United States of America

[5 ]Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, United States of America

National Institute of Agronomic Research, FRANCE

Author notes

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: KKB TD. Performed the experiments: TD DS VEH. Analyzed the data: KKB TD JMG VEH LNP RJK ASG. Contributed reagents/materials/analysis tools: KKB TD DS JMG VEH LNP RJK ASG. Wrote the paper: KKB TD.

* E-mail: kbuhman@ 123456purdue.edu

Article

Publisher ID: PONE-D-14-50570

DOI: 10.1371/journal.pone.0126823

PMC ID: 4436333

PubMed ID: 25992653

SO-VID: 7fb84d1f-c561-44e2-b3d1-c8bbe33cd312

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

History

Date received : 10 November 2014

Date accepted : 8 April 2015

Page count

Figures: 6, Tables: 2, Pages: 19

Funding

This project was supported by the Indiana Clinical and Translational Sciences Institute, by Grant # UL1TR001108 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award (KKB), Grant # 7-13-IN-05 by the American Diabetes Association, Innovation Award (KKB), Grant # R01 DK098606 from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (KKB, ASG), Grant # P30 DK046200 National Institutes of Health, NIDDK (ASG), Grant # U01 ES020958 National Institutes of Health, National Institute of Environmental Health Science (ASG), and the Purdue Research Foundation, Graduate Fellowship Award (KKB, TD). This material is based upon work supported by the U.S. Department of Agriculture, under agreement No. 58-1950-4-003 (ASG). Any opinions, findings, conclusion, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Data Availability All relevant data are within the paper and its Supporting Information files.

Characterization of the Proteome of Cytoplasmic Lipid Droplets in Mouse Enterocytes after a Dietary Fat Challenge

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Most cited references 55

Activities at the Universal Protein Resource (UniProt)

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

Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes.

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