title: ARAP2 promotes GLUT1-mediated basal glucose uptake through regulation of sphingolipid metabolism.
abstract: Lipid droplet formation, which is driven by triglyceride synthesis, requires several droplet-associated proteins. We identified ARAP2 (an ADP-ribosylation factor 6 GTPase-activating protein) in the lipid droplet proteome of NIH-3T3 cells and showed that knockdown of ARAP2 resulted in decreased lipid droplet formation and triglyceride synthesis. We also showed that ARAP2 knockdown did not affect fatty acid uptake but reduced basal glucose uptake, total levels of the glucose transporter GLUT1, and GLUT1 levels in the plasma membrane and the lipid micro-domain fraction (a specialized plasma membrane domain enriched in sphingolipids). Microarray analysis showed that ARAP2 knockdown altered expression of genes involved in sphingolipid metabolism. Because sphingolipids are known to play a key role in cell signaling, we performed lipidomics to further investigate the relationship between ARAP2 and sphingolipids and potentially identify a link with glucose uptake. We found that ARAP2 knockdown increased glucosylceramide and lactosylceramide levels without affecting ceramide levels, and thus speculated that the rate-limiting enzyme in glycosphingolipid synthesis, namely glucosylceramide synthase (GCS), could be modified by ARAP2. In agreement with our hypothesis, we showed that the activity of GCS was increased by ARAP2 knockdown and reduced by ARAP2 overexpression. Furthermore, pharmacological inhibition of GCS resulted in increases in basal glucose uptake, total GLUT1 levels, triglyceride biosynthesis from glucose, and lipid droplet formation, indicating that the effects of GCS inhibition are the opposite to those resulting from ARAP2 knockdown. Taken together, our data suggest that ARAP2 promotes lipid droplet formation by modifying sphingolipid metabolism through GCS.
authors.affiliation: Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden; Sahlgrenska University Hospital, Gothenburg, Sweden.
authors.forename: Aditi
authors.initials: A
authors.lastname: Chaudhari
authors.affiliation: Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden; Sahlgrenska University Hospital, Gothenburg, Sweden.
authors.forename: Liliana
authors.initials: L
authors.lastname: Håversen
authors.affiliation: Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden; Sahlgrenska University Hospital, Gothenburg, Sweden.
authors.forename: Reza
authors.initials: R
authors.lastname: Mobini
authors.affiliation: Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden; Sahlgrenska University Hospital, Gothenburg, Sweden.
authors.forename: Linda
authors.initials: L
authors.lastname: Andersson
authors.affiliation: Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden; Sahlgrenska University Hospital, Gothenburg, Sweden.
authors.forename: Marcus
authors.initials: M
authors.lastname: Ståhlman
authors.affiliation: Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden; Sahlgrenska University Hospital, Gothenburg, Sweden.
authors.forename: Emma
authors.initials: E
authors.lastname: Lu
authors.affiliation: Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden; Sahlgrenska University Hospital, Gothenburg, Sweden.
authors.forename: Mikael
authors.initials: M
authors.lastname: Rutberg
authors.affiliation: Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden; Sahlgrenska University Hospital, Gothenburg, Sweden.
authors.forename: Per
authors.initials: P
authors.lastname: Fogelstrand
authors.affiliation: Zora BioSciences Oy, Espoo, Finland.
authors.forename: Kim
authors.initials: K
authors.lastname: Ekroos
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors.forename: Adil
authors.initials: A
authors.lastname: Mardinoglu
authors.affiliation: Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden; Sahlgrenska University Hospital, Gothenburg, Sweden.
authors.forename: Malin
authors.initials: M
authors.lastname: Levin
authors.affiliation: Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden; Sahlgrenska University Hospital, Gothenburg, Sweden.
authors.forename: Rosie
authors.initials: R
authors.lastname: Perkins
authors.affiliation: Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden; Sahlgrenska University Hospital, Gothenburg, Sweden. Electronic address: jan.boren@wlab.gu.se.
authors.forename: Jan
authors.initials: J
authors.lastname: Borén
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1 / Q000378:metabolism*; D005963:Glucosylceramides / Q000378:metabolism; D066292:Lipid Droplets / Q000378:metabolism; D050356:Lipid Metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D000070539:Pleckstrin Homology Domains; D000072417:Protein Domains; D020543:Proteome / Q000378:metabolism; D040901:Proteomics; D013107:Sphingolipids / Q000378:metabolism*; D014280:Triglycerides / Q000096:biosynthesis
chemical_list: D000089982:ADP-Ribosylation Factor 6; D020690:GTPase-Activating Proteins; D051272:Glucose Transporter Type 1; D005963:Glucosylceramides; D020543:Proteome; C495678:Slc2a1 protein, mouse; D013107:Sphingolipids; D014280:Triglycerides; C000715727:Arf6 protein, mouse; D005947:Glucose
keywords: ARAP2; GLUT1; Glucosylceramide synthase; lipid droplets; triglyceride synthesis