Browsing by Subject "LIPID DROPLETS"

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  • Kentala, Henriikka; Koponen, Annika; Kivelä, Annukka M.; Andrews, Robert; Li, ChunHei; Zhou, You; Olkkonen, Vesa M. (2018)
    ORP2 is implicated in cholesterol transport, triglyceride metabolism, and adrenocortical steroid hormone production. We addressed ORP2 function in hepatocytes by generating ORP2-knockout (KO) HuH7 cells by CRISPR-Cas9 gene editing, followed by analyses of transcriptome, F-actin morphology, migration, adhesion, and proliferation. RNA sequencing of ORP2-KO cells revealed >2-fold changes in 579 mRNAs. The Ingenuity Pathway Analysis (IPA) uncovered alterations in the following functional categories: cellular movement, cell-cell signaling and interaction, cellular development, cellular function and maintenance, cellular growth and proliferation, and cell morphology. Many pathways in these categories involved actin cytoskeleton, cell migration, adhesion, or proliferation. Analysis of the ORP2 interactome uncovered 109 putative new partners. Their IPA analysis revealed Ras homolog A (RhoA) signaling as the most significant pathway. Interactions of ORP2 with SEPT9, MLC12, and ARHGAP12 were validated by independent assays. ORP2-KO resulted in abnormal F-actin morphology characterized by impaired capacity to form lamellipodia, migration defect, and impaired adhesion and proliferation. Rescue of the migration phenotype and generation of typical cell surface morphology required an intact ORP2 phosphoinositide binding site, suggesting that ORP2 function involves phosphoinositide binding and transport. The results point at a novel function of ORP2 as a lipid-sensing regulator of the actin cytoskeleton, with impacts on hepatocellular migration, adhesion, and proliferation.-Kentala, H., Koponen, A., Kivela, A. M., Andrews, R., Li, C., Zhou, Y., Olkkonen, V. M. Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation.
  • Pashay Ahi, Ehsan; A. Lecaudey, Laurène; Ziegelbecker, Angelika; Steiner, Oliver; A. Glabonjat, Ronald; Goessler, Walter; Lass, Achim; Sefc, Kristina M. (2020)
    Background Carotenoids contribute significantly to animal body coloration, including the spectacular color pattern diversity among fishes. Fish, as other animals, derive carotenoids from their diet. Following uptake, transport and metabolic conversion, carotenoids allocated to body coloration are deposited in the chromatophore cells of the integument. The genes involved in these processes are largely unknown. Using RNA-Sequencing, we tested for differential gene expression between carotenoid-colored and white skin regions of a cichlid fish, Tropheus duboisi "Maswa", to identify genes associated with carotenoid-based integumentary coloration. To control for positional gene expression differences that were independent of the presence/absence of carotenoid coloration, we conducted the same analyses in a closely related population, in which both body regions are white. Results A larger number of genes (n = 50) showed higher expression in the yellow compared to the white skin tissue than vice versa (n = 9). Of particular interest was the elevated expression level of bco2a in the white skin samples, as the enzyme encoded by this gene catalyzes the cleavage of carotenoids into colorless derivatives. The set of genes with higher expression levels in the yellow region included genes involved in xanthophore formation (e.g., pax7 and sox10), intracellular pigment mobilization (e.g., tubb, vim, kif5b), as well as uptake (e.g., scarb1) and storage (e.g., plin6) of carotenoids, and metabolic conversion of lipids and retinoids (e.g., dgat2, pnpla2, akr1b1, dhrs). Triglyceride concentrations were similar in the yellow and white skin regions. Extracts of integumentary carotenoids contained zeaxanthin, lutein and beta-cryptoxanthin as well as unidentified carotenoid structures. Conclusion Our results suggest a role of carotenoid cleavage by Bco2 in fish integumentary coloration, analogous to previous findings in birds. The elevated expression of genes in carotenoid-rich skin regions with functions in retinol and lipid metabolism supports hypotheses concerning analogies and shared mechanisms between these metabolic pathways. Overlaps in the sets of differentially expressed genes (including dgat2, bscl2, faxdc2 and retsatl) between the present study and previous, comparable studies in other fish species provide useful hints to potential carotenoid color candidate genes.
  • Long, Maeve; Sanchez-Martinez, Alvaro; Longo, Marianna; Suomi, Fumi; Stenlund, Hans; Johansson, Annika; Ehsan, Homa; Salo, Veijo T.; Montava-Garriga, Lambert; Naddafi, Seyedehshima; Ikonen, Elina; Ganley, Ian G.; Whitworth, Alexander J.; McWilliams, Thomas G. (2022)
    Mitophagy removes defective mitochondria via lysosomal elimination. Increased mitophagy coincides with metabolic reprogramming, yet it remains unknown whether mitophagy is a cause or consequence of such state changes. The signalling pathways that integrate with mitophagy to sustain cell and tissue integrity also remain poorly defined. We performed temporal metabolomics on mammalian cells treated with deferiprone, a therapeutic iron chelator that stimulates PINK1/PARKIN-independent mitophagy. Iron depletion profoundly rewired the metabolome, hallmarked by remodelling of lipid metabolism within minutes of treatment. DGAT1-dependent lipid droplet biosynthesis occurred several hours before mitochondrial clearance, with lipid droplets bordering mitochondria upon iron chelation. We demonstrate that DGAT1 inhibition restricts mitophagy in vitro, with impaired lysosomal homeostasis and cell viability. Importantly, genetic depletion of DGAT1 in vivo significantly impaired neuronal mitophagy and locomotor function in Drosophila. Our data define iron depletion as a potent signal that rapidly reshapes metabolism and establishes an unexpected synergy between lipid homeostasis and mitophagy that safeguards cell and tissue integrity.
  • Saarinen, Jukka; Sõzeri, Erkan; Fraser-Miller, Sara; Peltonen, Leena; A. Santos, Helder; Isomäki, Antti; Strachan, Clare J. (2017)
    We have used coherent anti-Stokes Raman scattering (CARS) microscopy as a novel and rapid, label-free and non-destructive imaging method to gain structural insights into live intestinal epithelial cell cultures used for drug permeability testing. Specifically we have imaged live Caco-2 cells in (bio)pharmaceutically relevant conditions grown on membrane inserts. Imaging conditions were optimized, including evaluation of suitable membrane materials and media solutions, as well as tolerable laser powers for non-destructive imaging of the live cells. Lipid structures, in particular lipid droplets, were imaged within the cells on the insert membranes. The size of the individual lipid droplets increased substantially over the 21-day culturing period up to approximately 10% of the volume of the cross section of individual cells. Variation in lipid content has important implications for intestinal drug permeation testing during drug development but has received limited attention to date due to a lack of suitable analytical techniques. CARS microscopy was shown to be well suited for such analysis with the potential for in situ imaging of the same individual cell-cultures that are used for permeation studies. Overall, the method may be used to provide important information about cell monolayer structure to better understand drug permeation results.
  • Koponen, Annika; Arora, Amita; Takahashi, Kohta; Kentala, Henriikka; Kivelä, Annukka M.; Jääskeläinen, Eeva; Peränen, Johan; Somerharju, Pentti; Ikonen, Elina; Viitala, Tapani; Olkkonen, Vesa M. (2019)
    ORP2 is a sterol-binding protein with documented functions in lipid and glucose metabolism, Akt signaling, steroidogenesis, cell adhesion, migration and proliferation. Here we investigate the interactions of ORP2 with phosphoinositides (PIPs) by surface plasmon resonance (SPR), its affinity for cholesterol with a pull-down assay, and its capacity to transfer sterol in vitro. Moreover, we determine the effects of wild-type (wt) ORP2 and a mutant with attenuated PIP binding, ORP2(mHHK), on the subcellular distribution of cholesterol, and analyze the interaction of ORP2 with the related cholesterol transporter ORP1L. ORP2 showed specific affinity for PI(4,5)P-2, PI(3,4,5)P-3 and PI(4)P, with suggestive K-d values in the mu M range. Also binding of cholesterol by ORP2 was detectable, but a K-d could not be determined. Wt ORP2 was in HeLa cells mainly detected in the cytosol, ER, late endosomes, and occasionally on lipid droplets (LDs), while ORP2(mHHK) displayed an enhanced LD localization. Overexpression of wt ORP2 shifted the D4H cholesterol probe away from endosomes, while ORP2(mHHK) caused endosomal accumulation of the probe. Although ORP2 failed to transfer dehydroergosterol in an in vitro assay where OSBP is active, its knock-down resulted in the accumulation of cholesterol in late endocytic compartments, as detected by both D4H and filipin probes. Interestingly, ORP2 was shown to interact and partially co-localize on late endosomes with ORP1L, a cholesterol transporter/sensor at ER-late endosome junctions. Our data demonstrates that ORP2 binds several phosphoinositides, both PI(4)P and multiply phosphorylated species. ORP2 regulates the subcellular distribution of cholesterol dependent on its PIP-binding capacity. The interaction of ORP2 with ORP1L suggests a concerted action of the two ORPs. (C) 2018 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.
  • Olkkonen, Vesa M.; Koponen, Annika; Arora, Amita (2019)
    Oxysterol-binding protein (OSBP)-related proteins (ORPs) constitute a family of intracellular lipid-binding/transport proteins (LTPs) in eukaryotes. They typically have a modular structure comprising a lipid-binding domain and membrane targeting determinants, being thus suited for function at membrane contact sites. Among the mammalian ORPs, ORP2/OSBPL2 is the only member that only exists as a 'short' variant lacking a membrane-targeting pleckstrin homology domain. ORP2 is expressed ubiquitously and has been assigned a multitude of functions. Its OSBP-related domain binds cholesterol, oxysterols, and phosphoinositides, and its overexpression enhances cellular cholesterol efflux. Consistently, the latest observations suggest a function of ORP2 in cholesterol transport to the plasma membrane (PM) in exchange for phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)), with significant impacts on the concentrations of PM cholesterol and PI4,5P(2). On the other hand, ORP2 localizes at the surface of cytoplasmic lipid droplets (LDs) and at endoplasmic-reticulum-LD contact sites, and its depletion modifies cellular triglyceride (TG) metabolism. Study in an adrenocortical cell line further suggested a function of ORP2 in the synthesis of steroid hormones. Our recent knock-out of ORP2 in human hepatoma cells revealed its function in hepatocellular PI3K/Akt signaling, glucose and triglyceride metabolism, as well as in actin cytoskeletal regulation, cell adhesion, migration and proliferation. ORP2 was shown to interact physically with F-actin regulators such as DIAPH1, ARHGAP12, SEPT9 and MLC12, as well as with IQGAP1 and the Cdc37-Hsp90 chaperone complex controlling the activity of Akt. Interestingly, mutations in OSBPL2 encoding ORP2 are associated with autosomal dominant non-syndromic hearing loss, and the protein was found to localize in cochlear hair cell stereocilia. The functions assigned to ORP2 suggest that this protein, in concert with other LTPs, controls the subcellular distribution of cholesterol in various cell types and steroid hormone synthesis in adrenocortical cells. However, it also impacts cellular TG and carbohydrate metabolism and F-actin-dependent functions, revealing a bewildering spectrum of activities.
  • Kentala, Henriikka; Koponen, Annika; Vihinen, Helena; Pirhonen, Juho; Liebisch, Gerhard; Pataj, Zoltan; Kivelä, Annukka; Li, Shiqian; Karhinen, Leena; Jääskeläinen, Eeva; Andrews, Robert; Meriläinen, Leena; Matysik, Silke; Ikonen, Elina; Zhou, You; Jokitalo, Eija; Olkkonen, Vesa M. (2018)
    ORP2 is a ubiquitously expressed OSBP-related protein previously implicated in endoplasmic reticulum (ER)lipid droplet (LD) contacts, triacylglycerol (TG) metabolism, cholesterol transport, adrenocortical steroidogenesis, and actin-dependent cell dynamics. Here, we characterize the role of ORP2 in carbohydrate and lipid metabolism by employing ORP2-knockout (KO) hepatoma cells (HuH7) generated by CRISPR-Cas9 gene editing. The ORP2-KO and control HuH7 cells were subjected to RNA sequencing, analyses of Akt signaling, carbohydrate and TG metabolism, the extracellular acidification rate, and the lipidome, as well as to transmission electron microscopy. The loss of ORP2 resulted in a marked reduction of active phosphorylated Akt(Ser473) and its target Glycogen synthase kinase 3(Ser9), consistent with defective Akt signaling. ORP2 was found to form a physical complex with the key controllers of Akt activity, Cdc37, and Hsp90, and to co-localize with Cdc37 and active Akt(Ser473) at lamellipodial plasma membrane regions, in addition to the previously reported ER-LD localization. ORP2-KO reduced glucose uptake, glycogen synthesis, glycolysis, mRNA-encoding glycolytic enzymes, and SREBP-1 target gene expression, and led to defective TG synthesis and storage. ORP2-KO did not reduce but rather increased ER-LD contacts under basal culture conditions and interfered with their expansion upon fatty acid loading. Together with our recently published work (Kentala et al. in FASEB J 32:1281-1295, 2018), this study identifies ORP2 as a new regulatory nexus of Akt signaling, cellular energy metabolism, actin cytoskeletal function, cell migration, and proliferation.
  • Combot, Yoann; Salo, Veijo T.; Chadeuf, Gilliane; Holttä, Maarit; Ven, Katharina; Pulli, Ilari; Ducheix, Simon; Pecqueur, Claire; Renoult, Ophelie; Lak, Behnam; Li, Shiqian; Karhinen, Leena; Belevich, Ilya; Le May, Cedric; Rieusset, Jennifer; Le Lay, Soazig; Croyal, Mikael; Tayeb, Karim Si; Vihinen, Helena; Jokitalo, Eija; Tornquist, Kid; Vigouroux, Corinne; Cariou, Bertrand; Magre, Jocelyne; Larhlimi, Abdelhalim; Ikonen, Elina; Prieur, Xavier (2022)
    Deficiency of the endoplasmic reticulum (ER) protein seipin results in generalized lipodystrophy by incompletely understood mechanisms. Here, we report mitochondrial abnormalities in seipin-deficient patient cells. A subset of seipin is enriched at ER-mitochondria contact sites (MAMs) in human and mouse cells and localizes in the vicinity of calcium regulators SERCA2, IP3R, and VDAC. Seipin association with MAM calcium regulators is stimulated by fasting-like stimuli, while seipin association with lipid droplets is promoted by lipid loading. Acute seipin removal does not alter ER calcium stores but leads to defective mitochondrial calcium import accompanied by a widespread reduction in Krebs cycle metabolites and ATP levels. In mice, inducible seipin deletion leads to mitochondrial dysfunctions preceding the development of metabolic complications. Together, these data suggest that seipin controls mitochondrial energy metabolism by regulating mitochondrial calcium influx at MAMs. In seipin-deficient adipose tissue, reduced ATP production compromises adipocyte properties, contributing to lipodystrophy pathogenesis.