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Dispersed lipid droplets: an intermediate site for lipid transport and metabolism in primary human adipocytes

Open AccessPublished:April 15, 2020DOI:https://doi.org/10.1194/jlr.ILR120000808
      Mature, primary adipocytes contain a central lipid droplet, which accounts for most of the cellular volume (>95%). As recently reported (
      • Morén B.
      • Hansson B.
      • Negoita F.
      • Fryklund C.
      • Lundmark R.
      • Göransson O.
      • Stenkula K.G.
      EHD2 regulates adipocyte function and is enriched at cell surface-associated lipid droplets in primary human adipocytes.
      ), primary adipocytes also hold small lipid droplets scattered throughout the cytosol, which rarely have been recognized due to technical limitations in performing image acquisition and analysis of these cells. Mitochondria connect to lipid droplets in tissues with high fatty acid storage and oxidative capacity (
      • Benador I.Y.
      • Veliova M.
      • Mahdaviani K.
      • Petcherski A.
      • Wikstrom J.D.
      • Assali E.A.
      • Acín-Pérez R.
      • Shum M.
      • Oliveira M.F.
      • Cinti S.
      Mitochondria bound to lipid droplets have unique bioenergetics, composition, and dynamics that support lipid droplet expansion.
      ), where they play an important role for energy transfer. This image illustrates mitochondria (white) organized in close vicinity to small lipid droplets (green), on the background of the large central lipid droplet (red), dispersed at the cell surface in a human primary adipocyte. An inverted grayscale image of each channel is provided to clearly illustrate the appearance and organization of both lipid droplets and mitochondria. We have confirmed this phenotype in both isolated adipocytes and intact adipose tissue using electron microscopy. We hypothesize that these small droplets serve as an intermediate stage in lipid transfer to and from the large central lipid droplet, and therefore significantly contribute to adipocyte metabolism. In support of this, we have detected the lipid transport protein CIDEC to be located to the droplets. Also, the fact that the small lipid droplets are spatially organized together with mitochondria supports that they are interconnected with energy metabolism. Primary adipocytes were isolated from subcutaneous human adipose tissue removed during reconstructive surgery. After washing in Krebs-Ringer Bicarbonate HEPES (KRBH) buffer containing 1% albumin, cells were incubated with MitoTracker for 30 min at 37°C. Next, cells were fixed with 4% paraformaldehyde in KRBH without albumin and stained with Bodipy. Cells were imaged on a Nikon A1+ confocal using 60× magnification objective, NA 1.40, and imaged as described (
      • Lizunov V.A.
      • Matsumoto H.
      • Zimmerberg J.
      • Cushman S.W.
      • Frolov V.A.
      Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells.
      ) using Mattek glass bottom dishes. Because the smallest droplets are difficult to visualize due to optical effects of the central lipid droplet, a limited z-stack was acquired using 8× averaging and a very limited pinhole size. Projections were made using Fiji.
      EQUIPMENT: Nikon A1 plus confocal microscope, 60× Apo DIC oil immersion objective, NA 1.40 (Nikon Instruments Inc.)
      REAGENTS: Bodipy (D3922), MitoTracker (RedCMXRos) from Molecular Probe (Waltham, MA)

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