Decoration of myocellular lipid droplets with perilipins as a marker for in vivo lipid droplet dynamics: A super-resolution microscopy study in trained athletes and insulin resistant individuals

Lipid droplets are lipid storage organelles found in nearly all cell types from adipocytes to cancer cells. Although increasingly implicated in disease, current methods to study lipid droplets in vertebrate models rely on static imaging or the use of fluorescent dyes, limiting investigation of their rapid in vivo dynamics. To address this, we created a lipid droplet transgenic reporter in whole animals and cell culture by fusing tdTOMATO to Perilipin-2 (PLIN2), a lipid droplet structural protein. Expression of this transgene in transparent casper zebrafish enabled in vivo imaging of adipose depots responsive to nutrient deprivation and high-fat diet. Simultaneously, we performed a large-scale in vitro chemical screen of 1280 compounds and identified several novel regulators of lipolysis in adipocytes. Using our Tg(-3.5ubb:plin2-tdTomato) zebrafish line, we validated several of these novel regulators and revealed an unexpected role for nitric oxide in modulating adipocyte lipid droplets. Similarly, we expressed the PLIN2-tdTOMATO transgene in melanoma cells and found that the nitric oxide pathway also regulated lipid droplets in cancer. This model offers a tractable imaging platform to study lipid droplets across cell types and disease contexts using chemical, dietary, or genetic perturbations.

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An in vivo reporter for tracking lipid droplet dynamics in transparent zebrafish

10.1101/2020.11.09.375667 ◽

2020 ◽

Author(s):

Dianne Lumaquin ◽

Eleanor Johns ◽

Joshua Weiss ◽

Emily Montal ◽

Olayinka Ooladipupo ◽

...

Keyword(s):

Nitric Oxide ◽

Lipid Droplet ◽

High Fat Diet ◽

Lipid Droplets ◽

Cell Types ◽

Structural Protein ◽

High Fat ◽

Droplet Dynamics ◽

Perilipin 2

AbstractLipid droplets are lipid storage organelles found in nearly all cell types from adipocytes to cancer cells. Although increasingly implicated in disease, current methods to study lipid droplets require fixation or static imaging which limits investigation of their rapid in vivo dynamics. To address this, we created a lipid droplet transgenic reporter in whole animals and cell culture by fusing tdTOMATO to Perilipin-2 (PLIN2), a lipid droplet structural protein. Expression of this transgene in transparent casper zebrafish enabled in vivo imaging of adipose depots responsive to nutrient deprivation and high-fat diet. Using this system, we tested novel regulators of lipolysis, revealing an unexpected role for nitric oxide in modulating adipocyte lipid droplets. Similarly, we expressed the PLIN2-tdTOMATO transgene in melanoma cells and found that the nitric oxide pathway also regulated lipid droplets in cancer. This model offers a tractable imaging platform to study lipid droplets across cell types and disease contexts.

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Imaging cytoplasmic lipid droplets in vivo with fluorescent perilipin 2 and perilipin 3 knock-in zebrafish

eLife ◽

10.7554/elife.66393 ◽

2021 ◽

Vol 10 ◽

Author(s):

Meredith H Wilson ◽

Stephen C Ekker ◽

Steven Arthur Farber

Keyword(s):

Lipid Droplet ◽

Lipid Droplets ◽

Cultured Cells ◽

Droplet Dynamics ◽

Associated Proteins ◽

Perilipin 2 ◽

Cytoplasmic Lipid Droplets ◽

Endogenous Loci

Cytoplasmic lipid droplets are highly dynamic storage organelles that are critical for cellular lipid homeostasis. While the molecular details of lipid droplet dynamics are a very active area of investigation, this work has been primarily performed in cultured cells. Taking advantage of the powerful transgenic and in vivo imaging opportunities available in zebrafish, we built a suite of tools to study lipid droplets in real-time from the subcellular to the whole organism level. Fluorescently tagging the lipid-droplet-associated proteins, perilipin 2 and perilipin 3, in the endogenous loci permits visualization of lipid droplets in the intestine, liver, and adipose tissue. Using these tools, we found that perilipin 3 is rapidly loaded on intestinal lipid droplets following a high-fat meal and later replaced by perilipin 2. These powerful new tools will facilitate studies on the role of lipid droplets in different tissues, under different genetic and physiological manipulations, and in a variety of human disease models.

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Identification of Perilipin-2 as a lipid droplet protein regulated in oocytes during maturation

Reproduction Fertility and Development ◽

10.1071/rd10091 ◽

2010 ◽

Vol 22 (8) ◽

pp. 1262 ◽

Cited By ~ 35

Author(s):

Xing Yang ◽

Kylie R. Dunning ◽

Linda L.-Y. Wu ◽

Theresa E. Hickey ◽

Robert J. Norman ◽

...

Keyword(s):

Lipid Droplet ◽

Lipid Content ◽

Lipid Droplets ◽

Intracellular Lipids ◽

Epidermal Growth ◽

Novel Method ◽

Perilipin 2 ◽

Lipid Droplet Protein

Lipid droplet proteins regulate the storage and utilisation of intracellular lipids. Evidence is emerging that oocyte lipid utilisation impacts embryo development, but lipid droplet proteins have not been studied in oocytes. The aim of the present study was to characterise the size and localisation of lipid droplets in mouse oocytes during the periovulatory period and to identify lipid droplet proteins as potential biomarkers of oocyte lipid content. Oocyte lipid droplets, visualised using a novel method of staining cumulus–oocyte complexes (COCs) with BODIPY 493/503, were small and diffuse in oocytes of preovulatory COCs, but larger and more centrally located after maturation in response to ovulatory human chorionic gonadotrophin (hCG) in vivo, or FSH + epidermal growth factor in vitro. Lipid droplet proteins Perilipin, Perilipin-2, cell death-inducing DNA fragmentation factor 45-like effector (CIDE)-A and CIDE-B were detected in the mouse ovary by immunohistochemistry, but only Perilipin-2 was associated with lipid droplets in the oocyte. In COCs, Perilipin-2 mRNA and protein increased in response to ovulatory hCG. IVM failed to induce Perilipin-2 mRNA, yet oocyte lipid content was increased in this context, indicating that Perilipin-2 is not necessarily reflective of relative oocyte lipid content. Thus, Perilipin-2 is a lipid droplet protein in oocytes and its induction in the COC concurrent with dynamic reorganisation of lipid droplets suggests marked changes in lipid utilisation during oocyte maturation.

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