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

2010 ◽  
Vol 22 (8) ◽  
pp. 1262 ◽  
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.

scholarly journals INFLUENCE OF NANOPARTICLES OF HIGHLY DISPERSED SILICA ON MORPHOLOGY AND INTRACYTOPLASMIC LOCALIZATION OF LIPID DROPLETS IN PORCINE OOCYTES

2017 ◽  
Vol 53 ◽  
pp. 284-292 ◽  
Author(s):  
D. A. Novichkova ◽  
T. I. Kuzmina ◽  
O. V. Shcherbak ◽  
N. P. Galagan ◽  
O. A. Epishko
Keyword(s):  
Growth Phase ◽  
Lipid Droplets ◽  
Nile Red ◽  
Intracellular Lipids ◽  
Highly Dispersed Silica ◽  
Highly Dispersed ◽  
Before And After ◽  
Further Development

Based on the visualization by the fluorescent probe (Nile red) of intracellular lipids in porcine oocytes that have finished growth phase in vivo or in vitro morphology and distribution of lipid drops in oocytes before and after cultivation with nanoparticles of highly dispersed silica (0.001% of HDS) have been characterized. In the cultivation of oocytes with HDS the level of oocytes that have finished growth phase in vitro with lipid droplets in the form of granules and diffuse type of distribution increases in comparison with the above-indicated markers in the oocytes of the other studied groups. The results of the experiments make it possible to interpret the obtained data on the form of lipids in the form of granules, as a form that determines the high potencies of oocytes for further development and assume that the transformation of granules into clusters during cultivation is considered as a predictor of subsequent destructive changes in the oocyte.

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

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Dianne Lumaquin ◽  
Eleanor Johns ◽  
Emily Montal ◽  
Joshua M Weiss ◽  
David Ola ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Large Scale ◽  
Fluorescent Dyes ◽  
Cell Types ◽  
Structural Protein ◽  
Droplet Dynamics

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.

91 A NOVEL TECHNIQUE FOR EVALUATION OF THE LIPID CONTENT IN PIG EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 126 ◽  
Author(s):  
M. Romek ◽  
B. Gajda ◽  
E. Krzysztofowicz ◽  
Z. Smorag
Keyword(s):  
Lipid Droplet ◽  
Lipid Content ◽  
Lipid Droplets ◽  
Nile Red ◽  
Total Lipid Content ◽  
Volume Density ◽  
Molecular Probes ◽  
New Method ◽  
Stages Of Development

A high level of lipids, mainly triglycerides and fatty acids, present in embryo cells in the form of lipid droplets is the major factor associated with low cryopreservation of porcine embryos. Previous results demonstrated that the low tolerance of pig embryos to cryopreservation can be increased through reduction of lipid droplet contents. Therefore, in order to improve cryopreservation techniques of porcine embryos, it is fundamental to establish proper culture conditions which ultimately will enable a decrease in lipid content. Unfortunately, there are no precise and efficient methods to evaluate the lipid contents of single pig embryos. Previously used stereological analysis combined with physical serial sectioning (Romek et al. 2007 Reprod. Domest. Anim. in press) is time-consuming, and measurement of triglyceride levels based on enzymatic hydrolysis eliminates other types of lipids from the analysis. Taking the above problems into account, we have developed a new method for evaluation of total lipid content in pig embryos. It is based on visualization of lipid droplets using the specific fluorescent dye Nile red and applying confocal scanning microscopy followed by the Cavalieri method. This method enables measurement of several stereological parameters, especially the volume density of lipid droplets per unit volume of cytoplasm Vv(fat,c), which quantifies most precisely the amount of intracellular lipid. The experiment was carried out on 2- to 4-cell and 8- to 16-cell pig embryos, morulae, blastocysts, and late blastocysts cultured in vitro. Embryos were developed from in vivo-produced zygotes to appropriate stages of development in North Carolina State University (NCSU) 23 medium. For each stage, ten of the embryos were examined. Embryos were denuded and fixed with 2% glutaraldehyde and 2% formaldehyde, stained with 100 nm Nile red (Molecular Probes, Leiden, The Netherlands), and analyzed by means of a confocal microscope LSM 510 Meta (Carl Zeiss MicroImaging GmbH, G�ttingen, Germany). Serial optical sections of each individual embryo were measured by the point counting method, and then the Cavalieri method was used to estimate Vv(fat,). Vv(fat,c) values calculated for embryos at different stages of development were compared by one-way analysis of variance and Tukey's intervals. For cultured pig embryos, volume density of lipid droplets Vv(fat,c) significantly decreased during cleavage from 0.55 µm3 µm–3 at the 2- to 4-cell-embryo stage to 0.46 µm3 µm–3 at the blastocyst stage. The differences between lipid droplet volumes calculated for morulae, blastocysts, and late blastocysts were statistically significant. In conclusion, our new method is more precise, efficient, and quick in comparison to previously used ones. Moreover, we confirmed that the content of total lipids in cultured pig embryo is reduced during its development. This research was funded by the State Committee for Scientific Research (Project No. 2 P06D 003 26) and Net of Reproduction Biotechnology.

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

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.

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

eLife ◽  
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.

Toxicity and efficacy evaluation of multiple targeted polymalic acid conjugates for triple-negative breast cancer treatment

2020 ◽  
Author(s):  
Lungwani Muungo
Keyword(s):  
Triple Negative ◽  
Growth Factor Receptor ◽  
Engineered Nanoparticles ◽  
Efficacy Evaluation ◽  
Dual Action ◽  
Epidermal Growth ◽  
Laminin 411 ◽  
Immunologic Analysis

Engineered nanoparticles are widely used for delivery of drugs but frequently lack proof of safetyfor cancer patient's treatment. All-in-one covalent nanodrugs of the third generation have beensynthesized based on a poly(β-L-malic acid) (PMLA) platform, targeting human triple-negativebreast cancer (TNBC). They significantly inhibited tumor growth in nude mice by blockingsynthesis of epidermal growth factor receptor, and α4 and β1 chains of laminin-411, the tumorvascular wall protein and angiogenesis marker. PMLA and nanodrug biocompatibility and toxicityat low and high dosages were evaluated in vitro and in vivo. The dual-action nanodrug and singleactionprecursor nanoconjugates were assessed under in vitro conditions and in vivo with multipletreatment regimens (6 and 12 treatments). The monitoring of TNBC treatment in vivo withdifferent drugs included blood hematologic and immunologic analysis after multiple intravenousadministrations. The present study demonstrates that the dual-action nanoconju-gate is highlyeffective in preclinical TNBC treatment without side effects, supported by hematologic andimmunologic assays data. PMLA-based nanodrugs of the Polycefin™ family passed multipletoxicity and efficacy tests in vitro and in vivo on preclinical level and may prove to be optimizedand efficacious for the treatment of cancer patients in the future.

In vitro and in vivo study of the enhancement of carotenoid bioavailability in vegetables using excipient nanoemulsions: Impact of lipid content

2021 ◽  
Vol 141 ◽  
pp. 110162
Author(s):  
Kangfei Yao ◽  
David Julian McClements ◽  
Chang Yan ◽  
Jie Xiao ◽  
Han Liu ◽  
...  
Keyword(s):  
Lipid Content ◽  
In Vivo Study

scholarly journals Imaging-Guided Therapy Simultaneously Targeting HER2 and EpCAM with Trastuzumab and EpCAM-Directed Toxin Provides Additive Effect in Ovarian Cancer Model

Cancers ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 3939
Author(s):  
Tianqi Xu ◽  
Anzhelika Vorobyeva ◽  
Alexey Schulga ◽  
Elena Konovalova ◽  
Olga Vorontsova ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Growth Factor Receptor ◽  
Repeated Administration ◽  
Ovarian Cancer Cells ◽  
Her2 Positive ◽  
Efficient Treatment ◽  
Pseudomonas Exotoxin A ◽  
Epidermal Growth

Efficient treatment of disseminated ovarian cancer (OC) is challenging due to its heterogeneity and chemoresistance. Overexpression of human epidermal growth factor receptor 2 (HER2) and epithelial cell adhesion molecule (EpCAM) in approx. 30% and 70% of ovarian cancers, respectively, allows for co-targeted treatment. The clinical efficacy of the monoclonal antibody trastuzumab in patients with HER2-positive breast, gastric and gastroesophageal cancers makes it readily available as the HER2-targeting component. As the EpCAM-targeting component, we investigated the designed ankyrin repeat protein (DARPin) Ec1 fused to a truncated variant of Pseudomonas exotoxin A with reduced immunogenicity and low general toxicity (LoPE). Ec1-LoPE was radiolabeled, evaluated in ovarian cancer cells in vitro and its biodistribution and tumor-targeting properties were studied in vivo. The therapeutic efficacy of Ec1-LoPE alone and in combination with trastuzumab was studied in mice bearing EpCAM- and HER2-expressing SKOV3 xenografts. SPECT/CT imaging enabled visualization of EpCAM and HER2 expression in the tumors. Co-treatment using Ec1-LoPE and trastuzumab was more effective at reducing tumor growth and prolonged the median survival of mice compared with mice in the control and monotherapy groups. Repeated administration of Ec1-LoPE was well tolerated without signs of hepatic or kidney toxicity. Co-treatment with trastuzumab and Ec1-LoPE might be a potential therapeutic strategy for HER2- and EpCAM-positive OC.

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