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 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.

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.

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.

Effect of a high-fat diet on the incorporation of stored triacylglycerol into hepatic VLDL

1992 ◽  
Vol 263 (4) ◽  
pp. E615-E623 ◽  
Author(s):  
O. L. Francone ◽  
G. Griffaton ◽  
A. D. Kalopissis
Keyword(s):  
Endoplasmic Reticulum ◽  
High Fat Diet ◽  
Lipid Droplets ◽  
Control Diet ◽  
Oxidation Products ◽  
High Fat ◽  
Very Low Density Lipoproteins ◽  
Total Utilization ◽  
Fat Feeding

Triacylglycerol (TG) stored in cytoplasmic lipid droplets of hepatocytes was labeled by in vivo [1-(14)C]oleic acid injection to study the effect of a high-fat diet on its incorporation into very-low-density lipoproteins (VLDL). Compared with the control diet, hepatocytes of fat-fed rats 1) contained 7.6 times more cytoplasmic (floating fat) TG and 1.9 times more endoplasmic reticulum (microsomal) TG; 2) had 8 and 6 times lower TG specific activities in cytoplasm and endoplasmic reticulum, respectively; 3) incorporated 22% less 14C label into hepatocyte esterified lipids (TG, cholesterol, phospholipid); 4) secreted 48 and 33% less radioactive and total VLDL-TG, respectively; 5) oxidized more cytoplasmic TG-fatty acid (FA); and 6) showed a 50% decreased total utilization of stored TG-FA. With both diets, the lysosomal inhibitor chloroquine concomitantly decreased productions of labeled VLDL-TG, CO2, and acid-soluble oxidation products. The decreased incorporation of stored TG into VLDL-TG appreciably contributes to the overall inhibition of hepatic VLDL secretion by fat feeding. It appears to be related to the decreased mobilization rate of stored TG and its increased channelling toward oxidation.

scholarly journals High fat diet induces dysregulation of hepatic oxygen gradients and mitochondrial function in vivo

2008 ◽  
Vol 417 (1) ◽  
pp. 183-193 ◽  
Author(s):  
Sudheer K. Mantena ◽  
Denty Paul Vaughn ◽  
Kelly K. Andringa ◽  
Heather B. Eccleston ◽  
Adrienne L. King ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
High Fat Diet ◽  
Protein Modification ◽  
Control Diet ◽  
Critical Role ◽  
Liver Mitochondria ◽  
Mitochondrial Proteins ◽  
High Fat ◽  
Alcoholic Fatty Liver

NAFLD (non-alcoholic fatty liver disease), associated with obesity and the cardiometabolic syndrome, is an important medical problem affecting up to 20% of western populations. Evidence indicates that mitochondrial dysfunction plays a critical role in NAFLD initiation and progression to the more serious condition of NASH (non-alcoholic steatohepatitis). Herein we hypothesize that mitochondrial defects induced by exposure to a HFD (high fat diet) contribute to a hypoxic state in liver and this is associated with increased protein modification by RNS (reactive nitrogen species). To test this concept, C57BL/6 mice were pair-fed a control diet and HFD containing 35% and 71% total calories (1 cal≈4.184 J) from fat respectively, for 8 or 16 weeks and liver hypoxia, mitochondrial bioenergetics, NO (nitric oxide)-dependent control of respiration, and 3-NT (3-nitrotyrosine), a marker of protein modification by RNS, were examined. Feeding a HFD for 16 weeks induced NASH-like pathology accompanied by elevated triacylglycerols, increased CYP2E1 (cytochrome P450 2E1) and iNOS (inducible nitric oxide synthase) protein, and significantly enhanced hypoxia in the pericentral region of the liver. Mitochondria from the HFD group showed increased sensitivity to NO-dependent inhibition of respiration compared with controls. In addition, accumulation of 3-NT paralleled the hypoxia gradient in vivo and 3-NT levels were increased in mitochondrial proteins. Liver mitochondria from mice fed the HFD for 16 weeks exhibited depressed state 3 respiration, uncoupled respiration, cytochrome c oxidase activity, and mitochondrial membrane potential. These findings indicate that chronic exposure to a HFD negatively affects the bioenergetics of liver mitochondria and this probably contributes to hypoxic stress and deleterious NO-dependent modification of mitochondrial proteins.

scholarly journals Lipid droplet dynamics and insulin sensitivity upon a 5-day high-fat diet in Caucasians and South Asians

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Anne Gemmink ◽  
Leontine E. H. Bakker ◽  
Bruno Guigas ◽  
Esther Kornips ◽  
Gert Schaart ◽  
...  
Keyword(s):  
Insulin Sensitivity ◽  
Lipid Droplet ◽  
High Fat Diet ◽  
South Asians ◽  
High Fat ◽  
Droplet Dynamics

scholarly journals Reduced Liver Autophagy in High-Fat Diet Induced Liver Steatosis in New Zealand Obese Mice

Antioxidants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 501
Author(s):  
Ioanna Korovila ◽  
Annika Höhn ◽  
Tobias Jung ◽  
Tilman Grune ◽  
Christiane Ott
Keyword(s):  
Lipid Droplet ◽  
High Fat Diet ◽  
Lipid Droplets ◽  
Liver Steatosis ◽  
Cellular Protein ◽  
Normal Function ◽  
High Fat ◽  
Alcoholic Fatty Liver ◽  
Lysosomal System ◽  
High Calorie

Non-alcoholic fatty liver disease (NAFLD), as a consequence of overnutrition caused by high-calorie diets, results in obesity and disturbed lipid homeostasis leading to hepatic lipid droplet formation. Lipid droplets can impair hepatocellular function; therefore, it is of utmost importance to degrade these cellular structures. This requires the normal function of the autophagic-lysosomal system and the ubiquitin-proteasomal system. We demonstrated in NZO mice, a polygenic model of obesity, which were compared to C57BL/6J (B6) mice, that a high-fat diet leads to obesity and accumulation of lipid droplets in the liver. This was accompanied by a loss of autophagy efficiency whereas the activity of lysosomal proteases and the 20S proteasome remained unaffected. The disturbance of cellular protein homeostasis was further demonstrated by the accumulation of 3-nitrotyrosine and 4-hydroxynonenal modified proteins, which are normally prone to degradation. Therefore, we conclude that fat accumulation in the liver due to a high-fat diet is associated with a failure of autophagy and leads to the disturbance of proteostasis. This might further contribute to lipid droplet stabilization and accumulation.

scholarly journals Adipose Triglyceride Lipase protects the endocytosis of renal cells on a high fat diet in Drosophila

2020 ◽  
Author(s):  
Aleksandra Lubojemska ◽  
M. Irina Stefana ◽  
Lena Lampe ◽  
Azumi Yoshimura ◽  
Alana Burrell ◽  
...  
Keyword(s):  
Lipid Droplet ◽  
High Fat Diet ◽  
Lipid Droplets ◽  
Adipose Triglyceride Lipase ◽  
Peroxisome Proliferator ◽  
Lipid Uptake ◽  
Renal Cells ◽  
High Fat ◽  
Peroxisome Proliferator Activated Receptor ◽  
Triglyceride Lipase

AbstractObesity-related renal lipotoxicity and chronic kidney disease (CKD) are prevalent pathologies with complex aetiologies. One hallmark of renal lipotoxicity is the ectopic accumulation of lipid droplets in kidney podocytes and in proximal tubule cells. Renal lipid droplets are observed in human CKD patients and in high-fat diet rodent models but their precise role remains unclear. Here, we establish a high-fat diet model in Drosophila that recapitulates renal lipid droplets and several other aspects of mammalian CKD. Cell-type specific genetic manipulations show that lipid can overflow from adipose tissue and is taken up by renal cells called nephrocytes. A high-fat diet drives nephrocyte lipid uptake via the multiligand receptor Cubilin, leading to the ectopic accumulation of lipid droplets. These nephrocyte lipid droplets correlate with ER and mitochondrial deficits, as well as with impaired macromolecular endocytosis, a key conserved function of renal cells. Nephrocyte knockdown of diglyceride acyltransferase 1 (DGAT1), overexpression of adipose triglyceride lipase (ATGL) and epistasis tests together reveal that fatty acid flux through the lipid droplet triglyceride compartment protects the ER, mitochondria and endocytosis of renal cells. Strikingly, boosting nephrocyte expression of the lipid droplet resident enzyme ATGL is sufficient to rescue high-fat diet induced defects in renal endocytosis. Moreover, endocytic rescue requires a conserved mitochondrial regulator, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC1α). This study demonstrates that lipid droplet lipolysis counteracts the harmful effects of a high-fat diet via a mitochondrial pathway that protects renal endocytosis. It also provides a genetic strategy for determining whether lipid droplets in different biological contexts function primarily to release beneficial or to sequester toxic lipids.

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