scholarly journals Nile Red and 2-NBDG Are Incompatible for the Simultaneous Detection of Lipid and Glucose Accumulation

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Andrew M. Hogan ◽  
Viswanathan Swaminathan ◽  
Nikitha K. Pallegar ◽  
Sherri L. Christian
Keyword(s):  
Glucose Uptake ◽  
Lipid Accumulation ◽  
Mammalian Cells ◽  
Simultaneous Detection ◽  
Nile Red ◽  
Lipid Synthesis ◽  
Cell Types ◽  
Fluorescent Intensity ◽  
Fine Control ◽  
Glucose Accumulation

Glucose is the universal energy source and a critical substrate for lipid synthesis in mammalian cells. Analysis of both glucose and lipid in cells is important for the understanding of the regulation of lipid synthesis in many cell types, but especially adipocytes, the major storage cell for fat in mammals. The fluorescent 7-nitrobenz-2-oxa-1,3-diazole (NBD) derivative of glucose, 2-NBDG, is used to monitor glucose uptake and the lipid-selective fluorophore Nile red is used to monitor lipid accumulation. Previous reports have used NBD-based fluorophores and Nile red simultaneously despite the possibility of spectral overlap. In this study, we determined if these fluorophores were experimentally compatible in preadipocytes and adipocytes stained with 2-NBDG and Nile red separately or costained. We found that Nile red is detectable in the wavelengths necessary to excite and detect 2-NBDG. This interference was further increased by the solvatochromic effect of lipid-localized Nile red. In addition, we found a synergistic increase in fluorescent intensity when both fluorophores were present. Unfortunately, even fine control of the excitation or emission wavelengths did not identify wavelengths suitable for selective detection when cells were costained. Therefore, 2-NBDG and Nile red cannot be used simultaneously—but can likely be used sequentially—to assess glucose uptake and lipid accumulation in lipid-laden cells.

scholarly journals An investigation of glucose uptake in relation to steroidogenesis in rat testis and tumour Leydig cells

1988 ◽  
Vol 249 (3) ◽  
pp. 925-928 ◽  
Author(s):  
P Amrolia ◽  
M H Sullivan ◽  
D Garside ◽  
S A Baldwin ◽  
B A Cooke
Keyword(s):  
Luteinizing Hormone ◽  
Glucose Uptake ◽  
Mammalian Cells ◽  
Leydig Cells ◽  
Cytochalasin B ◽  
Cell Types ◽  
Facilitated Diffusion ◽  
Rat Testis ◽  
Diffusion Systems ◽  
Glucose Analogue

The mechanisms of the requirement of glucose for steroidogenesis were investigated by monitoring the uptake of the glucose analogue 2-deoxy-D-glucose by rat testis and tumour Leydig cells. The characteristics of glucose transport in both of these cell types were found to resemble those of the facilitated-diffusion systems for glucose found in most other mammalian cells. The Leydig cells took up 2-deoxy-D-glucose but not L-glucose, and the uptake was inhibited by both cytochalasin B and forskolin. In the presence of luteinizing hormone, the rate of 2-deoxy-D-glucose uptake by both cell types was increased by approx. 50%. In addition to D-glucose, it was shown that the Leydig cells could also utilize 3-hydroxybutyrate or glutamine to maintain steroidogenesis.

scholarly journals Dissection of stress-activated glucose transport from insulin-induced glucose transport in mammalian cells using wortmannin and ML-9

1995 ◽  
Vol 309 (3) ◽  
pp. 731-736 ◽  
Author(s):  
L F Barros ◽  
R B Marchant ◽  
S A Baldwin
Keyword(s):  
Glucose Uptake ◽  
Glucose Transport ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Metabolic Stress ◽  
Cell Types ◽  
Mitogen Activated Protein Kinase ◽  
Uptake Capacity ◽  
Mitogen Activated Protein ◽  
Stimulation Of

The signaling pathways responsible for the activation of glucose transport by insulin and by metabolic stress in mammalian cells were studied in Clone 9 cells and 3T3-L1 adipocytes. Exposure of both cell types to azide or insulin markedly increased their glucose uptake capacity (Vmax.) without affecting their apparent affinity for glucose (Km). The effects of azide and insulin were not additive. Wortmannin, a selective inhibitor of phosphatidylinositol (PI) 3-kinase, did not affect stimulation of transport by azide but inhibited insulin-induced glucose transport with a Ki of < 10 nM. ML-9, a putative mitogen-activated protein kinase inhibitor, was equipotent in its inhibition of azide- and insulin-stimulated glucose transport. These findings suggest that multiple signalling cascades are involved in the stimulation of glucose transport in mammalian cells and that PI 3-kinase, an essential link in the pathway by which insulin stimulates glucose transport, is not necessary for the activation of glucose uptake by metabolic stress.

Abstract 664: Effects of Eicosapentaenoic Acid Plus Docosapentaenoic Acid and Eicosapentaenoic Acid Alone on Fasting and Postprandial Monocyte Phenotypes

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Huaizhu Wu ◽  
Xiao-Yuan D Perrard ◽  
George Bobotas ◽  
Kevin C Maki ◽  
Christie M Ballantyne
Keyword(s):  
Fatty Acids ◽  
Eicosapentaenoic Acid ◽  
Lipid Accumulation ◽  
Nile Red ◽  
Docosapentaenoic Acid ◽  
Ethyl Esters ◽  
Omega 3 Fatty Acids ◽  
Omega 3 ◽  
Fluorescent Intensity ◽  
Men And Women

Background and objective: Fasting and postprandial (PP) hypertriglyceridemia is associated with increased lipid accumulation in circulating monocytes and monocyte phenotypic changes, e.g., increased expression of CD11c (a β2 integrin), which may increase risk for atherosclerosis. Omega-3 fatty acids lower circulating triglycerides (TG). This study examined whether treatment with omega-3 fatty acids improves fasting and PP monocyte phenotypes associated with hypertriglyceridemia. Methods: This was an open-label, randomized, crossover study of men and women with elevated TG (200-400 mg/dL) to evaluate the effects of MAT9001 (eicosapentaenoic acid [EPA] plus docosapentaenoic acid [DPA], Matinas BioPharma Inc., USA), compared to Vascepa (EPA-ethyl esters [EE] only, Amarin Pharma Inc., USA), administered at 4 g/d dosages for 15 d (≥28-d washout between treatments). Fasting and PP (4 and 6 h) TG and monocyte phenotypes (analyzed by flow cytometry) were examined prior to and on d 15 of each treatment. Results: MAT9001 and Vascepa lowered fasting TG from baseline (-32% and -19% respectively; both p≤0.04; n=26-29). Only MAT9001 significantly reduced PP TG area under the curve (AUC) from pre- to post-treatment (p<0.01). The reduction in TG AUC was greater for MAT9001 (-23%) vs. Vascepa (+4%) (p=0.01). Treatment with MAT9001 or Vascepa reduced fasting and PP CD11c mean fluorescent intensity (MFI) levels on classical (cM) and intermediate (iM) monocytes by 6.3-23.2% (no significant differences between treatments; Table). Only MAT9001 significantly reduced PP CD11c MFI on nonclassical monocytes (ncM) and lowered fasting nile red levels, indicating less lipid accumulation, in cM and iM (Table). Summary: Decreases in TG produced by MAT9001 or Vascepa in men and women with hypertriglyceridemia were associated with reduced fasting and PP CD11c on cM and iM. MAT9001, but not Vascepa, significantly reduced PP ncM CD11c and fasting cM and iM nile red staining for lipids.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

scholarly journals Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

2013 ◽  
Vol 79 (23) ◽  
pp. 7360-7370 ◽  
Author(s):  
John Seip ◽  
Raymond Jackson ◽  
Hongxian He ◽  
Quinn Zhu ◽  
Seung-Pyo Hong
Keyword(s):  
Yarrowia Lipolytica ◽  
Lipid Accumulation ◽  
Oleaginous Yeast ◽  
De Novo ◽  
Lipid Synthesis ◽  
Omega 3 ◽  
Wild Type ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Dry Cell Weight

ABSTRACTIn the oleaginous yeastYarrowia lipolytica,de novolipid synthesis and accumulation are induced under conditions of nitrogen limitation (or a high carbon-to-nitrogen ratio). The regulatory pathway responsible for this induction has not been identified. Here we report that the SNF1 pathway plays a key role in the transition from the growth phase to the oleaginous phase inY. lipolytica. Strains with aY. lipolyticasnf1(Ylsnf1) deletion accumulated fatty acids constitutively at levels up to 2.6-fold higher than those of the wild type. When introduced into aY. lipolyticastrain engineered to produce omega-3 eicosapentaenoic acid (EPA),Ylsnf1deletion led to a 52% increase in EPA titers (7.6% of dry cell weight) over the control. Other components of theY. lipolyticaSNF1 pathway were also identified, and their function in limiting fatty acid accumulation is suggested by gene deletion analyses. Deletion of the gene encoding YlSnf4, YlGal83, or YlSak1 significantly increased lipid accumulation in both growth and oleaginous phases compared to the wild type. Furthermore, microarray and quantitative reverse transcription-PCR (qRT-PCR) analyses of theYlsnf1mutant identified significantly differentially expressed genes duringde novolipid synthesis and accumulation inY. lipolytica. Gene ontology analysis found that these genes were highly enriched with genes involved in lipid metabolism. This work presents a new role for Snf1/AMP-activated protein kinase (AMPK) pathways in lipid accumulation in this oleaginous yeast.

scholarly journals EEA1, a Tethering Protein of the Early Sorting Endosome, Shows a Polarized Distribution in Hippocampal Neurons, Epithelial Cells, and Fibroblasts

2000 ◽  
Vol 11 (8) ◽  
pp. 2657-2671 ◽  
Author(s):  
Jean M. Wilson ◽  
Meltsje de Hoop ◽  
Natasha Zorzi ◽  
Ban-Hock Toh ◽  
Carlos G. Dotti ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mammalian Cells ◽  
Hippocampal Neurons ◽  
Cell Types ◽  
Early Endosomes ◽  
Filamentous Material ◽  
Endocytic Vesicles ◽  
Fibroblastic Cells ◽  
Darby Canine Kidney ◽  
Endocytic Pathways

EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of “basolateral-type” endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.

scholarly journals Divergent functions of endotrophin on different cell populations in adipose tissue

2016 ◽  
Vol 311 (6) ◽  
pp. E952-E963 ◽  
Author(s):  
Yueshui Zhao ◽  
Xue Gu ◽  
Ningyan Zhang ◽  
Mikhail G. Kolonin ◽  
Zhiqiang An ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Lipid Accumulation ◽  
Lysyl Oxidase ◽  
Stromal Vascular Fraction ◽  
Cell Types ◽  
Marker Genes ◽  
New Perspective ◽  
Collagen Genes ◽  
Different Cell Types

Endotrophin is a cleavage product of collagen 6 (Col6) in adipose tissue (AT). Previously, we demonstrated that endotrophin serves as a costimulator to trigger fibrosis and inflammation within the unhealthy AT milieu. However, how endotrophin affects lipid storage and breakdown in AT and how different cell types in AT respond to endotrophin stimulation remain unknown. In the current study, by using a doxycycline-inducible mouse model, we observed significant upregulation of adipogenic genes in the white AT (WAT) of endotrophin transgenic mice. We further showed that the mice exhibited inhibited lipolysis and accelerated hypertrophy and hyperplasia in WAT. To investigate the effects of endotrophin in vitro, we incubated different cell types from AT with conditioned medium from endotrophin-overexpressing 293T cells. We found that endotrophin activated multiple pathological pathways in different cell types. Particularly in 3T3-L1 adipocytes, endotrophin triggered a fibrotic program by upregulating collagen genes and promoted abnormal lipid accumulation by downregulating hormone-sensitive lipolysis gene and decreasing HSL phosphorylation levels. In macrophages isolated from WAT, endotrophin stimulated higher expression of the collagen-linking enzyme lysyl oxidase and M1 proinflammatory marker genes. In the stromal vascular fraction isolated from WAT, endotrophin induced upregulation of both profibrotic and proinflammatory genes. In conclusion, our study provides a new perspective on the effect of endotrophin in abnormal lipid accumulation and a mechanistic insight into the roles played by adipocytes and a variety of other cell types in AT in shaping the unhealthy microenvironment upon endotrophin treatment.

scholarly journals Channelling of intermediates in the biosynthesis of phosphatidylcholine and phosphatidylethanolamine in mammalian cells

1998 ◽  
Vol 334 (3) ◽  
pp. 511-517 ◽  
Author(s):  
Bellinda A. BLADERGROEN ◽  
Math J. H. GEELEN ◽  
A. Ch. Pulla REDDY ◽  
Peter E. DECLERCQ ◽  
Lambert M. G. VAN GOLDE
Keyword(s):  
Staphylococcus Aureus ◽  
Mammalian Cells ◽  
Glioma Cells ◽  
Rat Hepatocytes ◽  
Cell Types ◽  
General Phenomenon ◽  
Rat Glioma ◽  
Permeabilized Cells ◽  
Metabolic Compartmentation ◽  
Choline Incorporation

Previous studies with electropermeabilized cells have suggested the occurrence of metabolic compartmentation and Ca2+-dependent channeling of intermediates of phosphatidylcholine (PC) biosynthesis in C6 rat glioma cells. With a more accessible permeabilization technique, we investigated whether this is a more general phenomenon also occurring in other cell types and whether channeling is involved in phosphatidylethanolamine (PE) synthesis as well. C6 rat glioma cells, C3H10T½ fibroblasts and rat hepatocytes were permeabilized with Staphylococcus aureus α-toxin, and the incorporation of the radiolabelled precursors choline, phosphocholine (P-choline), ethanolamine and phosphoethanolamine (P-EA) into PC and PE were measured both at high and low Ca2+ concentrations. In glioma cells, permeabilization at high Ca2+ concentration did not affect [14C]choline or [14C]P-choline incorporation into PC. However, reduction of free Ca2+ in the medium from 1.8 mM to < 1 nM resulted in a dramatic increase in [14C]P-choline incorporation into permeabilized cells, whereas [14C]choline incorporation remained unaffected. Also, in fibroblasts, reduction of extracellular Ca2+ increased [14C]P-choline and [14C]P-EA incorporation into PC and PE respectively. In hepatocytes, a combination of α-toxin and low Ca2+ concentration severely impaired [14C]choline incorporation into PC. Therefore, α-toxin-permeabilized hepatocytes are not a good model in which to study channeling of intermediates in PC biosynthesis. In conclusion, our results indicate that channeling is involved in PC synthesis in glioma cells and fibroblasts. PE synthesis in fibroblasts is also at least partly dependent on channeling.

scholarly journals Nile Red Quantifier: a novel and quantitative tool to study lipid accumulation in patient-derived circulating monocytes using confocal microscopy

2017 ◽  
Vol 58 (11) ◽  
pp. 2210-2219 ◽  
Author(s):  
Johan G. Schnitzler ◽  
Sophie J. Bernelot Moens ◽  
Feiko Tiessens ◽  
Guido J. Bakker ◽  
Geesje M. Dallinga-Thie ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Lipid Accumulation ◽  
Nile Red ◽  
Quantitative Tool

The structural mechanics of cell division in Trypanosoma brucei

2008 ◽  
Vol 36 (3) ◽  
pp. 421-424 ◽  
Author(s):  
Sue Vaughan ◽  
Keith Gull
Keyword(s):  
Cell Division ◽  
Trypanosoma Brucei ◽  
Mammalian Cells ◽  
Reverse Genetics ◽  
Protozoan Parasite ◽  
Cell Types ◽  
Single Copy ◽  
Structural Mechanics ◽  
Sub Saharan Africa ◽  
Mammalian Host

Undoubtedly, there are fundamental processes driving the structural mechanics of cell division in eukaryotic organisms that have been conserved throughout evolution and are being revealed by studies on organisms such as yeast and mammalian cells. Precision of structural mechanics of cytokinesis is however probably no better illustrated than in the protozoa. A dramatic example of this is the protozoan parasite Trypanosoma brucei, a unicellular flagellated parasite that causes a devastating disease (African sleeping sickness) across Sub-Saharan Africa in both man and animals. As trypanosomes migrate between and within a mammalian host and the tsetse vector, there are periods of cell proliferation and cell differentiation involving at least five morphologically distinct cell types. Much of the existing cytoskeleton remains intact during these processes, necessitating a very precise temporal and spatial duplication and segregation of the many single-copy organelles. This structural precision is aiding progress in understanding these processes as we apply the excellent reverse genetics and post-genomic technologies available in this system. Here we outline our current understanding of some of the structural aspects of cell division in this fascinating organism.

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