Quantification of Lipid Droplets and Associated Proteins in Cellular Models of Obesity via High-Content/High-Throughput Microscopy and Automated Image Analysis

McDonough, Patrick M.; Agustin, Ramses M.; Ingermanson, Randall; Loy, Patricia A.; Buehrer, Benjamin M.; Nicoll, James B.; Prigozhina, Natalie L.; Mikić, Ivana Medvedec; Price, Jeffrey H.

doi:10.1089/adt.2009.0196

Cited by 44 publications

(59 citation statements)

References 97 publications

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“…DHA (30 M) treatment resulted in a more prominent increase in oil red O staining (Figure 5B) as expected [47]. For comparison, extensive basal lipid droplet staining was detected in HepG2 cells which have prominent lipid storage [41]. Except for the slight decrease in cell numbers, HUVECs exposed to elevated glucose were indistinguishable from cells grown in normal medium, and with the exception of slightly increased GLUT10 levels, their GLUT transporter mRNA levels were comparable to those in control cells ( Figure 5C).…”

Section: The Effect Of Elevated Glucose Levels On Glut Transporter Exsupporting

confidence: 52%

Transendothelial glucose transport is not restricted by extracellular hyperglycaemia

Tumova

Kerimi

Porter

et al. 2016

Vascular Pharmacology

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Endothelial cells are routinely exposed to elevated glucose concentrations post-prandially in healthy individuals and permanently in patients with metabolic syndrome and diabetes, and so we assessed their sugar transport capabilities in response to high glucose. In human umbilical vein (HUVEC), saphenous vein, microdermal vessels and aorta, GLUT1 (SLC2A1), GLUT3 (SLC2A3), GLUT6 (SLC2A6), and in microdermal vessels also GLUT12 (SLC2A12), were the main glucose transporters as assessed by mRNA, with no fructose transporters nor SGLT1 (SLC5A1). Uptake of 14 C-fructose was negligible. GLUT1 and GLUT3 proteins were detected in all cell types and were responsible for ~60% glucose uptake in HUVECs, where both GLUT1 and GLUT3, but not GLUT6 siRNA knock-down, reduced the transport. Under shear conditions, GLUT1 protein decreased, GLUT3 increased, and 14 C-deoxy-glucose uptake was attenuated. In high glucose, lipid storage was increased, cell numbers were lower, 14 C-deoxyglucose uptake decreased owing to attenuated GLUT3 protein and less surface GLUT1, and transendothelial transport of glucose increased due to cell layer permeability changes. We conclude that glucose transport by endothelial cells is relatively resistant to effects of elevated glucose. Cells would continue to supply it to the underlying tissues at a rate proportional to the blood glucose concentration, independent of insulin or fructose.

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Section: The Effect Of Elevated Glucose Levels On Glut Transporter Exsupporting

confidence: 52%

Transendothelial glucose transport is not restricted by extracellular hyperglycaemia

Tumova

Kerimi

Porter

et al. 2016

Vascular Pharmacology

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“…Accordingly, high-content imagining techniques could be developed to specifically screen for additional chemical material that promotes similar effects on the NS5A distribution. Indeed, such analyses were previously employed to examine the behaviors of LDs in response to various stimuli (15,41,65).…”

Section: Discussionmentioning

confidence: 99%

Small Molecules Targeting Hepatitis C Virus-Encoded NS5A Cause Subcellular Redistribution of Their Target: Insights into Compound Modes of Action

Targett-Adams

Graham

Middleton

et al. 2011

J Virol

117

121

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Hepatitis C virus (HCV) is a global health concern; recent estimates suggest that 2.2 to 3% of the world's population, equivalent to 130 to 170 million individuals, are chronically infected with the virus (13, 31). These patients are at risk of developing debilitating liver diseases such as cirrhosis and hepatocellular carcinoma (1). Furthermore, current models suggest that the burden of HCV-associated disease is set to rise for the next 20 years (6). There is no HCV vaccine; the current standard of care (SOC) involves lengthy treatments with ribavirin and injected pegylated interferon, which exhibit variable efficacies and are associated with severe, and sometimes lifethreatening, side effects. Encouragingly, many direct-acting antiviral (DAA) molecules are in clinical development, and the most advanced (telaprevir and boceprevir) will probably be used to treat HCV-infected patients in 2011 (19,29,42,43,61). However, caution should be employed against overoptimism; attrition rates are high during drug development, and the first drugs will be given in combination with, not instead of, the current SOC. Therefore, the continued development of additional treatments is needed, especially since it is widely acknowledged that to limit the emergence of drug-resistant viral variants, effective therapeutic strategies for HCV will consist of multiple DAAs (50).A multitude of screening campaigns has revealed many diverse and interesting chemical compounds capable of specifically inhibiting HCV RNA replication. Many of these compounds target the HCV-encoded nonstructural (NS) proteins (NS3, NS4A, NS4B, NS5A, and NS5B), which are required for HCV genome synthesis (3, 37). To instigate HCV genome replication, the NS proteins interact with viral genomes and certain host-encoded factors to form multiprotein assemblies termed "replication complexes" (RCs), which are sites of viral RNA synthesis derived from the endoplasmic reticulum (ER) (8,14,45,53). In HCV-infected cells, RCs are juxtaposed to intracellular lipid storage organelles termed lipid droplets (LDs), which are coated with the HCV capsid protein (core) and probably serve as platforms to accept replicated genomes from RCs to initiate virion assembly (26,44,53). Of considerable interest are inhibitors that target the HCV-encoded NS5A protein. These inhibitors were originally discovered from the screening of cells containing HCV subgenomic replicons against libraries of small molecules and were identified as NS5A inhibitors by utilizing a strategy termed "chemical genetics" (12, 32). NS5A-targeting inhibitors are notable for their unprecedented potency in cell-based HCV replication assays: 50% inhibitory concentrations (IC 50 s) in the low-picomolar

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“…29 To analyze the effects of miRNAs on LDs, Vala Sciences Lipid Droplet staining kit components and image analysis software (CyteSeer ® ) were used that have been validated on a variety of adipocyte and hepatocyte cell models. 26 Images were acquired from the cells using a 20× objective, and the images were analyzed using the Lipid Droplet Algorithm contained within Vala's CyteSeer ® cell image analysis program. The data parameter used for this study was Tii Li Lm (total integrated intensity of the lipid image, integrated over pixels that correspond to the lipid mask), which is an estimate of the total cellular LD content.…”

Section: Validation Of the Hca Assay For Lds In Huh-7 Cellsmentioning

confidence: 99%

Identification of MicroRNAs That Control Lipid Droplet Formation and Growth in Hepatocytes via High-Content Screening

et al. 2010

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Hepatic lipid droplets (LDs) are associated with metabolic syndrome, type 2 diabetes, hepatitis C, and both alcoholic and nonalcoholic fatty liver disease. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the level of translation. Approximately 1000 different miRNA species are encoded within the human genome, and many are differentially expressed by healthy and diseased liver. However, few studies have investigated the role of miRNAs in regulating LD expression. Accordingly, a high-content assay (HCA) was performed in which human hepatocytes (Huh-7 cells) were transiently transfected with 327 unique human miRNAs; the cells were then fixed, labeled for nuclei and lipid droplets, and imaged with an automated digital microscopy workstation. LD expression was analyzed on a cell-by-cell basis, using automated image analysis. Eleven miRNAs were identified that altered LDs. MiR-181d was the most efficacious inhibitor, decreasing LDs by about 60%. miRNA-181d was also confirmed to reduce cellular triglycerides and cholesterol ester via biochemical assays. Furthermore, a series of proteins was identified via miRNA target analysis, and siRNAs directed against many of these proteins also modified LDs. Thus, HCA-based screening identified novel miRNA and protein regulators of LDs and cholesterol metabolism that may be relevant to hepatic diseases arising from obesity and alcohol abuse. (Journal of Biomolecular Screening 2010:798-805)

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Quantification of Lipid Droplets and Associated Proteins in Cellular Models of Obesity via High-Content/High-Throughput Microscopy and Automated Image Analysis

Cited by 44 publications

References 97 publications

Transendothelial glucose transport is not restricted by extracellular hyperglycaemia

Transendothelial glucose transport is not restricted by extracellular hyperglycaemia

Small Molecules Targeting Hepatitis C Virus-Encoded NS5A Cause Subcellular Redistribution of Their Target: Insights into Compound Modes of Action

Identification of MicroRNAs That Control Lipid Droplet Formation and Growth in Hepatocytes via High-Content Screening

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