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Fatty liver disease (FLD), also known as non-alcoholic steatohepatitis (NASH), recently came to light as a condition which afflicts an estimated 20 to 35% of the general USA adult population, 75% of the population that is obese, and 70% of patients with type II diabetes (Reddy and Rao 2006; Moscatiello et al., 2007; Neuschwander-Tetri 2007; Targher et al., 2007). A similar condition can be caused by excessive alcohol consumption. FLD is one of the top 3 causes of cirrhosis (American Liver Foundation website; http://www.liverfoundation.org/ education/info/fattyliver/), and is the third most common cause for chronic liver disease (Sears 2007). Excessive lipid droplet formation within hepatocytes is the defining feature of FLD and furthering our understanding of hepatic lipid droplets formation is thus a matter of great interest to the biomedical community.

AML12 cells: AML12 cells, derived from murine hepatocytes, retain many features of normal liver cells. To investigate the mechanisms of hepatic lipid droplet formation, AML12 cells were cultured in 96-well dishes, and incubated overnight with 200 μM oleic acid. Esterification of free fatty acids to Coenzyme A (CoA) is critical to the generation of triglycerides destined to be incorporated into lipid droplets (Figure 1) and this step is catalyzed by long-chain acyl synthetase (ACS). Triacsin C, an inhibitor of ACS, has recently been demonstrated to prevent lipid droplet formation in HuH7 hepatocytes (Fujimoto et al., 2006; 2007). To characterize the effect of triacsin C on AML12 cells, cells were incubated in the presence of triacsin C concentrations ranging from 0.01 to 5.4 μM concurrently with oleic acid. The cells were then fixed and stained for neutral lipids utilizing Vala’s Lipid Droplet Analysis kit reagents and imaged utilizing a Beckman-Coulter IC100 image cytometer. Cellular lipid droplet content was analyzed in an automated fashion by Vala Sciences’ CyteSeer® image analysis software.

Hepatic lipid droplet metabolism.

Cells were cultured in 96-well dishes with 200 µM OA and/or TC overnight. Cells were then stained, imaged (20X objective), and analyzed in an automated fashion. A, B, and C show OA cells visualized for nuclei (blue), lipid (green), and the CyteSeer™ -derived lipid mask (red). D, E, and F, show cells treated with OA + 5.7 µM TC. Images in B and E were optimized equally for display. G, Lipid droplets/cell (TC in µM). H, Ratio of lipid mask to cell area. Z’s were calculated between columns 1 vs. 2 for G and H. Each bar is the mean ± SD for n=8 wells.

While lipid droplets were relatively uncommon in control cells (not shown), cells exposed to oleic acid displayed prominent lipid droplets, particularly in the perinuclear regions, and the lipid droplets were very well recognized by CyteSeer (Figure 2A-C). The effect of oleic acid on cellular lipid droplet formation was quantified with high fidelity by CyteSeer. For example, the number of lipid droplets per cell (Figure 2G) was increased by 4-fold, with a Z’ value (Zhang et al., 1999), of 0.74. The ratio of lipid droplet area to cell size was increased by an even greater extent (>35-fold) with a Z’ value of 0.86. The very high Z’ values indicate that the model system is highly suitable for high throughput screening. Triacsin C inhibited lipid droplet formation with an IC50 of approx. 1 μM (Figure 2 D- H) which is consistent with expectations for this compound (Fujimoto et al., 2007).

We have also utilized CyteSeer to quantify lipid droplets in primary hepatocytes. Primary rat hepatocytes, isolated by perfusing an adult rat liver, were cultured for 2 days on a 96-well dish. The cells were then treated overnight with 200 µM oleic acid. Oleic acid elicited a large increase in droplet formation (Figure 3A and 3B) and an overall 8-fold increase in cell lipid staining intensity compared to controls (Figure 3E). The cells were also labeled for ADFP and ADFP was observed in close proximity to the droplets (Figure 3B and 3D). Thus, primary hepatocytes can be cultured in a high content screening format and can serve as an excellent model system for oleic acid-induced lipid droplet formation and for visualization of ADFP.

A, B Control cells. C, D OA-treated cells. E. Effect of OA on total lipid intensity per cell. Bars are mean ± SD for n=3 wells for control, or mean ± range for n=2 wells for OA cells.

Overall the results illustrate that Vala Science’s lipid staining reagents and CyteSeer® can be used to identify and characterize the ability of compounds to modify hepatic lipid droplet formation, a phenomenon critically relevant to FLD. Also, primary human hepatocytes can be purchased from Zen-Bio

HeLa cells: Vala’s lipid droplet staining reagents and CyteSeer®’s Lipid Droplet Analysis algorithm have also proven to be highly useful in quantifying lipid droplets in other cell types, such as HeLa. For HeLa cells exposed to oleic acid, the intensity of the lipid staining increased strongly over a 24 hr period (Figure 4A-C); furthermore, rapamycin, an inhibitor of the mTOR pathway, proved to have an inhibitory effect on droplet formation (Figure 4D).

THP-1 cells: Coronary artery disease is caused by cholesterol transport into lipid droplets in macrophage cells at sites of injury in the arterial wall; excess lipid droplets leads to formation of the “foam cells”, cell death and deposition of cholesterol-containing plaques. THP-1 cells are a widely used monocyte-derived cell line that differentiates into macrophages on exposure to phorbol esters. To determine if CyteSeer® could accurately quantify lipid droplets in this model system, THP-1 cells were exposed to oleic acid and fixed and stained for lipid droplets.

    Lipid droplets formed in THP-1 in response to oleic acid in a dose-dependent manner (Figure 5A), and CyteSeer® very accurately identified the corresponded to lipid droplets (Figure 5A). Lipid droplets/cell and the ratio of intensities between the lipid droplets and neighboring cytoplasm (Figures 5B and 5C) strongly increased on exposure to oleic acid and these responses were quantified with excellent reproducibility by CyteSeer, yielding Z’ scores > 0.50. Thus, lipid staining reagents from Vala and Vala’s CyteSeer image analysis software can be utilized to quantify lipid droplets in macrophage cells.

A) HeLa cells stained for lipids (green) and nuclei (blue) following 24 hr exposure to 150 μM oleic acid. B) Mask (red) derived for the lipid channel by CyteSeer®. C) Time course of lipid accumulation in HeLa. Y-axis is the median difference in intensity between the lipid droplets and the cytoplasmic region. D) Inhibition of lipid droplet formation in HeLa cells by rapamycin. Each bar is the mean SD for n=16 wells. OA, oleic acid, μM; RAP, rapamycin, nM. * p < .05 vs. OA by itself, Student Newman Keuls analysis.

THP-1 cells were differentiated to macrophages by exposure to 100 nM PMA for 3 days. The cells were then exposed to the indicated concentrations of oleic acid (OA) overnight, then fixed and stained for neutral lipids. A, Left panels, images obtained from THP-1 cells stained for nuclei (blue) and for lipid (green); right panels, lipid masks (green) calculated by CyteSeer® for the images in the left panels. B, Quantification of lipid droplets per cell. C, Quantification of the ratio of staining intensities for the lipid droplets vs. the neighboring cytoplasm.

    As the above data demonstrate, Vala Sciences lipid droplet staining reagents and CyteSeer® software provide tools for investigating the mechanisms that control lipid droplet formation in cell contexts relevant to fatty liver disease, and are likely to be of great interest to biomedical researchers interested in performing quantitative analysis via high content microscopy-based techniques. Please contact us for additional information.

Fujimoto, Y., Onoduka, J., Homma, K. J., Yamaguichi, S., Mori, M., Higashi, Y., Makita, M., Kinoshita, T., Noda, J-I., Itabe, H., Takano, T. 2006. Long-chain fatty acids induce lipid droplets formation in a cultured human hepatocyte in a manner dependent of acyl-CoA synthetase. Biol. Pharm. Bull. 29:2174-2180.

Fujimoto, Y., Itabe, H., Kinoshita, T., Homma, K. J., Onoduka, et al. 2007. Involvement of ACSL in local synthesis of neutral lipids in cytoplasmic lipid droplets in human hepatocyte HuH7. J. Lipid Res. 48:1280-1292.

Moscatiello, S., Manini, R., Marchesini, G. 2007. Diabetes and liver disease: an ominous association. Nutrition, Metabolism & Cardiovascular Diseases 17:63-70.

Neuschwander-Tetri, B. A. 2007. Fatty liver and the metabolic syndrome. 2007. Current Opin.Gastroenterol. 23:193-198.

Reddy, J. K., Rao, M. S. 2006. Lipid metabolism and liver inflammation. II. Fatty liver disease and fatty acid oxidation. Am. J. Physiol. Gastrointest. Liver Physiol. 290:G852-G858.

Targher, G., Bertolini, L., Padovani, R., Rodella, S., Tessari, R., et al. 2007. Prevalence of nonalcoholic fatty liver disease and its association with cardiovascular disease among type 2 diabetic patients. Diabetes Care (published on line, Feb. 2, 2007).

Sears, Dawn, 2007. Fatty Liver. eMedicine, from WebMD (published on line, June 6, 2007)

Zhang, J. –H., Chung, T. D. Y., Oldenburg, K. R. 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomolecular Screening 4:67-73.

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