Quantify Lipid Droplets and Associated Proteins in Human Adipocytes
In 2002, Surgeon General David Satcher stated that “...obesity may soon cause as much preventable disease and death as cigarette smoking” (FDA website: http://www.fda.gov/fdac/features /2002/202_fat.html ). Today, there is indeed an alarming world-wide trend of increased body weight; approximately 30% of the adults in the US are classified as obese (body mass index 30) (Popkin and Gordon-Larsen 2004). Obesity increases the energetic costs of motility and body maintenance and can lead to heart failure and other cardiovascular problems (Lawrence and Kopelman 2004). Increased body weight is typically due to increased number and size of the triglyceride-storing adipocytes (fat cells). Reduced fat deposition can be a clinical problem; HIV patients treated with antiretroviral therapies often experience a redistribution of body fat (diminished subcutaneous fat, and sometimes, increased visceral fat), termed “lypodystrophy syndrome” (Penzak and Chuck 2002) due to certain HIV-specific protease inhibitors which suppress preadipocyte differentiation and promote adipocyte apoptosis (Dowell et al., 2000). Thus, adipogenesis (the formation and differentiation of adipocytes) is of great interest to the biomedical and pharmaceutical community. Vala Sciences Inc,. which specializes in the development of automated cell-based assays, and Zen-Bio, a company that isolates and cultures human adipocytes harvested from liposuction procedures, are excited to announce a new line of reagent kits and software for automated quantification of lipid droplets and associated proteins in human adipocytes. The software and reagent kits will enable high throughput screening of candidate pharmaceuticals for anti- or pro-adipogenic effects.
Figure 1
Lipid droplets in human adipocytes.
Lipids in Adipogenesis
To begin developing our methodology, human preadipocytes (obtained from Zen-Bio) were plated in 96-well, glass bottomed plates, then cultured for 14 days in the presence of rosiglitazone, a PPARγ agonist which promotes adipogenesis. The cells were then fixed and stained for lipids with a lipid-specific green channel fluorescent probe (Figure 1). Rosiglitazone elicited a large increase in the number of cells that expressed lipid droplets which were similar to “a cluster of grapes” within each cell. To quantify the number and size of the lipid droplets in these images, a proprietary algorithm (patent pending) was developed that segments circular staining patterns (Figure 2). The algorithm was then applied to images obtained from the lipid optical channel for cells exposed to different doses of rosiglitazone (Figure 3). The number of lipid droplets, as quantified by the algorithm, increased in a dose-dependent manner, with a maximal effect (6-fold increase) obtained with 1 μM rosiglitazone. The Z’ value for this assay was 0.47, indicating that the assay is robust (Zhang et al., 1999). Rosiglitazone increased the size of the lipid droplets as well. Incubation with 1 μM rosiglitazone increased the frequency of droplets > 21 μm2 area by 2-fold. The dose-response relationship for rosiglitazone vs. lipid droplet number was very similar to the dose-response relationship obtained for cellular triglyceride content, as determined by a traditional colormetric assay (Figure 3B). The adipogenesis-promoting effect of rosiglitazone was thus fully defined by the use of high throughput microscopy techniques and Vala Sciences CyteSeer® Lipid Droplet Analysis software.
Figure 2. Identification of lipid droplets.
Human adipocytes exposed to 100 nM rosi. A) original image. B) Binary mask of A to automatically quantify droplet number and size.
PAT Proteins In Adipogenesis
The role of proteins that associate with lipid droplets during adipogenesis and lipolysis is of great interest. Lipid droplets are contained in a phospholipid monolayer and a protein coat. PAT proteins (named after the founding members of the family: Perilipin, Adipophilin, and TIP47) associate with lipid droplets at different stages of their formation and maturation (Hickenbottom et al., 2004; Wolins et al., 2005; Robenek et al., 2005). It is likely that these proteins help package the triacylglycerol into the droplets (Figure 4). At early stages, the droplets are coated with adipophilin; however, during maturation, perilipin displaces adipophilin (Tansey et al., 2004). Lipolysis in adipocytes is mediated by at least two mechanisms. β-adrenergic receptor stimulation increases cAMP and activates protein kinase A (PKA). PKA-mediated phosphorylation of perilipin recruits hormone sensitive lipase (HSL) to the droplets (Sztalryd et al., 2003), leading to lipolysis. Lipolysis is also likely mediated by adipose triglyceride lipase (ATGL) a recently discovered lipase that is highly expressed in adipose tissue (Zimmermann et al., 2004). At present, the mechanisms that regulate ATGL expression and activity have not been determined. Thus, protein translocation to the lipid droplets occurs during adipogenesis (adipophilin and perilipin) and during lypolysis (HSL and likely ATGL).
Figure 3. The effects of rosiglitazone on lipid droplet number, size, and triglyceride content in human adipocytes.
Human preadipocytes were cultured in a 96-well dish and exposed to the indicated concentration of rosiglitazone for 14 days. The cells were then fixed and imaged for lipid droplets and nuclei utilizing a digital microscopy workstation equivalent to a Beckman-Coulter IC 100 (40X objective), with 4 images acquired per well. Cells cultured in an identical fashion were also analyzed for triglyceride content via use of bacterial lipase and a colormetric assay for glycerol. A) The dose-response relationship between rosiglitazone and lipid droplet number as quantified with Vala Sciences proprietary software. B) The dose-response relationship for triglyceride content. For A and B, each symbol represents the mean ± SD for n=4 wells.
Figure 4. Lipogenesis and lipolysis in adipocytes.
During early stages of droplet formation, the droplets are thought to be coated with adipophilin. As the droplets mature, perilipin becomes associated with the droplets. Perilipin helps attract HSL (hormone-sensitive lipase) to the droplets during cAMP- mediated increases in lipolysis. Adipocyte triglyceride lipase (ATGL) has also recently been identified as participating in lipolysis. BAR, beta adrenergic receptor.
Figure 5. Staining of human adipocytes for lipid droplet and associated proteins.
A), adipocytes that have been stimulated to promote adipogenesis by exposure to a PPARγ agonist visualized for lipid (green). B), corresponding field visualized for perilipin (red) and nuclei (blue). Note that every lipid droplet in A is coated by perilipin, resulting in a “perilipin ring” staining pattern in B. C), adipocytes maintained in the absence of the PPARγ agonist display fewer lipid droplets. D), the corresponding field stained for perilipin and nuclei. Note that the lipid droplet identified by an arrow in C and D is coated with perilipin whereas neighboring droplets are not perilipin coated.
Why High Content Microscopy?
Aside from electron microscopy, microscopy-based cell imaging is the only methodology for detecting the colocalization of proteins with lipid droplets, as the proteins and droplets can be visualized in the same field of view utilizing different optical channels. Accordingly, human adipocytes were cultured under maintenance or adipogenesis-inducing conditions and visualized for lipid droplets and perilipin. Adipocytes cultured with PPARγ agonists displayed numerous lipid droplets and the majority of the droplets were also associated with prominent staining for perilipin (Figure 5A and 5B). This was typically manifest as a “perilipin ring” around each lipid droplet. In contrast, for cells cultured in the absence of the PPARγ agonist, fewer lipid droplets were detected; furthermore, while most of the droplets were coated with perilipin, many cells expressed small lipid droplets that were not coated with perilipin (Figure 5C and 5D). Without the ability to see the colocalization of perilipin and lipid droplets, the information could not have been obtained.
Figure 6. Screen shots from CyteSeer’s Image Viewer.
The background image shows a field of view depicting rosiglitazone-treated human adipocytes stained for lipid droplets and for nuclei; the inset shows the lipid droplet mask calculated by CyteSeer from the images. White arrows identify the same cell in the background and inset images.
Vala Sciences’ CyteSeer
Software engineers at Vala Sciences have incorporated the Lipid Droplet Analysis and Perilipin Analysis algorithms into our Windows-compatible CyteSeer® Image Cytometry Software platform, which also features our previously developed Membrane Activation algorithm for analysis of membrane associated proteins including Protein Kinase C, N-Cadherin, E-Cadherin, and VE-cadherin (Figure 6). CyteSeer® can be used with images collected from 96-well dishes (and higher formats) with high content digital microscopy workstations, and can also import and analyze individual images obtained by researchers acquiring images with standard fluorescence microscopes and digital cameras. CyteSeer® features a convenient user-interface and performs a sophisticated suite of measurements on each cell in the field, providing the user with a set of unprecedented tools to help interpret the effects of test compounds on the adipogenesis pathway. The Lipid Droplet Analysis algorithm currently calculates the mean and standard deviation for an impressive 37 separate metrics for each cell in the field of view; the perilipin algorithm performs the same calculations for the lipid droplets, and, calculates 48 additional measurements specific for perilipin and lipid droplet:perilipin colocalization (examples include adaptations of the Pearsons’ and Mander’s coefficients of colocalization – Manders et al., 1993).
Results
To further demonstrate the quantification of lipid droplets and perilipin expression and localization afforded by CyteSeer®, human preadipocytes obtained from Zen-Bio were exposed to different doses of rosiglitazone for 9 days, a time point at which there are relatively few small droplets as it is early in the adipogenesis differentiation process. The cells were stained for lipid droplets and perilipin and visualized using a Beckman IC 100 high content microscopy workstation utilizing a 10X objective. Data parameters from these images calculated by CyteSeer® were then graphed vs. rosiglitazone concentration (Figure 7). Exposure to rosiglitazone strongly increased the number of cells containing lipid droplets, the number of droplets per cell, the average diameter of the lipid droplets, the average intensity of the droplets and the area of the cell corresponding to perilipin staining (Figure 7A-E). Also, for the parameters listed, the Z’ values were all > 0.5, indicating that the assay is very suitable for high throughput screening applications. CyteSeer® precisely quantified the rosiglitazone-induced increases in lipid droplet number and size; furthermore, the results document, for perhaps the first time, the sensitivity of perilipin expression to PPARγ agonists in human preadipocytes.
Figure 7. Quantification of lipid droplets and perilipin in human adipcytes by CyteSeer.
Human preadipocytes were treated with the indicated concentrations of rosiglitazone for 9 days, then visualized for lipid droplets and perilipin using Vala Sciences reagents. A, The percentage of cells that expressed lipid droplets. B, The average number of lipid droplets per cell. C, Estimated average lipid droplet diameter . D, Average intensity of the lipid droplets. E, Perilipin Mask area per cell. Each bar is the mean ± SD
Conclusions
The above data demonstrate that Vala Sciences lipid droplet staining reagents and CyteSeer® software provide tools for investigating the mechanisms that control lipid droplet formation in adipogenesis and is of great interest to biomedical researchers interested in performing quantitative analysis via high content microscopy-based techniques. Contact us if you are interested in learning more.
References
Dowell, P., Flexner, C., Kwiterovich, P. O., Lane, M. D. 2000. Suppression of preadipocyte differentiation and promotion of adipocyte death by HIV protease inhibitors. J. Biol. Chem. 275:41325-41332.
Hickenbottom, S. J., Kimmel, a. R., Londos, C., Hurley, J. H. 2004. Structure of a lipid droplet protein: the PAT family member TIP47. Structure 12:1199-1207.
Lawrence, V. J., Kopelman, P. G. 2004. Medical consequences of obesity. Clinics in Dermatology 22:296-302.
Manders, E. M., Verbeek, F. J., Afen, J. A. 1993. Measurement of colocalisation of objects in dual colour confocal microscopy. J. Microscopy 169:375-382
Penzak, S. R., Chuck, S. K. 2002. Management of protease inhibitor-associated hyperlipidemia. Am. J. Cardiovasc. Drugs 2:91-106.
Popkin, B. M., Gordon-Larsen, P. 2004. The nutrition transition: worldwide obesity dynamics and their determinants. 28:S2-S9.
Robenek, H., Robenek, M. J., Troyer, D. 2005. PAT family proteins pervade lipid droplet cores. J Lipid Res.46:1331-1338.
Sztalryd, C., Xu, G., Dorward, H., Tansey, J. T. et al., 2003. Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation. J. Cell Biol. 161:1093-1103.
Tansey, J. T., Sztalryd, C., Hlavin, E. M., Kimmel, A. R., Londos, C. 2004. The central role of perilipin A in lipid metabolism and adipocyte lipolysis. IUBMB Life 56:379-385.
Wolins, N. E., Quaynor, B. K., Skinner, J. R., et al. 2005. S3-12, adipophilin, and TIP47 package lipid in adipocytes. J. Biol. Chem. 280:19146-19155.
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.
Zimmermann, R., Strauss, J. G., Haemmerle, G., et al., 2004. Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase. Science 306:1383-1386.
Vala Sciences Contact Information
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