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Tutorials : Aug 1, 2005 ( )
New Tool for Metabolic Disease Research
Novel Fluorescence Assay for Fatty Acid Uptake!--h2>
Defects in fatty acid metabolism have been linked to several pathological states, including insulin desensitization, Type 2 diabetes, obesity, and cardiovascular disease. Specifically, uptake of long chain fatty acids (LCFAs) plays an important role in the absorption of dietary lipids as well as the delivery of metabolic energy to a variety of tissues (Figure 1).
Recent findings have also directly implicated elevated intracellular levels of LCFAs in the obesity-associated insulin desensitization of skeletal muscle and liver. LCFA uptake in adipocytes is mainly facilitated by membrane proteins, particularly members of the recently discovered fatty acid transporter protein (FATPs/SLC27) family. Once LCFAs are bound by accessory proteins such as CD36, and then transported into the cell by the FATPs, they are acylated and subsequently bound by fatty acid binding proteins (FABPs) and acy-CoA binding protein (ACBP) to prevent rapid efflux.
The identification of this fatty acid transporter family and other fatty acid uptake enhancing proteins, such as CD36, has allowed a better understanding of the mechanisms and regulation of LCFA transport on a cellular level, yielding insight into the control of energy homeostasis and its dysregulation in diseases such as diabetes and obesity. In addition, these cell surface proteins represent new targets for the inhibition of LCFA uptake.
The development of fatty acid uptake regulators as potential drugs is dependent upon having a rapid assay that can be used in high throughput screening. Conventional assays using radioisotopes or flow cytometry are expensive and time-consuming and have limited the ability of drug researchers to identify potential new drugs via high throughput screening.
The ideal assay for LCFA transport would be homogeneous, nonradioactive, kinetic, and physiologically relevant (i.e., nontoxic and cell-based). Here, we report the development of a fluorescence assay based on the extracellular quenching of a fluorescent fatty acid analogue, which can be quantified in real time by fluorescent plate readers or standard fluorescent microscopy.
The QBT Fatty Acid Uptake Assay combines Molecular Devices' (Sunnyvale, CA) patented quench technology and cell-based assay expertise with a previously validated BODIPY-labeled fatty acid analog.
Using this assay, we measured differentiation- and hormone-induced changes in LCFA uptake by 3T3-L1 cells, a well-established model system for glucose and fatty acid uptake. Using the same assay in combination with an automated microscopy system, we were able to visualize and quantify cellular fatty acid uptake and accumulation in different subcellular localizations (data not shown).
Fatty acid transport into cells is facilitated by the class of proteins known as Fatty Acid Transport Proteins, or FATPs. A significant proportion of the transport occurs through this mechanism, especially in cells that are insulin-sensitive.
The QBT Fatty Acid Uptake assay has been used to measure LCFA uptake in the important 3T3-L1 model. Figure 2 shows the configuration and representative data of the QBT fatty acid uptake assay. In panel A, the fluorescent fatty acid analogue BODIPY-FA (green dots) and a quenching agent (red dots) are in contact with adipocytes that can actively take up the fluorescent fatty acid but not the quencher.
Only the BODIPY-FA that is transported into the cells is detected in the bottom-reading fluorescent plate reader; fluorescence in the solution is quenched by the quencher. Panel B of Figure 2 shows kinetic readings with a Molecular Devices' Flexstation plate reader of LCFA uptake by 3T3-L1 adipocytes (A), undifferentiated 3T3-L1 cells (B), and no cells (C).
Panel C of Figure 2 shows an insulin dose response curve of LCFA uptake in 3T3-L1 cells. Traces A to F correspond to adipocytes with 160, 16, 8, 1.6, 0.16, and 0 nM insulin, respectively; traces G and H correspond to fibroblasts with 160 and 0 nM insulin, respectively.
It is also possible to study fatty acid uptake in human adipocytes. In Figure 3, we show the results of using adipocytes from primary preadipocyte cultures.
In panel A, the QBT assay was carried out on human preadipocytes, or cohorts that were differentiated to adipocytes (HAd), in a 96-well plate. Panel B shows that insulin responsiveness can be assessed in this system. In panel C, a known inhibitor of LCFA uptake, 16-bromopalmitate, shows strong inhibition in human adipocytes at 125 M.
We have shown that the QBT Fatty Acid Uptake Assay works to measure fatty acid uptake in two well-defined and validated models of fatty acid metabolismdifferentiated 3T3-L1 cells and human adipocytes. The assay reproduces known biological changes in uptake in these systems.
Further, the fact that it is HTS-compatible and works in a human cell model system suggests that it is applicable to developing human therapeutic agents. Applications of this technology to other cell systems, such as skeletal muscle and liver cells, and to other transporter systems are currently being investigated.
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