Abstract
Lipases constitute an important class of water-soluble enzymes that catalyze the hydrolysis of hydrophobic triacylglycerol (TAG). Their enzymatic activity is typically measured using multistep procedures involving isolation and quantification of the hydrolyzed products. We report here a new fluorescence method to measure lipase activity in real time that does not require the separation of substrates from products. We developed this method using adipose triglyceride lipase (ATGL) and lipoprotein lipase (LpL) as model lipases. We first incubated a source of ATGL or LpL with substrate vesicles containing nitrobenzoxadiazole (NBD)-labeled TAG, then measured increases in NBD fluorescence, and calculated enzyme activities. Incorporation of NBD-TAG into phosphatidylcholine (PC) vesicles resulted in some hydrolysis; however, incorporation of phosphatidylinositol into these NBD-TAG/PC vesicles and increasing the ratio of NBD-TAG to PC greatly enhanced substrate hydrolysis. This assay was also useful in measuring the activity of pancreatic lipase and hormone-sensitive lipase. Next, we tested several small-molecule lipase inhibitors and found that orlistat inhibits all lipases, indicating that it is a pan-lipase inhibitor. In short, we describe a simple, rapid, fluorescence-based triacylglycerol hydrolysis assay to assess four major TAG hydrolases: intracellular ATGL and hormone-sensitive lipase, LpL localized at the extracellular endothelium, and pancreatic lipase present in the intestinal lumen. The major advantages of this method are its speed, simplicity, and elimination of product isolation. This assay is potentially applicable to a wide range of lipases, is amenable to high-throughput screening to discover novel modulators of triacylglycerol hydrolases, and can be used for diagnostic purposes.
Highlights
Supplementary key words lipids triacylglycerol adipose triglyceride lipase (ATGL) enzyme activity NBD-labeled TAG (NBD-TAG) lipoprotein lipase lipolysis orlistat hormone-sensitive lipase high-throughput screenin
We used well-described methods of separating TAGs from free fatty acid (FFA) based on differential solubility in polar and nonpolar solvents [52] that were previously used in radiolabel assays to measure ATGL activity [25]
We studied the effect of different inhibitors on lipoprotein lipase (LpL) activity and found that atglistatin had no effect, but orlistat and hormone-sensitive lipase inhibitor (HSLi) (SC206238) potently inhibited LpL activity (Fig. 7D)
Summary
Substrate vesicles (10 μl, 880 pmol of NBD-TAG) were incubated with 100 μl of buffer K containing different amounts of enzyme source. To measure the inhibition of PTL activity, pancreatic tissue homogenates with 80 ng of colipase in solution were incubated with NBD-TAG vesicles in the presence and absence of the three inhibitors. To measure ATGL activity, we incubated different amounts of cell lysates with fixed amounts of NBD-TAG-containing PC vesicles (prepared using a 1:6 M ratio of NBD-TAG to PC; Fig. 2B) and extracted FFAs after 1 h.
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