Abstract 31: A Novel Fluorescence-Based Assay is Used to Investigate the Triglyceride Hydrolytic Activity of Lipases and to Identify Synthetic Peptides With Strong Lipase-Stimulating Activities
Hypertriglyceridemia is estimated to affect about one third of Americans and is an independent risk factor for cardiovascular disease, pancreatitis and xanthomatosis. Lipoprotein lipase (LPL) is a key enzyme involved in the hydrolysis and clearance of triglycerides from plasma and its enzymatic activity is regulated by specific apolipoproteins; apoC-III, which acts as a potent inhibitor and apoC-II and apoA-V, which act as stimulators. Their importance in hypertriglyceridemia is underscored by GWAS analyses, which have identified the APOA1/C3/A4/A5 gene cluster (chromosome 11q23) as an important determinant of dylipidemia. We have developed a fluorescence-based lipase assay that utilizes a fluorogenic triglyceride analog (EnzChek®, Molecular Probes) and facilitates the rapid analysis of lipase enzyme kinetics in a microplate format. This assay has made it possible for us to undertake the screening of the Prestwick (1,200 FDA-approved drugs) and Natural Product (~3,000) libraries at the University of Illinois high-throughput screening facility, in order to identify lipase activating compounds. To validate the assay we have characterized the influence of heparin, different metals and apolipoproteins on the hydrolytic activities of LPL in vitro, and hepatic lipase in HepG2 cell culture. As expected, enzyme activities were supported by heparin and calcium, and both apoC-II and apoC-III when complexed with VLDL, stimulated and inhibited lipase activity, respectively. Interestingly, the heparin/Fe2+ complex was a strong inhibitor of lipase activity, possibly explaining the high incidence of hypertriglyceridemia associated with iron supplementation to treat iron-deficiency. Finally, using a synthetic peptide approach we have localized apoA-V’s lipase stimulating and heparin binding domains to a short amphipathic, alpha-helical peptide. When included in the assays this peptide could increase lipase hydrolytic rates by 3-6 fold both in vitro, and in cell culture. Given that on a molar basis apoA-V is about 1200-fold less abundant than apoC-II, we anticipate that apoA-V will provide valuable sequence information for the development of potent triglyceride lowering therapeutics.
Author Disclosures: J.D. Janizek: None. K. Munro: None. M.H. Davidson: None. J.B. Ancsin: None.
- © 2014 by American Heart Association, Inc.