Lipoprotein lipase (LPL) and hepatic lipase (HL) are important enzymes in lipoprotein metabolism and in the metabolism of nutrients absorbed in gastrointestinal tract. LPL hydrolyses chylomicron and very low density lipoprotein triacylglycerols to regulate the production of low density lipoprotein (LDL) and high density lipoprotein (HDL). The fatty acids liberated are available to tissues as sources of metabolic energy or for re-esteri-fication and storage. HL catalyzes the hydrolysis of tri-, di-, and monoacylglycerol, acyl-CoA thioesters, and phospholipids. It may be important in the production of HDK3 from HDL2, and possibly in the generation of LDL from intermediate density lipoprotein. Familial LPL deficiency is an autosomal recessive disorder, which, in the homozygous state, manifests as hyperchylomicronemia and pancreatitis, often accompanied by hepatosplenomegaly. We have cloned the cDNAs for human and mouse LPL and human and rat HL. In this application, we propose to study the structural and functional basis of LPL and HL action. (i) We will study the regulation of LPL expression in the mouse cell line 3T3-L1 or 3T3-F422A preadipocyte. We will examine the effects of adipocyte differentiation on LPL enzyme activity, rate of synthesis, mRNA levels, and LPL gene transcription. Similarly, the effects of insulin in fully differentiated adipocytes will be investigated. Insulin is an important hormone regulating LPL in intact animals. Our studies in vitro provide insight into the mechanism of insulin action on LPL expression. (ii) We will determine the structures of the cloned normal human LPL and HL genes. We will then determine the structural basis for homozygous LPL deficiency in three patients with the disorder. LPL mRNA structure will be determined in the RNA extracted from fat biopsies obtained from two of these patients. The genomic structures will be determined from their cloned genes, and also by the polymerase chain reaction. Finally, we will investigate the cis- and trans-acting elements regulating LPL and HL gene expression. (iii) The cis-acting elements will be investigated by the use of hydrid DNA-reporter gene constructs and transfection assays, as well as transgenic mice experiments. We are interested mainly in the organ-specific expression of the HL gene, and the differentiation-dependent expression of the LPL gene in 3T3-L1 preadipocytes/adipocytes. (iv) Trans-acting factors that bind to some of these cis-acting elements will be isolated and characterized. The structure of some of these factors will be determined by direct cloning and sequencing. Our overall goal is to obtain an understanding of LPL and HL expression at the molecular level, and to delineate the molecular defect underlying LPL deficiency in three affected homozygous patients.
Showing the most recent 10 out of 87 publications
