Hepatic monoacylglycerol acyltransferase (MGAT) is a tissue- specific and developmentally-regulated enzyme whose specific activity in the neonate is 700-fold higher than in the adult. MGAT catalyzes the acylation of 2-monoacylglycerol to sn-1,2- diacylglycerol and provides an alternate pathway of diacylglycerol synthesis in the neonate. Since diacylgylcerol is both an intermediate of glycerolipid biosynthesis and the intracellular activator of protein kinase C, MGAT may function in glycerolipid recycling and/or in regulating cellular diacylglycerol levels. Both of these roles would be important in perinatal hepatocytes which are proliferting, differentiating biochemically, rapidly accummulating membranes and organelles, and secreting increasing amounts of glycerolipid in bile and lipoproteins. The proposed studies will focus on the role of MGAT and its regulation in perinatal hepatocytes. Using radio-labeled substrates, we will examine MGAT's role in recycling triacylglycerol to synthesize phospholipids. We will test the hypothesis that MGAT can regulate cell diacylglycerol levels and alter basal protein kinase C activity. Cells will be incubated with compounds that should selectively increase diacylglycerol levels only in hepatocytes that contain MGAT activity. We will then measure diacylglycerol levels and determine whether protein kinase C is activated. In order to study developmental regulation, we will complete the purification of MGAT and clone the cDNA. Antibody will be used to evaluate protein levels and turnover. cDNA probes will be used to examine mRNA levels and translation in both normal developing liver and in cultured neonatal hepatocytes after hormonal induction or repression of MGAT activity. Additionally, microsomal diacylglycerol lipase, the putative supplier of MGAT's monoacylglycerol substrate, will be characterized and its topography and ontogeny will be studied. We will construct perinatal-hepatocyte/hepatoma hybrids in order to study regulation of MGAT in a permanent cell line. These studies will provide new information on the regulation of glycerolipid metabolism during an important developmental period that has received relatively little attention.
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