Monoacylglycerol acyltransferase (MGAT) activity is a tissue-specific microsomal enzyme that is expressed at high levels in the neonatal period, in hibernating mammals, and in migrating birds. In each of these conditions, high rates of lipolysis are accompanied by an increased need for essential fatty acids; in each condition, essential fatty acids are preferentially conserved. Hepatic MGAT specific activity also increases in streptozotocin-induced diabetes, a pathological state characterized by high rates of beta-oxidation. We have provided conceptual and experimental evidence that MGAT plays a critical role in the conservation of essential fatty acids during normal physiological periods with high rates of lipolysis. MGAT may contribute, as well, to diabetes- associated changes in membrane fatty acids. This proposal represents a unified plan to use biochemical and molecular techniques to study the physiological significance, regulation, and structure of MGAT. Using a mixed micellar assay, we will analyze the stoichiometry of MGAT's substrates and its 1,2-diacylglycerol and phospholipid activators. We will test the hypothesis directly that MGAT functions in hepatocytes and in differentiated 3T3-L1 adipocytes to retain essential fatty acids. We will determine whether MGAT's activity and character are regulated by its membrane microenvironment. In order to study both developmental regulation and the relationship between structure and function in membranes, we will clone and sequence MGAT cDNA, study mRNA abundance in differentiating tissues and cells, and use transfected cDNA to study cellular diacylglycerol trafficking. In addition to providing novel regulatory and structural information about this critical enzyme of glycerolipid synthesis, these studies will provide tools with which to investigate diacylglycerol trafficking within cells, the regulation of essential fatty acid homeostasis and of energy metabolism, and the link between triacylglycerol synthesis and VLDL and chylomicron assembly.
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