Determination of the intracellular localisation of the DGAT enzymes and the effect on metabolic function in different cell types

• Main Author: Wurie, Haja R.
• Published: University of Warwick 2012
• Subjects: 612.3; QP Physiology
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.690470

Triglycerides (TAG) synthesis and secretion have major influences on postprandial and fasting triglyceridaemia and on hepatic steatosis, parameters that affect the development of insulin resistance and type-2 diabetes. Two main enzymes (diacylglycerol acyltransferases – DGATs) that catalyse the final reaction of TAG synthesis have been identified, DGAT1 and DGAT2. They play non-redundant roles in different tissues, highlighted by the very different phenotypes of Dgat1-/- and Dgat2-/- mice. Using three inhibitors with different actions on these enzymes, the differential localisation of DGAT2 (cytosolic aspect) and DGAT1 (both cytosolic and luminal aspects) activities within the endoplasmic reticular membranes of rat liver microsomes, HepG2 and Caco-2 cells and their relative contributions to total DGAT activity were determined. Subsequently, the sensitivity to these inhibitors on the processes of the incorporation of preformed (exogenously supplied) fatty acids, de novo synthesised fatty acids, and glycerol into the acyl and glyceroyl moieties of the TAG molecules was determined. Inhibition of DGAT2 affected primarily the incorporation of de novo synthesised fatty acids and of nascent diglycerides (into which glycerol was newly incorporated), whilst inhibition of DGAT1 affected primarily the retention of the glyceroyl moiety within the TAG fraction stoichiometrically with the incorporation of preformed fatty acid, but not with that of fatty acids derived from de novo lipogenesis. In spite of its modest contribution toward overall cellular DGAT activity, inhibition of DGAT2 was rate-limiting for TAG synthesis, indicating that DGAT2 and DGAT1 act in series with DGAT2 being upstream of DGAT1. Similar conclusions were drawn from data obtained with Caco-2 cells, with important differences owing to the expression of other enzymes expressing DGAT activity. The dual topology of DGAT1 in the ER membrane is suggested to enable the synthesis of TAG on either aspect of the membrane with DAG connecting the two pools of TAG in the cytosolic and ER luminal cellular compartments.