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The two diacylglycerol acyltransferases, DGAT1 and DGAT2, are known to have non-redundant functions, in spite of catalysing the same reaction and being present in the same cell types. The basis for this distinctiveness, which is reflected in the very different phenotypes of Dgat1(-/-) and Dgat2(-/-) mice, has not been resolved. Using selective inhibitors of human DGAT1 and DGAT2 on HepG2 cells and gene silencing, we show that, although DGAT2 activity accounts for a modest fraction (< 20%) of overall cellular DGAT activity, inhibition of DGAT2 activity specifically inhibits (and is rate-limiting for) the incorporation of de novo synthesized fatty acids and of glycerol into cellular and secreted triglyceride to a much greater extent than it affects the incorporation of exogenously added oleate. By contrast, inhibition of DGAT1 affects equally the incorporation of glycerol and exogenous (preformed) oleate into cellular and secreted triacylglycerol (TAG). These data indicate that DGAT2 acts upstream of DGAT1, largely determines the rate of de novo synthesis of triglyceride, and uses nascent diacylglycerol and de novo synthesized fatty acids as substrates. By contrast, the data suggest that DGAT1 functions in the re-esterification of partial glycerides generated by intracellular lipolysis, using preformed (exogenous) fatty acids. Therefore, we describe distinct but synergistic roles of the two DGATs in an integrated pathway of TAG synthesis and secretion, with DGAT2 acting upstream of DGAT1., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Triacylglycerol (TAG) synthesis and secretion are important functions of the liver that have major impacts on health, as overaccumulation of TAG within the liver (steatosis) or hypersecretion of TAG within very low density lipoproteins (VLDL) both have deleterious metabolic consequences. Two diacylglycerol acyltransferases (DGATs 1 and 2) can catalyze the final step in the synthesis of TAG from diacylglycerol, which has been suggested to play an important role in the transfer of the glyceride moiety across the endoplasmic reticular membrane for (re)synthesis of TAG on the lumenal aspect of the endoplasmic reticular (ER) membrane (Owen, M., Corstorphine, C. C., and Zammit, V. A. (1997) Biochem. J. 323, 17-21). Recent topographical studies suggested that the oligomeric enzyme DGAT1 is exclusively lumen facing (latent) in the ER membrane. By contrast, in the present study, using two specific inhibitors of human DGAT1, we present evidence that DGAT1 has a dual topology within the ER of HepG2 cells, with approximately equal DGAT1 activities exposed on the cytosolic and lumenal aspects of the ER membrane. This was confirmed by the observation of the loss of both overt (partial) and latent (total) DGAT activity in microsomes prepared from livers of Dgat1-/- mice. Conformational differences between DGAT1 molecules having the different topologies were indicated by the markedly disparate sensitivities of the overt DGAT1 to one of the inhibitors. These data suggest that DGAT1 belongs to the family of oligomeric membrane proteins that adopt a dual membrane topology. [ABSTRACT FROM AUTHOR]