Evidence That Diacylglycerol Acyltransferase 1 (DGAT1) Has Dual Membrane Topology in the Endoplasmic Reticulum of HepG2 Cells

Abstract

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.Hypertriglyceridemia is a key biomarker for the metabolic/ insulin resistance syndrome and for associated morbidities, including type-2 diabetes and cardiovascular disease (1). Similarly, excessive accumulation of triglycerides in cytoplasmic lipid droplets results in hepatic steatosis, now recognized as being associated, possibly causatively, with whole body insulin resistance, and which may progress to nonalcoholic fatty liver or steatohepatitis (2, 3). Fasting hypertriglyceridemia is primarily due to the hypersecretion of triglyceride (TAG) 3 by the liver, within very low density lipoproteins (VLDL). Therefore, an understanding of the enzymology involved in triglyceride synthesis, remodeling, storage, and assembly into secreted VLDL is essential for the design of pharmacological strategies aimed at managing dyslipidaemia without the exacerbation of hepatic steatosis, and vice versa.Diacylglycerol acyltransferases (DGATs) catalyze the final reaction of TAG synthesis. Two distinct gene products, DGAT1 and DGAT2, that catalyze most of tissue TAG synthesis have been described (4, 5) but remain relatively poorly characterized, and their respective, nonredundant functions are still to be elucidated. In the present study, we have used two specific inhibitors (which belong to different chemical classes of compounds) of human DGAT1, in combination with the selective permeabilization of the plasma membrane and the ER membrane of whole hepatocytes, to study the sidednes...

The use of stable isotope-labeled glycerol and oleic acid to differentiate the hepatic functions of DGAT1 and -2

“…The remaining DGAT activity in HepG2 cells appears to be DGAT2-mediated (20-30%). Our data are consistent with previously reported data on DGAT1 activity in HepG2 cells ( 15 ). To further defi ne potential differences in the hepatic functions of DGAT1 and DGAT2, we investigated TG synthesis in human HepG2 cells and in C57BL/6J mice using stable The most-intriguing fi nding from these studies relates to the ability of DGAT1 inhibitors to block incorporation of exogenously added oleate but not to disrupt incorporation of exogenously added glycerol into TG, and the opposite is true for the selective DGAT2 inhibitors.…”

Hepatitis C Virus Entry Is Impaired by Claudin-1 Downregulation in Diacylglycerol Acyltransferase-1-Deficient Cells

“…In fact, DGAT1 recycles the products of triglyceride hydrolysis, which are partial glycerides, for triglyceride synthesis. In addition, DGAT1 inhibition decreases the intracellular lipid pool comprised of triglycerides, partial glycerides, and free fatty acids in human liver-derived cells (10,41). In the present study, a low-dose exogenous palmitic acid treatment prevented the downregulation of CLDN1 and HNF4␣ expression in DGAT1-silenced cells.…”

Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core

“…Wurie et al (31) used two different DGAT1 inhibitors (including iA used in this study) with differential effects on overt and latent DGAT1 activities. As iA only inhibits overt DGAT1 activity at high concentrations and high concentrations of the drug are required to inhibit HCV infection and localization of HCV proteins to LDs (11), HCV may have evolved to specifically exploit overt DGAT1 activity in cells.…”