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.
[Show abstract][Hide abstract] ABSTRACT: Ectopic fat accumulation has been linked to lipotoxic events, including the development of insulin resistance in skeletal muscle. Indeed, intramyocellular lipid storage is strongly associated with the development of type 2 diabetes. Research during the last two decades has provided evidence for a role of lipid intermediates like diacylglycerol and ceramide in the induction of lipid-induced insulin resistance. However, recently novel data has been gathered that suggest that the relation between lipid intermediates and insulin resistance is less straightforward than has been previously suggested, and that there are several routes towards lipid-induced insulin resistance. For example, research in this field has shifted towards imbalances in lipid metabolism and lipid droplet dynamics. Next to imbalances in key lipogenic and lipolytic proteins, lipid droplet coat proteins appear to be essential for proper intramyocellular lipid storage, turnover and protection against lipid-induced insulin resistance. Here, we discuss the current knowledge on lipid-induced insulin resistance in skeletal muscle with a focus on the evidence from human studies. Furthermore, we discuss the available data that provides supporting mechanistic information.
Progress in lipid research 11/2011; 51(1):36-49. DOI:10.1016/j.plipres.2011.11.003 · 10.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride (TG) synthesis. There are two isoforms, DGAT1 and DGAT2, with distinct protein sequences and potentially different physiological functions. To date, the ability to determine clear functional differences between DGAT1 and DGAT2, especially with respect to hepatic TG synthesis, has been elusive. To dissect the roles of these two key enzymes, we pretreated HepG2 hepatoma cells with (13)C(3)-D(5)-glycerol or (13)C(18)-oleic acid, and profiled the major isotope-labeled TG species by liquid chromatography tandem mass spectrometry. Selective DGAT1 and DGAT2 inhibitors demonstrated that (13)C(3)-D(5)-glycerol-incorporated TG synthesis was mediated by DGAT2, not DGAT1. Conversely, (13)C(18)-oleoyl-incorporated TG synthesis was predominantly mediated by DGAT1. To trace hepatic TG synthesis and VLDL triglyceride (VLDL-TG) secretion in vivo, we administered D(5)-glycerol to mice and measured plasma levels of D(5)-glycerol-incorporated TG. Treatment with an antisense oligonucleotide (ASO) to DGAT2 led to a significant reduction in D(5)-glycerol incorporation into VLDL-TG. In contrast, the DGAT2 ASO had no effect on the incorporation of exogenously administered (13)C(18)-oleic acid into VLDL-TG. Thus, our results indicate that DGAT1 and DGAT2 mediate distinct hepatic functions: DGAT2 is primarily responsible for incorporating endogenously synthesized FAs into TG, whereas DGAT1 plays a greater role in esterifying exogenous FAs to glycerol.
The Journal of Lipid Research 04/2012; 53(6):1106-16. DOI:10.1194/jlr.M020156 · 4.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Overproduction of apolipoprotein B (apoB)-containing lipoproteins by the liver and the intestine is 1 of the hallmarks of insulin resistance and type 2 diabetes and a well-established risk factor of cardiovascular disease. The assembly of apoB lipoproteins is regulated by the availability of lipids that form the neutral lipid core (triacylglycerol and cholesteryl ester) and the limiting lipoprotein monolayer (phospholipids and cholesterol). Although tremendous advances have been made over the past decade toward understanding neutral lipid and phospholipid biosynthesis and neutral lipid storage in cytosolic lipid droplets (LDs), little is known about the mechanisms that govern the transfer of lipids to the lumen of the endoplasmic reticulum for apoB lipidation. ApoB-synthesizing organs can deposit synthesized neutral lipids into at least 3 different types of LDs, each decorated with a subset of specific proteins: perilipin-decorated cytosolic LDs, and 2 types of LDs formed in the lumen of the endoplasmic reticulum, the secretion-destined LDs containing apoB, and resident lumenal LDs coated with microsomal triglyceride transfer protein and exchangeable apolipoproteins. This brief review will address the current knowledge of lumenal lipid metabolism in the context of apoB assembly and lipid storage.
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