The biosynthesis and storage of triglyceride (TG) is an important cellular process conserved from yeast to man. Most mammalian cells accumulate TG in lipid droplets, most prominent in adipocytes, which are specialized to store large amounts of the TG over long periods. In this study, we followed TG biosynthesis and targeting by fluorescence imaging in living 3T3-L1 adipocytes and COS7 fibroblasts. Key findings were (i) not only TG but also its direct metabolic precursor diacylglycerol, DG, accumulates on lipid droplets; (ii) the essential enzyme diacylglycerol acyltransferase 2 associates specifically with lipid droplets where it catalyzes the conversion of DG to TG and (iii) individual lipid droplets within one cell acquire TG at very different rates, suggesting unequal access to the biosynthetic machinery. We conclude that at least part of TG biosynthesis takes place in the immediate vicinity of lipid droplets. In vitro assays on purified lipid droplets show that this fraction of the biosynthetic TG is directly inserted into the growing droplet. All organisms store metabolic energy to satisfy needs when nutrient levels decrease. Mammals store most of this energy as triglyceride (TG) in the lipid droplets of adipocytes (1). Adipocytes are specialists with high rates of metabolite uptake and synthesis of TG, which is stored in large lipid droplets (10-to 100-mm diameter) and mobilized upon demand by the co-ordinated action of several lipases (2). Although essentially all cell types contain small lipid droplets, most non-adipocytes store only little TG and do not form large droplets. However, a basal ability to esterify fatty acids and glycerol is important for all cells because even low amounts of free fatty acid are toxic (3).Little is known about mechanisms by which cells package newly synthesized TG into storage droplets (1). Lipid droplets in animals, plants and yeast consist of a hydrophobic core made from neutral lipids surrounded by a phospholipid monolayer with proteins attached both integrally and peripherally (4,5). Biogenesis of neutral lipids is assumed to occur at the endoplasmic reticulum, ER. In mammals, the pathway starts by acylation of glycerol-3-phosphate with two fatty acids to give phosphatidic acid, PA. Subsequent dephosphorylation yields diacylglycerol (DG), the precursor for synthesis of both phospholipids and, by a third acylation, TG. Two enzymes diacylglycerol acyltransferase (DGAT)1 (6) and DGAT2 (7) catalyze the TG synthesis. Recently, microscopic analysis of plant cells overexpressing tagged DGAT1 and DGAT2 pinpointed their localization to distinct regions of the ER, excluding each other (8). Also in mammalian cells, overexpressed, tagged DGAT2 localized to the ER (9). Analysis of phenotypes of knockout mice point to a complex physiological organization of TG synthesis. Mice lacking the ubiquitously expressed DGAT1 have reduced body fat, a lactation defect and are resistant to diet-induced obesity (10). Interestingly, they show normal TG levels in plasma and adipose tissue. DGAT2 is...
Phosphatidylcholine (PC) is synthesized by two different pathways, the Lands cycle and the Kennedy pathway. The recently identified key enzymes of the Lands cycle, lysophosphatidylcholine acyltransferase 1 and 2 (LPCAT1 and -2), were reported to localize to the endoplasmic reticulum and to function in lung surfactant production and in inflammation response. Here, we show in various mammalian cell lines that both enzymes additionally localize to lipid droplets (LDs), which consist of a core of neutral lipids surrounded by a monolayer of phospholipid, mainly PC. This dual localization is enabled by the monotopic topology of these enzymes demonstrated in this study. Furthermore, we show that LDs have the ability to locally synthesize PC and that this activity correlates with the LPCAT1 and -2 expression level. This suggests that LPCAT1 and -2 have, in addition to their known function in specialized cells, a ubiquitous role in LD-associated lipid metabolism.
BackgroundLipids are stored within cells in lipid droplets (LDs). They consist of a core of neutral lipids surrounded by a monolayer of phospholipids, predominantly phosphatidylcholine (PC). LDs are very dynamic and can rapidly change in size upon lipid uptake or release. These dynamics require a fast adaptation of LD surface. We have recently shown that two Lands cycle PC synthesizing enyzmes, LPCAT1 and LPCAT2 can localize to the LD surface.ResultsHere, we show that knock-down of both enzymes leads to an increase in LD size without changes in the total amount of neutral lipids, while interference with the de-novo Kennedy pathway PC biosynthesis is associated with changes in triacylglyceride synthesis. We show that function of LPCAT1 and 2 is conserved in Drosophila melanogaster by the ortholog CG32699. Furthermore we demonstrate that modulation of the LD pool by LPCAT1 influences the release of lipoprotein from liver cells.ConclusionActivity of the Kennedy pathway regulates the balance between phospholipids and neutral lipids, while the Lands cycle regulates lipid droplet size by regulating surface availability and influencing surface to volume ratio. Differences in lipid droplet size may account for differences in lipid dynamics and be relevant to understand lipid overload diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-014-0043-3) contains supplementary material, which is available to authorized users.
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