A Phospholipase D-dependent Process Forms Lipid Droplets Containing Caveolin, Adipocyte Differentiation-related Protein, and Vimentin in a Cell-free System

Abstract

We developed a microsome-based, cell-free system that assembles newly formed triglyceride (TG) into spherical lipid droplets. These droplets were recovered in the d < 1.055 g/ml fraction by gradient ultracentrifugation and were similar in size and appearance to those isolated from rat adipocytes and 3T3-L1 cells. Caveolin 1 and 2, vimentin, adipocyte differentiation-related protein, and the 78-kDa glucose regulatory protein were identified on the droplets from the cell-free system. The caveolin was soluble in 1% Triton X-100, as was the caveolin on lipid droplets from 3T3-L1 cells. The lipid droplets from the cell-free system, like those from 3T3-L1 cells, contained TG, diacylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The assembly of these TGcontaining structures was dependent on the rate of TG biosynthesis and required an activator present in the 160,000 ؋ g supernatant from homogenized rat adipocytes. The activator induced phospholipase D (PLD) activity, and its effect on the release of the TG-containing structures from the microsomes was inhibited by 1-butanol (but not 2-butanol) or 2,3-diphosphoglycerate. The activator could be replaced by a constitutively active PLD or phosphatidic acid. These results indicate that PLD and the formation of phosphatidic acid are important in the assembly of the TG-containing structures.Within the cell, triglycerides (TG) 1 are stored in cytosolic lipid droplets. Little is known about how these structures are assembled. Several proteins have been identified on their surface, including adipocyte differentiation-related protein (ADRP or adipophilin), perilipins (reviewed in Refs. 1 and 2), and caveolin (see Refs. 3-5 and reviewed in Refs. 2 and 6), but their roles in lipid droplet assembly are unknown.ADRP and perilipins are abundant on lipid droplets. ADRP is found mostly on smaller droplets; when overexpressed, it stimulates lipid droplet formation (7), suggesting a role in the assembly process. Perilipins are present on large lipid droplets (7) and have a central role in the turnover of TG within these structures (1, 8, 10), perhaps affording protection against hormone-sensitive lipase (11). Perilipin-null mice have smaller lipid droplets and a higher rate of basal lipolysis than wild-type mice and are resistant to diet-induced obesity (12).Caveolin is a 21-kDa membrane protein with a hairpin structure whose N and C termini face the cytosol (13, 14). In mammals, there are three caveolin genes. Caveolin 1 and 2 are expressed in adipocytes, whereas caveolin 3 is present in muscle. Caveolin plays a key role in intracellular lipid transport (15, 16), binding fatty acid (17), and transporting cholesterol (16) in a manner reminiscent of the way plasma apolipoproteins transport lipids in the blood (16,18).Phospholipase D (PLD) catalyzes the conversion of phosphatidylcholine to phosphatidic acid (PA) and appears to be important in intracellular transport and sorting processes (19 -22), either as an intracellular messenger or as a cone-shaped l...

Not Just Fat: The Structure and Function of the Lipid Droplet

“…By depletion of adipocyte differentiation-related protein (ADRP) or by inhibition of group IVA cytosolic phospholipase A 2 , TG levels in LDs decreased and shifted to the microsome Gubern et al 2008). An in vitro experiment suggested the involvement of phospholipase D in the LD formation process (Marchesan et al 2003). The results suggest that LD formation does not occur spontaneously because of lipid ester accumulation but require some active mechanism.…”

Role of ADP Ribosylation Factor 1 in the Assembly and Secretion of ApoB-100–Containing Lipoproteins

“…We have recently presented evidence that PLD activity is involved in the assembly of cytosolic lipid droplets. 28 Thus, PLD 1 may promote the formation of the lipoproteins, but this effect may be counteracted by a diversion of triglycerides into cytosolic lipid droplets.…”

“…We have recently presented evidence that PLD activity is involved in the assembly of cytosolic lipid droplets. 28 Thus, PLD 1 may promote the formation of the lipoproteins, but this effect may be counteracted by a diversion of triglycerides into cytosolic lipid droplets.Our findings in this study, together with previously published results, can be summarized in a hypothesis for the role of ARF1 in the assembly of VLDL 1 (Figure 5). The assembly process starts with the cotranslational lipidation of apoB-100, resulting in a primordial "pre-VLDL" particle.…”

Adipose morphology and metabolic disease

“…The surface layers of LDs are coated with a phospholipid monolayer, and phospholipid metabolism was a shared feature of the genes identified from this yeast screen (Walther and Farese, 2009). Phosphatidic acid (PA), a cone-shaped lipid common in phospholipids that alters the curvature of membranes and promotes membrane fusion events, was a key factor in the formation of supersized LDs (Fei et al, 2011;Marchesan et al, 2003). Further, supersized LDs could be formed by PA-stimulated fusion of LDs (Fei et al, 2011), suggesting that the phospholipid monolayer of LDs is important for LD growth and may mediate LD fusion to facilitate LD hypertrophy.…”