Biochem Biophys Res Commun. 2016 Nov;480(4):641-647. doi: 10.1016/j.bbrc.2016.10.109.

Saturated fatty acid in the phospholipid monolayer contributes to the formation of large lipid droplets. 

Kotoko Arisawa1, Haruka Mitsudome1, Konomi Yoshida1, Shizuka Sugimoto1, Tomoko Ishikawa2, Yoko Fujiwara2,3, and Ikuyo Ichi2,3

1 Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo 112-8610, Japan

2 Institute for Human Life Innovation, Ochanomizu University, Tokyo 112-8610, Japan

3 Natural Science Division, Faculty of Core Research, Ochanomizu University, Tokyo 112-8610, Japan

 

Abstract

The degree of saturation of fatty acid chains in the bilayer membrane structure is known to control membrane fluidity and packing density. However, the significance of fatty acid composition in the monolayers of lipid droplets (LDs) has not been elucidated. In this study, we noted a relationship between the size of LDs and the fatty acid composition of the monolayer. To obtain large LDs, we generated NIH3T3 cells overexpressing fat-specific protein 27 (FSP27). This induced the fusion of LDs, resulting in larger LDs in FSP27-overexpressing cells compared with LDs in control cells. Moreover, the lipid extracts of LDs from FSP27-overexpressing cells reconstituted large-droplet emulsions in vitro, implying that the lipid properties of LDs might affect the size of LDs. FSP27-overexpressing cells had more saturated fatty acids in the phospholipid monolayer of the LDs compared with control cells. To further investigate the effects of the degree of phospholipid unsaturation on the size of LDs, we synthesized artificial emulsions of a lipid mixed with DPPC (di16:0-PC), DSPC (diC18:0-PC), DOPC (diC18:1n-9-PC), or DLPC (diC18:2n-6-PC) and compared the sizes of the resulting LDs. The emulsions prepared from saturated PC had larger droplets than those prepared from unsaturated PC. Our results suggest that saturated fatty acid chains in phospholipid monolayers might establish the form and/ or stability of large LDs.

PMID: 27983976

 

Supplement

The present work highlights the significance of fatty acid chain of phospholipid on lipid droplets (LDs). We focused on the difference in the fatty acid composition of monolayer between small and large LDs, and demonstrated that saturated fatty acid of phospholipids in the LD monolayer has biophysical potential for the formation of large LDs [1].

Mature adipocytes store excess lipids in intracellular LDs, which are made up of a hydrophobic core of triacylglycerol and cholesterol ester surrounded by a monolayer of phospholipids studded with a variety of LD proteins. Recent studies show that the type of head group and the amount of phospholipids on the surface of LDs affect the size of individual LDs [2, 3]. Although these facts have suggested that physical properties of surface phospholipids are crucially important for the decision of the LD size, it is poorly understood whether the properties of hydrophobic region of phospholipids on LD monolayer impact the size of LDs. Our previous study reported that the fatty acid composition of phospholipid monolayer remarkably changed during the differentiation of 3T3-L1 adipocytes whereas the fatty acid composition of phospholipid bilayer did not change as much as that of phospholipid monolayer [4]. However, it is still uncertain that the change of fatty acid in the monolayer could play a role in LD expansion. The degree of unsaturation of fatty acid chains in the bilayer membrane structure is known to control membrane physical properties such as fluidity, curvature, and packing density. Therefore, the fatty acid compositions in monolayer of LDs might be also important for physical properties of LDs, then it is possible that these physical properties affect the size of LDs. Thus we examined the relationship between the fatty acid composition of phospholipid monolayer and the LD expansion.

First, we checked the differences in fatty acid composition between small and large LDs. To obtain large LDs, we generated NIH3T3 cells overexpressing fat specific protein 27 (FSP27), which induces the fusion of LDs. It was observed that FSP27-overexpressing cells had larger LDs and more saturated fatty acids (such as C16:0 and C18:0) in the phospholipid monolayer of the LDs compared with control cells. The amount of triacylglycerol (TAG) and phospholipid such as phosphatidylcholine (PC) was similar between FSP27 cells and control cells, even though FSP27-overexpressing cells had notably large LDs.

To examine that the lipid component of large LDs by themselves had the biophysical potential for forming large droplet, we reconstructed LD-like oil-in-water emulsions using the lipid extract of isolated LDs from each cell. The droplet size of emulsions reconstituted from lipid extracts of FSP27 cells was larger than that of control cells. This experiment showed that the lipids from large LDs by themselves had the physical properties required to form large LDs.

Then, we investigated the effects of the degree of fatty acid unsaturation on the size of artificial emulsions. The droplet size of emulsions containing saturated PC (DPPC, diC16:0-PC and DSPC, diC18:0-PC) was larger than that of emulsions containing unsaturated PC (DOPC, diC18:1n-9-PC and DLPC diC18:2n-6-PC) (Fig.1). Moreover, in case of using dilenoleoylphosphatidylcholine (DLPC, diC18:2n-6-PC), which includes two polyunsaturated acyl chain, the size of their emulsions were even smaller than that of DOPC emulsions. These results suggested that the degree of saturation of fatty acid in the phospholipid monolayer acted on the formation of large LDs.

Furthermore, we revealed that the monolayer based on saturated fatty acids had less fluidity than the layer based on unsaturated fatty acid by measuring the packing density of surface phospholipid monolayers in emulsions by using the fluorescence probe Laurdan. Low fluidity of membrane caused by saturated fatty acids of PC might restrict lateral diffusion of phospholipids and more frequently occur the lipid packing defects. Such condition induces the exposure of the core TAG of emulsions to the aqueous phase. To eliminate this unstable condition, it is possible that saturated phospholipid emulsions attempt to coalesce together and remedy the defects by supplying sufficient phospholipids. As a result of these processes, stabilized large emulsions might be generated.

Our results showed that the differences in fatty acid composition of phospholipid monolayer between small and large LDs, and revealed that the existence of saturated fatty acids in phospholipid monolayer is a factor that controls the size of LDs.

 

 

Figure 1. Preparation of artificial emulsions by using the various types of PC.

Artificial emulsions were stained with Nile Red; TAG and PC were mixed in a molar ratio of 20:1 to reflect the composition of intracellular LDs. Four types of artificial PC were used: dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), containing two saturated fatty acids (C16:0 and C18:0, respectively); dioleoylphosphatidylcholine (DOPC), containing two monounsaturated fatty acids (C18:1n-9); and dilenoleoylphosphatidylcholine (DLPC), containing two polyunsaturated fatty acids (C18:2n-6).

 

References:

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