Am J Physiol Cell Physiol 303: C713–C714, 2012;
doi:10.1152/ajpcell.00250.2012.
Editorial Focus
Fretting about fat: a new look at the lipid droplet surface and the roundabout
role of Plin2 in cellular lipid storage. Focus on “Direct interaction of Plin2
with lipids on the surface of lipid droplets: a live cell FRET analysis”
Jeffrey S. Elmendorf
Departments of Cellular and Integrative Physiology and Biochemistry and Molecular Biology, Indiana University School of
Medicine, Centers for Diabetes Research, Membrane Biosciences, and Vascular Biology and Medicine, Indianapolis, Indiana
Address for reprint requests and other correspondence: J. S. Elmendorf,
Depts. of Cellular & Integrative Physiology and Biochemistry & Molecular
Biology, Indiana Univ. School of Medicine, Centers for Diabetes Research,
Membrane Biosciences, and Vascular Biology and Medicine, Indianapolis, IN
46202 (e-mail: [email protected]).
http://www.ajpcell.org
cient FRET proximity to Plin2 to indicate direct contact (Fig.
1). Careful mathematical considerations by the authors predict
that the fluorescent lipids interacting with Plin2 were within the
monolayer or bound to a protein at the monolayer surface.
Previous work indicated that Plin2 interacted with lipids;
however, the limits of optical resolution (2,200 Å) did not
permit determining whether the Plin2-lipid interactions on the
lipid droplet surface were direct. The likely physical association was also suggested in previous fluorescent ligand saturation binding assays by McIntosh and colleagues (9). Here,
however, their use of FRET shows for the first time the direct
binding of Plin2 with lipid droplet surface lipids. The authors
envision that the direct Plin2-lipid interactions on the lipid
droplet surface likely regulate lipid exchange. Interestingly,
beyond Plin2 forming direct physical association with lipids,
the authors also observed that Plin2 overexpression altered
levels of key enzymes involved in lipolysis and lipogenesis.
For example, expression levels of enzymes involved in triacylglycerol and phospholipid synthesis were significantly upregulated while those associated with lipolysis were either
decreased or remained unaffected by Plin2 overexpression. In
line with the promotion of lipogenesis, these Plin2-overexpressing cells also displayed elevated levels of triacylglycerols,
fatty acids, cholesteryl esters, and phospholipids. The new
finding that Plin2 overexpression increased phospholipid con-
Fig. 1. FRET analysis of Plin2-lipid interactions. A cyan variant of green
fluorescent protein (CFP) was chosen for the CFP-Plin2 expression construct
because CFP forms a strong FRET interaction with commercially available
NBP-labeled lipids that target to lipid droplets (e.g., phosphatidylcholine,
cholesterol, sphingomyelin) due to strong overlap of the emission spectra of
the cyan fluorescence (408 nm excitation, 475 nm emission) with the NBD
excitation spectra (488 nm excitation, 530 nm emission). In addition, a strong
overlap of CFP emission with the excitation of the lipid droplet stain Nile red
(NR; 568 nm excitation, 598 nm emission) provided a control that was beyond
FRET proximity. The solid nonbolded squiggly lines represent the 408 nm
excitation, the dashed lines represent 475 nm CFP emissions, and the solid
bolded squiggly lines represent the 530 nm NBD emissions or FRET signals.
0363-6143/12 Copyright © 2012 the American Physiological Society
C713
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of fat storage remains a
fundamental challenge in biology and a significant issue in
medicine. Essentially all eukaryotic cells have lipid droplet
structures that store neutral lipids (mainly triglycerides and
cholesterol esters) within a monolayer of phospholipids (1).
Residing on the lipid droplet phospholipid monolayer are
several types of proteins; most notable are proteins of the
perilipin family that maintain lipid droplet structure and function (7). There are five perilipin family members (Plin1–Plin5;
reviewed in Ref. 1).
Plin1 regulates the mobilization of fatty acids from triglycerols stored in large lipid droplets. This lipolysis is initiated by
activation of protein kinase A, which phosphorylates Plin1 and
hormone-sensitive lipase (HSL). The phosphorylation of Plin1
releases comparative gene identification-58 (CGI-58) from the
CGI-58-Plin1 complex. The released CGI-58 then activates
adipose triglyceride lipase (ATGL) and recruits phosphorylated HSL to the lipid droplet surface. Together, these lipases
mobilize neutral lipids to provide metabolic energy and lipids
for membrane synthesis. Interestingly, this functional aspect
appears to be specific to Plin1 as evidenced by the observation
that, in Plin1-null mice, Plin2 replaces Plin1 on adipocyte lipid
droplets but does not functionally compensate for loss of Plin1
with respect to regulating lipolysis. In fact, several studies have
shown that increased expression of Plin2 promotes cellular
lipid accumulation, as opposed to lipolysis (reviewed in Ref. 1).
How Plin2 physically functions to promote lipid accumulation
has been difficult to visualize, and was the aim of the study by
McIntosh and colleagues (10), published in this issue of American Journal of Physiology-Cell Physiology. The authors used
live cell Förster resonance energy transfer (FRET) to examine
live cell interactions of Plin2 with lipids. This technique
allowed for the detection of Plin2-lipid interactions on the lipid
droplet surface inside live cells and permitted estimations of
intermolecular distances between Plin2 and lipid molecules to
within 10 –100 Å. These analyses demonstrated binding of
Plin2 to lipid droplet surface lipids such as phosphatidylcholine, cholesterol, sphingomyelin, and stearic acid (Fig. 1). In
support of direct molecular contact, the high FRET efficiencies
between the labeled Plin2 and lipids indicated molecular distances on the order of 44 –57 Å. The specificity of these
Plin2-lipid interactions on the surface of the lipid droplets was
confirmed by observing that Nile red, another fluorescently
labeled molecule that targets lipid droplets, was not in suffiUNDERSTANDING THE BASIC BIOLOGY
Editorial Focus
C714
such as the liver, lower Plin2 levels could be beneficial,
whereas in others such as skeletal muscle, higher Plin2 levels
could be protective. A potential for complexity therefore exists
as Plin2 physiology is investigated and its role in the global
pandemic of obesity and comorbidities is defined.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author.
AUTHOR CONTRIBUTIONS
J.S.E. prepared the figure, drafted the manuscript, edited and revised the
manuscript, and approved the final version of the manuscript.
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AJP-Cell Physiol • doi:10.1152/ajpcell.00250.2012 • www.ajpcell.org
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tent, in addition to the already appreciated effect of this
overexpression on triglyceride and cholesterol ester levels,
illuminates an accessory role of Plin2 in increasing the lipid
droplet membrane size to accommodate the gain in neutral
lipids.
Observing that Plin2 forms a direct physical association with
lipids involved in lipid droplet structure and function, and that
Plin2 promotes lipogenesis, is complementary to siRNA silencing and knockdown studies, showing that Plin2 deficiency
results in decreased lipid droplet size and number (1, 8). In this
context, evidence through the years has assigned Plin2 the role
of accumulating cellular lipids either normally or abnormally.
In the adipocyte, Plin2 was first identified by the Serrero group
as an RNA transcript significantly induced during differentiation (6). Perplexing for some time was that Plin2 protein
decreases as adipocyte differentiation progresses. This has
been, at least in part, clarified now with McIntosh and Atshaves’ findings that Plin2 functions to build fat stores. Further,
mature adipocytes and steroidogenic cells express Plin1 (as
discussed above) that functions to mobilize stored lipids for
provision of metabolic fuel substrates and steroid hormone
synthesis, respectively. Other differentiated cells, however,
including pancreatic ␤ cells, retinal epithelial cells, and dendritic cells, rely on Plin2 expression for lipid-dependent processes such as insulin secretion, vision, and antigen presentation, respectively (3–5).
The current results from the Atshaves laboratory are an
important step in understanding how intracellular lipid levels
are tightly controlled. Whereas appropriate intracellular lipid
levels are essential for various cellular functions, fat storage
exceeding intracellular demand may result in lipotoxic events.
In obesity, for example, lipids may accumulate ectopically in
skeletal muscle and contribute to insulin resistance. A particularly exciting aspect of skeletal muscle Plin2 function may be
to enhance the partitioning of excess fatty acids toward triacylglycerol storage in lipid droplets, thereby blunting lipotoxicity-associated insulin resistance (2, 11). Given that obesity
has various deleterious effects on other tissues including the
brain, liver, and vasculature, and is an established risk factor
for many cancers, further analyses of the role of Plin2 in
various diseases such as Alzheimer’s disease, nonalcoholic
fatty liver disease, cardiovascular disease, and cancer is clearly
warranted. Although Plin2 loss of function studies have been
associated with reduced lipid accumulation in the liver and
macrophage foam cells, interpretation of these data is complicated by the subsequent finding that the Plin2-deficient mouse
line unexpectedly expresses an amino-terminal truncation of
Plin2 that may partially replace the function of full-length
Plin2 (1). Nonetheless, these data suggest that in some tissues