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of contacted LD with neutral lipid transferred from the smaller LD to the larger one
(
Gong et al., 2011
). Importantly, we have recently observed that Plin1, a major LD
surface protein in adipocytes, interacts with Fsp27 and facilities its activity (
Sun
et al., 2013
). The cooperative action between Fsp27 and Plin1 likely contributes
to the formation of unilocular LDs in white adipocytes.
In the following sections, we summarize our methods to quantitatively measure
LD sizes, neutral lipid exchange between LDs, and Fsp27-mediated LD fusion.
These methods will greatly facilitate our understanding of molecular pathways gov-
erning LD growth in adipocytes.
14.1
QUANTITATIVE MEASUREMENT OF LD SIZE
LDs have variable sizes and tend to cluster with each other in many different cell
types. The heterogeneity of LD sizes roots in the dynamic regulation of lipid metab-
olism on individual LD and inter-LD neutral lipid flux (
Gong et al., 2011; Wilfling
et al., 2013
) and may have profound effect on lipid metabolism (
Nishino et al., 2008;
Toh et al., 2008
). To evaluate the effect of genetic, pharmacological, and nutrient
manipulation on LD sizes, we developed a method to quantitatively measure the sizes
of heterogeneous population of LDs. Briefly, TAG in the LDs are metabolically labeled
with Bodipy 558/568-conjugated fluorescent fatty acids, which confers the LD staining
with higher photostability in comparison with Bodipy-FL. The automatic image anal-
ysis software is used to identify and measure the sizes of labeled LDs.
14.1.1
Measuring LD sizes in 3T3-L1 preadipocytes or mature
adipocytes
14.1.1.1
Materials
3T3-L1 preadipocytes
Six-well cell culture plate
4% paraformaldehyde in PBS
DAPI (4
0
,6-diamidino-2-phenylindole), 10 mM in deionized water
Mounting media
Acid-washed cover slips and microscope slides
10 mM oleic acid coupled to BSA (10 mg/ml)
Bodipy 558/568 C12 fatty acid analogue (1 mg/ml in ethanol, Life Technologies)
Microscope equipped with fluorescence and optical sectioning
Lipofectamine 2000 (Life Technologies)
14.1.1.2
Experimental procedure
1.
0.4
10
6
3T3-L1 preadipocytes are seeded to six-well culture plate containing
cover slip (24
24 mm) in DMEM containing 10% FBS.
2.
After overnight culture, cells are transfected with specific plasmid DNA or
siRNA using Lipofectamine 2000 according to the manufacturer's instruction.
3.
4-6 h after transfection, cells are washed with DMEM and incubated in fresh
DMEM containing 10% FBS, 200
M of oleic acids complexed with BSA, and
m
0.2-1
g/ml Bodipy 558/568 C12 fatty acids.
m
