Biology Reference
In-Depth Information
organelles are working. As fractionations are never pure, it is always recommended
for each preparation to be assessed for the level of specific organelle markers in the
LD fraction. We also used two additional members of the LD protein, perilipin 5, a
LD-associated protein previously shown to be nutritionally regulated and also to be a
major cardiac LD protein, as well as perilipin 3, another member of the perilipin pro-
tein family whose expression by contrast has been shown not affected by fed and
fasted conditions (
Dalen et al., 2007
). Both perilipin 5 and perilipin 3 are termed ex-
changeable LD-associated proteins as they can be in the cytosol or bound the surface
of LDs (
Brasaemle & Bickel, 2006
). In contrast, perilipin 2 is only found bound to
LDs and thus its expression can be used as a LD-specific marker (
Straub et al., 2013;
Xu et al., 2005
). These organelle-specific markers show that this method produces
highly enriched in LDs from heart tissue (
Fig. 8.2
). Perilipin 2 signal is only detected
in the LD fraction, while perilipin 5 and specifically perilipin 3 can be detected in all
three fractions. Perilipin 5 content increases highly with starvation; however, there is
no change in the detected signals for calnexin and LDH in the other fractions.
ATPase is found essentially in the pellet but is also found associated with the LD
fraction in fasting conditions. We and others have previously shown that perilipin
5 mediates physical interactions between LDs and mitochondria by a yet unknown
mechanism (
Pollak et al., 2013; Wang et al., 2013
). The increased presence of mi-
tochondria proteins in the LD fraction may be due to increased presence of perilipin 5
in this fraction.
To normalize sample loading and allow comparison between cardiac LDs samples
isolated from different physiological conditions, samples loaded on the gel can be
normalized by the amount of proteins measured in the samples or the amount of start-
ing tissue when volume used remained in equal proportions. In our hands, we have
experienced more variation from sample to sample when measuring the amount of
proteins using a commercial Bradford commercial assay, likely due to the high lipid
content. We are now routinely normalizing our samples by initial heart weights and
maintaining volumes in proportion to allow comparison between samples (
Fig. 8.2
).
Here, we demonstrate the feasibility to isolate cardiac LDs, a particularly chal-
lenging task due to the low abundance of LDs and the difficulty to isolate them due to
the fibrous nature of the tissue. This methodology is compatible with secondary as-
says such as proteomics, lipids, and proteins determination that will provide neces-
sary information to understand the role of the LDs in the heart.
8.2
ANALYSIS OF CARDIAC LDS BY 2D ANALYSIS OF LDs
BY CONVENTIONAL TEM
Light microscopy analysis of LDs in heart histological sections has been fairly lim-
ited to visualize the total amount of neutral lipid content by using common lipid
stains such as Oil Red O (
Christoffersen et al., 2003; Kuramoto et al., 2012;
Sharma et al., 2004
). Recently, visualization by histocytochemistry of a very number
of LD-associated proteins, restricted to the LD coat proteins perilipins 2, 3, and 5,
