Biology Reference
In-Depth Information
7.2
METHODS
7.2.1
Isolation of CLDs from mouse liver
Before we start: all samples and solutions throughout the preparation of LDs should
be kept on ice, and make sure that protease inhibitor cocktail is freshly added to all
buffers used during the preparation.
7.2.1.1
Mice, diets, and feeding states
In order to have a successful (purer) CLD fractionation, the amount of lipid present
in the liver should be considered before starting any LD isolation procedures. As a
general rule, the larger amount of lipid in the tissue, the higher the chance of con-
tamination of LDs with other organelles. Therefore, three key interrelated issues
must be taken into account before isolating CLDs from mouse livers: (i) the diet,
(ii) the fasting time, and (iii) the amount of TG present in the tissue. First, high-fat
diets increase the amount of TG in the liver but also make the tissue much more
brittle thereby increasing the likelihood of obtaining contaminated LD fractions. On
the other hand, low-fat diets may result in livers with less TG, but CLDs become
easier to isolate since the chances of contamination with other intracellular mem-
branes are minimized. Second, and equally important, is the feeding state of the
animals: it is known that fasting induces the accumulation of hepatic TG and there-
fore CLD number, size, and also protein composition vary depending on the feed-
ing states of the animals. Fasting times longer than 24 h are not recommended for
the mouse, since the animal enters into a starvation mode at such a prolong fasting
period, and the proteome does not represent a normal physiological state. On the
other hand, we determined that refeeding times (after a 24 h fast) should be no lon-
ger than 6 h if one is to study CLD composition in animals with defined nutritional
states. This is as a mean of synchronicity: after a 24 h fast, animals are eager to eat,
thus all mice feed at a fairly constant pace for at least 5 h. From 6 h on, mice start to
eat less and at a different pace from each other, which has an impact on CLD size,
number, and protein/lipid composition. Refeeding times shorter than 6 h might re-
sult in a significant overlap in CLD protein composition between fasting and
refeeding states. Third, the amount of lipid present in the liver may be first eval-
uated taking into account the genetic background of the animal, since defined liver
lipid phenotypes have been described for different strains of mice. For instance,
Ces1
/
mice present with obesity and mild steatosis (
Quiroga et al., 2012
). Sim-
ilarly, virus-induced knockdown of Atgl also results in hepatic steatosis (
Ong,
Mashek, Bu, Greenberg, & Mashek, 2011
).
7.2.1.2
CLD fractionation
CLDs are isolated by methods developed by
Brasaemle and Wolins (2006)
with
some modifications specific to the liver tissue (
Table 7.1
). Mice should be anesthe-
tized by inhalation with isoflurane and exsanguinated by cardiac puncture. Quickly
but carefully, livers are harvested, perfused with ice-cold PBS, and cleaned from any
