Biology Reference
In-Depth Information
Technically, the study of CLD metabolism in the liver should not represent a
problem, provided mild homogenization protocols are followed that preserve the in-
tegrity of intracellular organelles and avoid contamination of CLDs with lumenal
TG-rich lipoproteins and LLDs. Therefore, the study of hepatic CLDs components
has added complexity compared to CLDs from other cells and tissues. A few studies
have been performed to describe the proteome of hepatic CLDs (
Fujimoto et al.,
2004; Turro et al., 2006
). An early method for isolating hepatic CLDs involved dis-
continuous density gradient centrifugation and yielded six discrete bands of lipid par-
ticles, rich in TG and cholesterol (
Ontko, Perrin, & Horne, 1986
). Unfortunately, due
to the lack of knowledge of CLD-associated proteins (PAT family of proteins was
discovered nearly a decade later), the purity and protein composition of the various
fractions method has not been adequately validated.
Here, we describe a method for isolating CLDs from the liver, based on amethod by
Brasaemle and Wolins
(2006)
, and a method to isolate LLDs. The use of a soft tissue
homogenization is crucial to preservation of CLD integrity of LDs. Simple two-step
low-speed centrifugation and a single ultracentrifugation step using a discontinuous
density gradient yield highly purified CLD preparations. We have used this method
to analyze a CLDproteome in fasted and re-fed conditions. The CLDproteome changes
dramatically depending on the feeding state of the mouse and therefore it is highly
advisable that CLDs are prepared from animals in a controlled metabolic state.
It is important to note that this method of CLD isolation can be applied to evaluate
a wide range of metabolic processes such as lipid metabolism in different feeding
states, biochemical determinations such as enzyme activities, particle size, etc.
The analysis of the dynamic nature of these organelles not only provides the tools
for the understanding of molecular mechanisms involved in CLD formation and mo-
bilization, but also paves the road to development of novel therapies for treatment of
pathological conditions.
The research on LLDs has been challenging for multiple reasons: (1) it is dif-
ficult to resolve LLDs from apoB-containing VLDL and its precursors; (2) it is
difficult to compromise the integrity of the microsomal membranes without af-
fecting the integrity of LLDs; (3) LLDs are present in low abundance; (4) con-
tamination from ER-resident proteins needs to be avoided; (5) LLDs are too
small to be visualized by light microscopy, and there had been only limited suc-
cess with electron microscopy in studying LLDs. The protocol we developed
overcomes most of these difficulties and represents a practical and effective
waytopurifyLLDs.
To separate LLDs from apoB-containing VLDL and its precursors, we used im-
munoprecipitation to remove apoB-containing particles, which was proved success-
ful since apoB was absent from the post-IP fraction, while the LLD-associated
carboxylesterase 3/TGH was recovered in this fraction (
Wang et al., 2007
). How-
ever
, care should be taken to maintain the integrity of particles during immunopre-
cipitation procedure; immunoprecipitation should be performed in the absence of
detergents, as they would destroy lipid particles, and/or change protein and lipid
composition of isolated LLDs. Similarly, the method using high pH carbonate
