Biology Reference
In-Depth Information
VLDL is assembled: the lumenal apoB-free lipid droplets (LLDs) and the
apoB-containing particles (VLDL and its precursors). LLDs have been proposed to
provide TG source for the bulk lipidation of VLDL precursors. Because the hepatic
CLDs, LLDs, and VLDL are important contributors to whole body energy homeosta-
sis, it has become important to characterize these intracellular TG storage entities.
The most challenging issue in characterization of CLDs, LLDs, and the nascent VLDL
is a reliable method to separate these very distinct LDs. Added complication is the rap-
idly changing composition of LD-associated proteins with various metabolic states of
the hepatocyte (feeding/fasting, stress induced by viral infection, etc.). It is therefore
important to take into consideration these factors when studying hepatic LDs.
CLDs are composed of a lipid core, mainly TG, cholesterol esters (CE), and
retinyl esters, surrounded by an amphipathic lipid monolayer (phospholipids and free
cholesterol) decorated with LD-associated proteins including the PAT (
P
erilipin1,
A
DRP/Perilipin2,
T
IP47/Perilipin3) protein family (
Bickel, Tansey, & Welte,
2009; Farese & Walther, 2009; Kuhnlein, 2012; Yang et al., 2012
). Hepatic CLDs
also contain numerous proteins that are also found on CLDs in adipocytes and other
cell types, such as ER-resident proteins, Rab GTPases, and cytoskeleton
components, indicating that LDs might share similar regulation in all cell types.
To date, only a few studies have been performed investigating protein composition
of hepatic CLDs. One of these studies was performed in rat liver following partial
hepatectomy, where 50 proteins were identified, including perilipin 2, ER-resident
proteins, lipid and vitamin metabolism enzymes, cytoskeletal components, cell
signaling and cell activation regulation proteins, and a number of proteins that par-
ticipate in diverse intracellular trafficking pathways and exocytosis (
Turro et al.,
2006
). Another work performed in the HuH7 hepatoma cell line identified 17 pro-
teins, including perilipin 2 and lipid and steroid metabolism enzymes as the most
abundant proteins (
Fujimoto et al., 2004
). Except for the presence of PAT protein
family, hydroxysteroid dehydrogenases, and Rab5 GTPase, no similarities in
protein contents between the two LD compositions were observed, possibly reflect-
ing the differences between human hepatoma cell line (HuH7) and the liver, and/or
differences in the metabolic state of the cells. Lack of overlap between the
proteomes published by different groups also to a certain degree reflects the various
degree of contamination present in all subcellular organelles isolated by density
centrifugation. Even though a large amount of contaminants are removed
while LDs partition across layers of a density gradient, it is impossible to remove
all, especially since most common contaminations arise from hydrophobic
proteins nonspecifically interacting with LDs, as well as from abundantly
expressed proteins. It is also likely that some LDs are in the continuum with the
ER (the site of LD origin), thus a small amount of the ER proteome may copurify
with LDs.
Similarly, studies investigating hepatic LLDs are limited, mainly because they
have proved very challenging. Light microscopy is not suitable for observing LLDs
since their size is within or below the range of lipoproteins (ranging from 7 to
200 nm) and is under the detection limit of conventional light microscopy. Even with
the state-of-the-art super
resolution microscopy techniques,
it
is difficult
to
