Biology Reference
In-Depth Information
7.2.3 Use of BODIPY Fatty Acids for Visualization of CLD Dynamics ........... 119
7.2.3.1 Preparation of Mouse Hepatocytes Grown on the Cover Slip or
Glass Bottom Dish................................................................................119
7.2.3.2 Oleic Acid and BODIPY Fatty Acid Incubations of Hepatocytes ..120
7.2.3.3 Microscope Setup and Imaging .................................................121
7.2.3.4 Image Processing and Analysis .................................................122
7.3 Discussion....................................................................................................... 122
Conclusion ............................................................................................................. 125
Acknowledgments ................................................................................................... 125
References ............................................................................................................. 126
Abstract
The liver plays an important role in triacylglycerol (TG) metabolism. It can store
large amounts of TG in cytosolic lipid droplets (CLDs), or it can package TG into
very-low density lipoproteins (VLDL) that are secreted from the cell. TG packaged
into VLDL is derived from TG stored within the endoplasmic reticulum in lumenal
lipid droplets (LLDs). Therefore, the liver contains at least three kinds of LDs that
differ in their protein composition, subcellular localization, and function. Hepatic
LDs undergo tremendous changes in their size and protein composition depending
on the energetic (fasting/feeding) and pathological (viral infection, nonalcoholic
fatty liver disease, etc.) states. It is crucial to develop methodologies that allow
the isolation and analyses of the various hepatic LDs in order to gain insight into
the differential metabolism of these important lipid storage/transport particles in
health and disease. Here, we present detailed protocols for the isolation and analysis
of CLDs and LLDs and for monitoring CLD dynamics.
INTRODUCTION AND RATIONALE
All organisms and cell types investigated so far can store triacylglycerols (TGs), ste-
rol esters in lipid droplets (LDs). Until recently, LDs were largely considered to be
inert organelles, functioning only as lipid-storing bodies. However, it is now clear
that LDs are active and dynamic organelles that play multiple roles in lipid
metabolism, signal transduction, protein storage, and lipid trafficking (
Olofsson
et al., 2009; Walther & Farese, 2012
).
The liver plays a central role in lipid metabolism and storage. In addition to the
critical role of this organ in TG synthesis, storage, and provision of substrates for
b
-oxidation and ketogenesis, the liver is a specialized organ that secretes TG into
the blood in apolipoprotein B (apoB)-containing very-low density lipoprotein
(VLDL) particles. Therefore, hepatocytes contain several types of LDs, each with
a specific subcellular localization and distinct protein composition. In addition to
cytosolic LDs (CLDs) that are present in most other cell types, hepatocytes contain
at least two more types of LDs in the lumen of the endoplasmic reticulum (ER) where
