In Vitro Analysis of the Very‐Low Density Lipoprotein Export from the Trans‐Golgi Network

Abstract

The movement of mature VLDL particles from the TGN to the plasma membrane (PM) is a complex physiological process that plays a critical role in hepatic lipid homeostasis. However, the molecular mechanisms regulating these intracellular transport events had not been studied until recently because of the lack of appropriate molecular assays and techniques. This unit provides a detailed description of cell‐free approaches and techniques to study the TGN‐to‐PM transport of the mature VLDL at the molecular level. A major emphasis is placed on the preparation and purification of sub‐cellular organelles because the success of in vitro assays for the vesicle formation and fusion depends on the quality of the isolated TGN, hepatic PM and hepatic cytosol. A number of critical factors that control the formation of mature VLDL‐containing vesicle, the PG‐VTV, from the TGN and their subsequent targeting to and fusion with the hepatic PM have been discussed. © 2015 by John Wiley & Sons, Inc.

Cathepsin B regulates hepatic lipid metabolism by cleaving liver fatty acid–binding protein

“…For the preparation of ER and Golgi, McA-RH7777 cells were washed once with buffer A (136 mM NaCl, 11.6 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 , 7.5 mM KCl, 0.5 mM dithiothreitol, pH 7.2) and resuspended in 0.25 M sucrose in 10 mM Hepes containing 50 mM EDTA and protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany) and homogenized using Parr cell disruption vessel at 1,100 p.s.i. for 40 min followed by isolation and purification of the ER and Golgi using a sucrose step gradient as described previously (25,44).…”

“…Cytosol was prepared from McA-RH7777 cells using the same procedure as reported earlier (25,44)). In brief, McA-RH7777 cells were washed once with buffer B (25 mM Hepes, 125 mM KCl, 2.5 mM MgCl 2 , 0.5 mM DTT, and protease inhibitors, pH 7.2) and homogenized using a Parr cell disruption vessel at 1,100 p.s.i.…”

Silencing of Small Valosin-containing Protein-interacting Protein (SVIP) Reduces Very Low Density Lipoprotein (VLDL) Secretion from Rat Hepatocytes by Disrupting Its Endoplasmic Reticulum (ER)-to-Golgi Trafficking

“…Metabolic Labeling of Hepatocytes and Isolation of ER and Golgi-Hepatocytes were metabolically labeled using [ 3 H]oleic acid, and the ER containing [ 3 H]triacylglycerol (TAG) was prepared using the same procedure as described previously (11,30). Briefly, freshly isolated primary hepatocytes or McARH7777 cells were washed once with buffer A (136 mM NaCl, 11.6 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 , 7.5 mM KCl, 0.5 mM dithiothreitol, pH 7.2) and resuspended in buffer A supplemented with [ 3 H]oleic acid (100 Ci) complexed with bovine serum albumin (BSA) and incubated at 37°C for 35-40 min.…”

“…Hepatocytes were then resuspended in 0.25 M sucrose in 10 mM Hepes containing 50 mM EDTA and protease inhibitors (Roche Diagnostics GmbH) and homogenized using a Parr cell disruption vessel at 1,100 p.s.i. for 40 min followed by isolation and purification of the ER and Golgi using a sucrose step gradient as reported earlier (8,30,31).…”

“…Preparation of Hepatic Cytosol-Hepatic cytosol was prepared utilizing the same methodology as reported previously (8,30). In brief, freshly harvested hepatocytes were washed once with buffer B (25 mM Hepes, 125 mM KCl, 2.5 mM MgCl 2 , 0.5 mM DTT, protease inhibitors, pH 7.2) and homogenized using a Parr cell disruption vessel at 1,100 p.s.i.…”