The long term objective of the proposed studies is to develop a model for the assembly of very low density lipoproteins (VLDL) by the liver. The prevailing model for VLDL assembly is a two step model in which the formation of a small, dense, phospholipid- rich, apoB-containing particle in the rough endoplasmic reticulum (ER) (step 1) is followed by the addition of bulk triglyceride within the ER (step 2). Studies from our laboratory have suggested that assembly is not complete within the ER, and have led to our working hypothesis: VLDL is assembled in the liver in a three-step process with the first two steps occurring in the ER and the final step, the addition of triglyceride and phospholipid, occurring in route to or within the Golgi apparatus. To test this working hypothesis we propose the following specific aims:
Specific Aim #1. To define the role of the Golgi apparatus in the third step of VLDL assembly. These studies will utilize both in vivo and in vitro approaches to define further the mechanism of lipidation of nascent VLDL within the Golgi apparatus and to evaluate the role of microsomal triglyceride transfer protein (MTP) in this process.
Specific Aim #2. To evaluate the contributions to VLDL assembly during vesicular transport from the endoplasmic reticulum to the Golgi apparatus. It is our hypothesis that assembly continues during the trafficking of nascent particles from the ER to the Golgi apparatus. These studies will utilize an in vitro vesicle generation system to assess assembly during transport.
Specific Aim #3. To define the role of apoE in the assembly of VLDL. ApoE deficiency decreases hepatic triglyceride secretion by 50%, which corresponds to our estimates of the mass of triglyceride added to forming VLDL post ER. These studies will test the hypothesis that apoE modulates the intracellular distribution of triglyceride and affects the lipidation of nascent lipoproteins. Elevated concentrations of low density lipoproteins (LDL) in the plasma represent a primary risk factor for atherosclerotic heart disease. Since DL are formed in the plasma compartment exclusively through the intravascular catabolism of VLDL, an understanding of the intracellular events in the assembly pathway of VLDL may prove important in developing new treatment strategies for patients with elevated levels of LDL and at risk for atherosclerotic heart disease.
