ROLE OF APOLIPOPROTEIN A-V IN TRIGLYCERIDE METABOLISM

ApoA-V, a 366 amino acid protein synthesized by the liver, was identified in 2001 by comparative sequence analysis. The APOA5 gene is located downstream of the AI/CIII/AIV gene cluster on human chromosome 11q23. Apolipoprotein A-V (apoA-V) plasma concentrations are low (~100-200 µg/L) in comparison to other apolipoproteins, yet exert significant effects on triglyceride homeostasis. To examine the basis for its low concentration in plasma, the secretion efficiency of apoA-V was measured in stably transfected McA-RH7777 rat hepatoma cells. Pulse-chase analysis revealed that only ~20% of newly synthesized apoA-V was secreted into culture medium within two hours post-synthesis. Similar results were obtained in transfected nonhepatic (CHO) cells. In neither cell system was apoA-V found associated with cell surface heparin sulfate proteoglycans. Similar results were also obtained when endogenous mouse apoA-V was analyzed in primary hepatocytes. In addition to its low secretion efficiency, the electrophoretic banding pattern of apoA-V suggested that the protein is susceptible to intracellular proteolysis with ~60% undergoing presecretory turnover within 2 hours post-synthesis. To study the regulation of apoA-V by lipid synthesis, stably transfected McA-RH7777 cells were treated with 0.8 mM sodium oleate. The resulting increase in triglyceride synthesis resulted in a dramatic decline in apoA-V secretion, a corresponding increase in cell-associated apoA-V, but no change in total apoA-V recovery. Reduced secretion was accompanied by movement of apoA-V onto cytosolic lipid droplets, as evidenced by apoA-V's colocalization with ADRP, a lipid droplet marker, and its flotation during sucrose gradient ultracentrifugation. The possible intracellular role of apoA-V in the regulation of apoB and TG secretion was examined by generating stably transfected doxycycline-inducible McA-RH7777 cells. Upon induction of apoA-V expression, a 32% decrease in apoB-100 secretion and a 21% decrease in triglyceride secretion were observed. In addition, apoA-V expression caused a 57% decrease in the apoA-V in the d<1.006 g/ml VLDL density fraction during equilibrium density gradient ultracentrifugation. These results indicate that 1) apoA-V inefficiently traffics within the secretory pathway but that its intracellular itinerary may be regulated by changes in TG synthesis and/or accumulation and 2) part of the effect of apoA-V on TG metabolism could be exerted at the level of hepatic TG mobilization and secretion.

Grantor dc:publisher

Wake Forest University

Year dc:date.issued

2009

Author dc:creator

• Blade, Anna

• Apolipoprotein

Language dc:language.iso

en_US

Handle dc:identifier.uri

http://hdl.handle.net/10339/14862

OAI identifier oai:identifier

oai:wakespace.lib.wfu.edu:10339/14862