Here, we report the involvement of autophagy in ER stress–induced degradation of apoB and provide evidence for a significant role of autophagy in regulating apoB biogenesis in primary hepatocyte systems. Induction of ER stress following short-term glucosamine treatment of McA-RH7777 cells resulted in significantly increased
colocalization of apoB with click here green fluorescent protein–microtubule-associated protein 1 light chain 3 (GFP-LC3), referred to as apoB-GFP-LC3 puncta, in a dose-dependent manner. Colocalization with this autophagic marker correlated positively with the reduction in newly synthesized apoB100. Treatment of McA-RH7777 cells with 4-phenyl butyric acid, a check details chemical ER stress inhibitor, prevented glucosamine- and tunicamycin-induced increases in GRP78 and phosphorylated eIF2α, rescued newly synthesized [35S]-labeled apoB100, and substantially blocked the colocalization of apoB with GFP-LC3. Autophagic apoB degradation was also observed in primary rat and hamster hepatocytes at basal conditions as well as upon the induction of ER stress.
In contrast, this pathway was inactive in HepG2 cells under ER stress conditions, unless proteasomal degradation was blocked with N-acetyl-leucinyl-leucinyl-norleucinal and the medium was supplemented with oleate. Transient transfection
of McA-RH7777 cells with a wild-type protein kinase R–like ER kinase (PERK) complementary DNA resulted in dramatic induction of apoB autophagy. In contrast, transfection with a kinase inactive mutant PERK gave rise to reduced apoB autophagy, suggesting that new apoB autophagy may occur via a PERK signaling–dependent mechanism. Conclusion: Taken together, these data suggest that induction of ER stress leads to markedly enhanced apoB autophagy in a PERK-dependent pathway, which can be blocked with the chemical chaperone 4-phenyl butyric acid. ApoB autophagy rather than proteasomal degradation may be a more pertinent physiological mechanism regulating hepatic lipoprotein production in primary hepatocytes. (HEPATOLOGY 2011;) Apolipoprotein B100 (apoB), the major protein component of low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL), is constitutively synthesized in the liver and regulated through cotranslational and posttranslational degradation.1, 2 Intracellular degradation of newly synthesized apoB has been shown to involve various mechanisms including endoplasmic reticulum (ER)-associated degradation (ERAD), ER60-associated degradation, LDL receptor–associated degradation, and autophagy.