Insulin-dependent apolipoprotein B degradation is mediated by autophagy and involves class I and class III phosphatidylinositide 3-kinases

Insulin acutely stimulates the degradation of apolipoprotein B (apo B) which decreases very low density lipoprotein (VLDL) secretion by liver. Insulin-dependent apo B degradation (IDAD) occurs following phosphatidylinositide 3-kinase (PI3K) activation and involves lysosomal degradation. Insulin suppression of apo B secretion is blocked by over-expression of phosphatase and tensin homologue (PTEN) in McArdle RH7777 (McA) cells suggesting the importance of Class I PI3K generated PI (3,4,5) triphosphate (PIP3) in IDAD. Classical autophagy inhibitors including 3-methyladenine, l-asparagine and bafilomycin A1 also blocked the ability of insulin to suppress apo B secretion by rat hepatocytes (RH) suggesting that IDAD occurs through an autophagy-related mechanism. IDAD is also blocked following over-expression in McA cells of a dominant negative kinase-defective Vps34, a class III PI3K that generates PI 3-monophosphate required for autophagy. Vps34 inhibition of IDAD occurs without altering insulin-dependent S473 phosphorylation of Akt indicating PI3K/PIP3/Akt signaling is intact. Cellular p62/SQSTM1, an inverse indicator of autophagy, is increased with insulin treatment consistent with the known ability of insulin to inhibit autophagy, and therefore the role of insulin in utilizing components of autophagy for apo B degradation is unexpected. Thapsigargan, an inducer of endoplasmic reticulum (ER) stress, and a recently demonstrated autophagy inhibitor, blocked apo B secretion which contrasted with other autophagy inhibitors and mutant Vps34 results which were permissive with respect to apo B secretion. Pulse chase studies indicated that intact B100 and B48 proteins were retained in cells treated with thapsigargan consistent with their accumulation in autophagosomal vacuoles. Differences between IDAD and ER stress-coupled autophagy mediated by thapsgargin suggest that IDAD involves an unique form of autophagy. Insulin action resulting in hepatic apo B degradation is novel and important in understanding regulation of hepatic VLDL metabolism.