The Phosphatidylinositol 3‐Kinase Inhibitors Wortmannin and LY294002 Inhibit Autophagy in Isolated Rat Hepatocytes

TLDR

Phosphatidylinositol 3‑kinase (PI3K) is essential for membrane‑dependent processes, and autophagy—a protein‑degrading pathway—relies on membrane flow. The study aimed to determine whether PI3K is required for autophagy by testing two PI3K inhibitors, wortmannin and LY294002. Researchers incubated isolated rat hepatocytes lacking amino acids with low concentrations of wortmannin or LY294002 and measured proteolysis and autophagic sequestration. Low concentrations of wortmannin (IC50 ≈ 30 nM) and LY294002 (IC50 ≈ 10 µM) strongly inhibited proteolysis and autophagic sequestration in amino‑acid‑deprived hepatocytes, with no further effect when autophagy was already maximally suppressed, and 3‑methyladenine was shown to inhibit PI3K, supporting the conclusion that PI3K activity is required for autophagy.

Abstract

Recent studies indicate that phosphatidylinositol 3‐kinase is essential in the regulation of many processes dependent on membrane flow. Autophagy is a complex pathway in which cell material, including proteins, can be degraded. Membrane flow plays a pivotal role in this process. To find out whether phosphatidylinositol 3‐kinase is also required for autophagy, we tested the effects on autophagy of two structurally unrelated phosphatidylinositol 3‐kinase inhibitors, wortmannin and 2‐(4‐morpholinyl)‐8‐phe‐nylchromone (LY294002). The addition of low concentrations of each of these inhibitors to incubations of hepatocytes in the absence of amino acids resulted in a strong inhibition of proteolysis. The antiproteolytic effect of wortmannin (IC 50 , 30 nM) and LY294002 (IC 50 10 μM) was accompanied by inhibition of autophagic sequestration and not by an increase in lysosomal pH or a decrease in intracellular ATP. No further inhibition of proteolysis by the two compounds was observed when autophagy was already maximally inhibited by high concentrations of amino acids. 3‐Methyladenine, which is commonly used as a specific inhibitor of autophagic sequestration, was an inhibitor of phosphatidylinositol 3‐kinase, thus providing a target for its action. It is proposed that phosphatidylinositol 3‐kinase activity is required for autophagy. 3‐Methyladenine inhibits autophagy by inhibition of this enzyme.