Autophagy is involved in inflammation, fibrosis, and carcinogenesis-A study using mice model

我们没有阐明肝脏炎症、纤维化和癌变与肝细胞特异性Pten缺陷(Pten KO)小鼠的自噬有关。取而代之的是，我们获得了关于自噬在I类PI3-激酶激活中的抑制机制的新发现。通过电子显微镜获得的形态数据显示，在亮胃素存在的情况下，Pten KO小鼠肝脏中积累的自噬空泡(自噬小体和自溶酶体)比野生型小鼠减少了约70%。此外，突变肝脏中的一些空泡具有腔结构，隔离成分缩小到空泡中心，表明自噬小体向自溶体的成熟受到抑制。14C-亮氨酸标记的长寿命肝细胞蛋白的降解数据表明，与野生型肝细胞相比，Pten KO肝细胞的自噬蛋白降解受到强烈抑制。因此，在Pten KO小鼠的肝脏中，自噬明显受到抑制，其中I类PI3-激酶/Akt信号通路被激活。然而，野生型和Pten KO小鼠肝脏中ATG12-ATG5结合物和脂化的自噬细胞膜标记物LC3-II的水平没有显著差异，提示Pten缺乏本身并不影响ATG结合反应本身。而Percoll密度梯度离心法分离的Pten KO肝脏经亮胃素抑制的自溶酶体对溶酶体(β-己糖苷酶和LGP85)、自噬小体(Lc3-II)和自溶酶体(P32)的表达与野生型肝脏相比有明显的抑制作用。结论：Pten缺乏症的I类PI3-K/Akt信号增强在自噬小体的形成和成熟阶段抑制自噬，但不抑制ATG结合反应。

We did not clarify that hepatic inflammation, fibrosis, and carcinogenesis were involved in affected autophagy in hepatocyte-specific Pten-deficient (Pten KO) mice. Instead, we got a new finding about inhibitory mechanisms of autophagy in the activation of class I PI3-kinase.Morphological data obtained by electron microscopy demonstrated that autophagic vacuoles (autophagosome plus autolysosome) accumulating in the presence of leupeptin decreased by about 70% in the liver of Pten KO mice compared to wild-type mice. Moreover, some vacuoles in mutant livers possessed luminal structures with sequestered components shrunken to the center of vacuoles, suggesting the inhibition of maturation from autophagosome to autolysosome. Data for the degradation of long-lived hepatocyte proteins labeled with 14C-leucine showed that the autophagic protein degradation was strongly suppressed in Pten KO hepatocytes compared to wild-type hepatocytes. Thus, autophagy was markedly inhibited in the liver of Pten KO mice in which class I PI3-kinase/Akt signaling pathway was activated. However, no significant difference was found in the levels of the ATG12-ATG5 conjugate and LC3-II, the lipidated form of LC3, an intrinsic autophagosomal membrane marker, between the livers of wild-type and Pten KO mice, suggesting that Pten deficiency did not affect ATG conjugation reactions per se. While, leupeptin-inhibited autolysosomes from Pten KO livers separated by Percoll density-gradient centrifugation possessed considerably inhibitory expression of lysosomal (beta-hexosaminidase and LGP85), autophagosomal (LC3-II), and autolysosomal (p32) markers compared to wild-type livers.We conclude that enhanced class I PI3-kinase/Akt signaling in Pten deficiency suppresses autophagy at the formation and maturation steps of autophagosomes, without inhibiting ATG conjugation reactions.