| Budget Amount *help |
¥4,010,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
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.
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