FP05-5 Temporary AKT Activation Contributes to Endoplasmic Reticulum Stress and Apoptosis in Hepatocytes Induced By Palmitate

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: FP05-Lipids: Regulation & Mechanism of Disease
Basic
Saturday, June 15, 2013: 11:00 AM-11:30 AM
Presentation Start Time: 11:20 AM
Room 133 (Moscone Center)

Poster Board SAT-726
Thiago Matos Ferreira de Araujo*1, Juliana de Almeida Faria2, Andrezza Pinheiro Kinote2, Licio Augusto Velloso3 and Gabriel Forato Anhe4
1University of Campinas - UNICAMP, Campinas, Brazil, 2University of Campinas - UNICAMP, 3University of Campinas, Campinas, Brazil, 4State Univeristy of Campinas, Campinas, SP, Brazil
Background and aims: The “lipotoxicity” caused by excess of fatty acids are known to contribute to nonalcoholic steatohepatitis (NASH) and cell death in hepatocytes due to activation of ER stress-related pathways. AKT is a serine/threonine protein kinase that is essential for regulating cell growth, proliferation, survival and the interaction with environmental stimuli. Classically, AKT is fully activated by phosphoinositide-dependent kinase 1 (PDK1) and mammalian Target of Rapamycin (mTOR) complex 2, leading to inhibition of apoptosis. Alternatively, transitory AKT activation was demonstrated to cause cell death through yet not described mechanisms. ER stress triggers the Unfolded Protein Response (UPR) that is comprised by three independent branches; the PKR-like ER kinase (PERK), the activating transcription factor (ATF)-6 and serine/threonine-protein kinase/endoribonuclease Ire1. The aim of this study is to demonstrate if short-term AKT activation by palmitate is linked to ER stress and apoptosis in HepG2 cells.

Materials and methods: HepG2 cells were treated with palmitate (0,5 mmol/l) pre-conjugated with albumin, rapamycin (500 nmol/l) and/or AKT Inhibitor (100 nmol/l). Cells were also transfected with siRNA targeted to Raptor or a siRNA targeted to Rictor. A scrambled siRNA was used as control. mTORC2/AKT pathway and UPR were assessed by Western blotting. Cell death was assessed by DNA fragmentation.

Results: Palmitate-induced apoptosis was maximal after 12h of treatment. CHOP, a marker of ER-stress-dependent apoptosis was also modulated by palmitate and peaked 12h after exposition to palmitate. Processed ATF6 was upregulated 3h after palmitate exposition and these levels were sustained up to 12h. Palmitate also activated PERK and increased ATF4 expression (respectively after 6 and 12h of treatment). The events were preceded by short-term and transitory AKT activation by palmitate. By inhibiting mTORC1/2 with rapamycin we observed a suppression of palmitate-induced UPR activation and apoptosis. Similarly inhibition of apoptosis and ER stress was also observed with the use of a specific inhibitor for AKT (“AKT inhibitor”) and Rictor knockdown with siRNA.

Conclusion: Our results demonstrated that palmitate induces a short-term and transitory AKT activation that leads to long term activation of UPR and apoptosis. This response is likely to be triggered by mTORC2 activation. All together, those results could help to the understanding of NASH physiopathology.

Nothing to Disclose: TMFD, JDAF, APK, LAV, GFA

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: São Paulo Research Foundation - FAPESP