Stearoyl-CoA Desaturase 1 Activity Is Required for Autophagosome Formation

Yuta Ogasawara,Eisuke Itakura,Nozomu Kono,Noboru Mizushima,Hiroyuki Arai,Atsuki Nara,Tamio Mizukami,Akitsugu Yamamoto

doi:10.1074/jbc.m114.591065

Abstract

Autophagy is one of the major degradation pathways for cytoplasmic components. The autophagic isolation membrane is a unique membrane whose content of unsaturated fatty acids is very high. However, the molecular mechanisms underlying formation of this membrane, including the roles of unsaturated fatty acids, remain to be elucidated. From a chemical library consisting of structurally diverse compounds, we screened for novel inhibitors of starvation-induced autophagy by measuring LC3 puncta formation in mouse embryonic fibroblasts stably expressing GFP-LC3. One of the inhibitors we identified, 2,5-pyridinedicarboxamide, N2,N5-bis[5-[(dimethylamino)carbonyl]-4-methyl-2-thiazolyl], has a molecular structure similar to that of a known stearoyl-CoA desaturase (SCD) 1 inhibitor. To determine whether SCD1 inhibition influences autophagy, we examined the effects of the SCD1 inhibitor 28c. This compound strongly inhibited starvation-induced autophagy, as determined by LC3 puncta formation, immunoblot analyses of LC3, electron microscopic observations, and p62/SQSTM1 accumulation. Overexpression of SCD1 or supplementation with oleic acid, which is a catalytic product of SCD1 abolished the inhibition of autophagy by 28c. Furthermore, 28c suppressed starvation-induced autophagy without affecting mammalian target of rapamycin activity, and also inhibited rapamycin-induced autophagy. In addition to inhibiting formation of LC3 puncta, 28c also inhibited formation of ULK1, WIPI1, Atg16L, and p62/SQSTM1 puncta. These results suggest that SCD1 activity is required for the earliest step of autophagosome formation.

Highlights

Autophagosome membranes are believed to have a high content of unsaturated fatty acids, but the roles of unsaturated fatty acids in autophagy are not clear
To determine whether 28c suppresses the signal transduction pathway leading to mTOR, we investigated whether 28c suppresses rapamycin-induced autophagy. 28c strongly suppressed GFP-LC3 puncta formation (Fig. 7, A and B) and conversion of LC3-I into LC3-II (Fig. 7C) upon rapamycin treatment
One of the effective inhibitors that we identified has a structure similar to that of a chemical previously described as an SCD1 inhibitor

Summary

Background

Autophagosome membranes are believed to have a high content of unsaturated fatty acids, but the roles of unsaturated fatty acids in autophagy are not clear. The molecular mechanisms underlying formation of this membrane, including the roles of unsaturated fatty acids, remain to be elucidated. Two well known inhibitors of autophagy are 3-methyladenine and wortmannin, both of which suppress autophagosome formation at the same step, production of PtdIns[3]P, by inhibiting PtdIns 3-kinase [23]. We identified several inhibitors of autophagy by screening a chemical library consisting of structurally diverse small molecules In this screen, we counted LC3 puncta after starvation in mouse embryonic fibroblasts stably expressing GFP-LC3 (GFP-LC3 MEFs). One of the inhibitors we identified, 2,5-pyridinedicarboxamide, N2,N5-bis[5-[(dimethylamino)carbonyl]-4-methyl-2thiazolyl], is structurally similar to a previously known stearoyl-CoA desaturase (SCD) 1 inhibitor [24] Another SCD1 inhibitor, 28c [25], inhibited autophagy. Ours is the first report that demonstrates a requirement for SCD1 activity in mammalian autophagy

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION