The MAP1-LC3 conjugation system is involved in lipid droplet formation

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Abstract

Lipid droplets (LDs) are ubiquitous in eukaryotic cells, while excess free fatty acids and glucose in plasma are converted to triacylglycerol (TAG) and stored as LDs. However, the mechanism for the generation and growth of LDs in cells is largely unknown. We show here that the LC3 lipidation system essential for macroautophagy is involved in LD formation. LD formation accompanied by accumulation of TAG induced by starvation was largely suppressed in the hepatocytes that cannot execute autophagy. Under starvation conditions, LDs in addition to autophagosomes were abundantly formed in the cytoplasm of these tissue cells. Moreover, LC3 was localized on the surface of LDs and LC3-II (lipidation form) was fractionated to a perilipin (LD marker)-positive lipid fraction from the starved liver. Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid metabolism via LD formation.

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Materials and methods

Antibodies. Antibodies to rat full-length LC3 [12] were prepared by immunizing the recombinant rat full-length LC3 protein. Antibodies were purified from anti-sera with affinity chromatography coupled with antigens. Anti-perilipin, anti-ApoB, and anti-GAPDH antibodies were purchased from Progen and Ambion.

Measurement of total triacylglycerol. Cells or tissues were lysed in 0.02% of SDS and total lipids were extracted using the method of Bligh and Dyer [13]. Triacylglycerol contents in the

LD formation is suppressed in hepatocytes by an Atg7 deficiency

LDs accumulate abundantly in the cytoplasm of hepatocytes when mice are starved for 12 to 48 h. We first examined whether LDs are normally formed in the liver of conditional Atg7-deficient mice (Atg7flox/flox: Albumin-Cre) at 22 days of age after weaning. LDs stained with 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY), an indicator for neutral lipids like TAG [18], were sparsely detected in hepatocytes of Atg7-deficient mouse livers under starvation conditions for 12 h,

Acknowledgments

The authors thank K. Ikeue, K. Ohta, A. Koseki and K. Isahara for technical assistance. This work was supported by a grant-in-aid for Creative Scientifc Research (to Y.U., M.K., M.S. and T.U.) from the Japan Society for the Promotion of Science.

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    These authors equally contributed to this work.

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