LC3, a microtubule-associated protein1A/B light chain3, is involved in cytoplasmic lipid droplet formation

doi:10.1016/j.bbrc.2010.01.121

Biochemical and Biophysical Research Communications

Volume 393, Issue 2, 5 March 2010, Pages 274-279

https://doi.org/10.1016/j.bbrc.2010.01.121 Get rights and content

Abstract

The cytoplasmic lipid droplet (LD) is one of organelles that has a neutral lipid core with a single phospholipid layer. LDs are believed to be generated between the two leaflets of the endoplasmic reticulum (ER) membrane and to play various roles, such as high effective energy storage. However, it remains largely unknown how LDs are generated and grow in the cytoplasm. We have previously shown that the Atg conjugation system that is essential for autophagosome formation is involved in LD formation in hepatocytes and cardiac myocytes. We show here that LC3 itself is involved in LD formation by using RNA interference (RNAi). All cultured cell lines examined, in which the expression of LC3 was suppressed by RNAi, showed reduced LD formation. Triacylglycerol, a major component of LDs, was synthesized and degraded in LC3 mRNA-knockdown cells as well as in control cells. Interestingly, potential of the bulk protein degradation in the knockdown-cells was also evident in the control cells. These findings indicate that LC3 is involved in the LD formation regardless of the bulk degradation, and that LC3 has two pivotal roles in cellular homeostasis mediated by autophagy and lipid metabolism.

Section snippets

Materials and methods

Antibodies. Antibodies to rat full-length LC3 were prepared by immunizing the recombinant rat full-length LC3 protein [13]. Anti-GAPDH and anti-perilipin antibodies were purchased from Ambion and Progen, respectively.

RNA interference of LC3. The oligonucleotide sequences synthesized for RNAi were listed in Table S1. Two complementary oligonucleotides were annealed and subcloned into pSuper_neo vector (Oligoengine, USA). RNAi vectors were transfected into various types of cells using

Induction of LDs in PC12 cells during cultures for 12 h.

Since the LC3 conjugation system is involved in LD formation in hepatocytes and cardiac myocytes [7], we further examined roles of LC3 in LD formation using cultured tumor cells such as PC12, HeLa, COS1, and HepG2 cells. It has been shown that LDs temporally accumulate in the cultured cell lines during proliferation [22]. We therefore analyzed whether LDs are produced during cultures of the cells in which they were seeded at a density of 70% confluence in culture dishes. When stained for

Discussion

Although in vivo tissue cells are different from proliferating cultured cells, an increase in LDs has also been shown in rodent peripheral tissue cells, especially in hepatocytes and cardiac myocytes when the animals were starved for 24 and 48 h [7]. Fat cells in adipose tissues release FFAs under starvation conditions, while hepatocytes and cardiac myocytes take up released FFAs and store them as LDs to some extent [24]. Thus, it seems likely that such response of peripheral tissue cells to

Acknowledgments

This work was supported by a Grant-in-aid for Creative Scientific Research (to Y.U., M.K., M.S. M.S. and T.U.) from the Japan Society for the Promotion of Science.

References (32)

S.A. Kuznetsov et al.
18 kDa microtubule-associated protein: identification as a new light chain (LC-3) of microtubule-associated protein 1 (MAP-1)
FEBS Lett.
(1987)
S.S. Mann et al.
Molecular characterization of light chain 3. A microtubule binding subunit of MAP1A and MAP1B
J. Biol. Chem.
(1994)
I. Tanida et al.
LC3 conjugation system in mammalian autophagy
Int. J. Biochem. Cell Biol.
(2004)
I. Tanida et al.
Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrates, GATE-16, GABARAP, and MAP-LC3, and facilitates the conjugation of hApg12p to hApg5p
J. Biol. Chem.
(2002)
M. Shibata et al.
The MAP1-LC3 conjugation system is involved in lipid droplet formation
Biochem. Biophys. Res. Commun.
(2009)
C. Londos et al.
Perilipins, ADRP, and other proteins that associate with intracellular neutral lipid droplets in animal cells
Semin. Cell Dev. Biol.
(1999)
J.P. Flatt
Use and storage of carbohydrate and fat
Am. J. Clin. Nutr.
(1995)
S. Jacob
Lipid droplet accumulation in the heart during fasting
Acta Histochem.
(1987)
S.C. Kalhan et al.
Glyceroneogenesis and the source of glycerol for hepatic triacylglycerol synthesis in humans
J. Biol. Chem.
(2001)
M. Koike et al.
Participation of autophagy in storage of lysosomes in neurons from mouse models of neuronal ceroid-lipofuscinoses (Batten disease)
Am. J. Pathol.
(2005)

J.J. Mrotek et al.

A method for the isolation of lipid droplet fractions from decapsulated rat adrenals

Steroids

(1981)

H. Green et al.

An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion

Cell

(1975)

E. Ogier-Denis et al.

Guanine nucleotide exchange on heterotrimeric Gi3 protein controls autophagic sequestration in HT-29 cells

J. Biol. Chem.

(1996)

D.A. Roncari

Fatty acid synthase from human liver

Methods Enzymol.

(1981)

C. Thiele et al.

Cell biology of lipid droplets

Curr. Opin. Cell Biol.

(2008)

H. Nakatogawa et al.

Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion

Cell

(2007)

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LC3, a microtubule-associated protein1A/B light chain3, is involved in cytoplasmic lipid droplet formation

Abstract

Section snippets

Materials and methods

Induction of LDs in PC12 cells during cultures for 12 h.

Discussion

Acknowledgments

FEBS Lett.

J. Biol. Chem.

Int. J. Biochem. Cell Biol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Semin. Cell Dev. Biol.

Am. J. Clin. Nutr.

Acta Histochem.

J. Biol. Chem.

Am. J. Pathol.

Steroids

Cell

J. Biol. Chem.

Methods Enzymol.

Curr. Opin. Cell Biol.

Cell