**4. Generation of LDs is inhibited in various LC3 mRNA-knockdown cultured cells**

The cytoplasmic LD is an organelle 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 efficiency energy storage [16]. However, it remains largely unknown how LDs are generated or grow in the cytoplasm. As has been shown previously, the Atg conjugation system that is essential for autophagosome formation is involved in LD formation in hepatocytes and cardiac myocytes. This tendency has also been confirmed in white adipose tissue of conditional Atg7-knockout mice that show less production fat bodies in the tissue [17].

154 Lipid Metabolism

positive; +/-, rare; WB, Western blot

lipid fraction

++, highly positive; +, positive

**Table 2.** Proteins detected in lipid fractions

metabolism via LD formation.

**cultured cells** 

hepatocytes cardiac myocytes

Liver heart

24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h ++ ++ ++ ++ +/- +/- ++ +

Starvation Apg LD Apg LD

LC3 (ICH) ++ ++ ++ ++ +/- - + +/- LC3-II (WB) ++ ++ ++ ++ ++ +/- + +/-

Mitophagy (EM) +/- + not detected Apg, autophagosome; EM, electron microscopy; h, hours; ICH, immunocytochemistry; ++, highly positive; +,

**Table 1.** Morphological changes detected in hepatocytes and cardiac myocytes after starvation

ADRP ++ ++ LC3-II + +

In fact, cytosolic LC3 is converted to membrane-bound LC3 (LC3-II) in both hepatocytes and cardiac myocytes 24 hours after the start of starvation. Electron microscopic morphometry reveals that the volume densities of autophagosomes/autolysosomes and LDs increase in hepatocytes 24 and 48 hours after the onset of starvation, whereas autophagosomes and autolysosomes are rarely found in cardiac myocytes and the volume density of LDs is only counted and significantly increased in them [9]. The amounts of TAG in hepatocytes and cardiac myocytes significantly increase after the onset of starvation, whereas the increase in TAG amount is much lower in cardiac myocytes than in hepatocytes and continues until 24 hours. Moreover, LC3 is localized on the surface of LDs and LC3-II (lipidated form) is fractionated into a perilipin and ADRP (LD marker)-positive lipid fraction from the starved liver and cardiac myocytes, respectively. In fact, the surface of such LDs obtained from the LD fraction is labeled by gold particles showing the antigenicity of LC3. Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid

**4. Generation of LDs is inhibited in various LC3 mRNA-knockdown** 

The cytoplasmic LD is an organelle 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 efficiency energy storage [16]. However, it remains largely unknown how LDs are generated or grow in the cytoplasm. As has been shown previously, the Atg conjugation system that is essential for autophagosome

Staining of LD in LD fraction from livers with anti-LC3 ++

It has been shown that LDs temporally accumulate in the cultured cell lines during proliferation [22]. We have confirmed that LDs are produced in cultured cells seeded at a density of 70% confluency [11]. Accordingly, it has been shown that LDs that are stained with BODIPY are significantly augmented in PC12 cells 12 hours after the start of cultures, while immunosignals for LC3 are colocalized with BODIPY-positive LDs [11]. By immunoelectron microscopy, gold particles indicating LC3 are found on the surface of LDs in PC12 cells. Moreover, by cell fractionation the membrane type of LC3 is demonstrated in the perilipin-positive LD fraction. It still remains unknown whether LC3 itself is involved in LD formation. Since LC3 is a substrate of Atg7, cultured cell lines such as HeLa cells, PC12 cells, HepG2 cells, and Cos-1 cells, were examined to check the relationship of LC3 and LD formation. Expression of LC3 was suppressed by the method of RNA interference (RNAi), and it was found that LD formation is largely inhibited in these cells. TAG, a major component of LDs, is synthesized and degraded in LC3 mRNA-knockdown cells as well as in control cells. Interestingly, the potential for bulk protein degradation in the knockdowncells is also evident in the control cells.

3T3 L1 cells, a progenitor cell line of adipocytes, accumulate LDs 12 hours after the start of cultures and LD formation is suppressed in the cells when mRNA of LC3 is knocked down [11]. Differentiation of L1 cells into adipocytes is confirmed by the mRNA expression of sterol regulatory element binding factor 1 (SREBF1) and peroxisome proliferator activated receptor (PPAR), adipose specific proteins. It takes 6days until the L1 cells differentiate, and as the cells differentiate, it is found that the amount of LC3 mRNA also increases. In this differentiated situations, the surface of LDs in L1 cells is covered with perilipin and LC3. In LC3 mRNA-knockdown L1 cells, however, BODIPYpositive LDs largely disappear.

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.

## **5. Connection between autophagy and lipid metabolism**

Recent studies provide supporting evidence for a connection between autophagy and lipid metabolism, both lipid storage and lipolysis. The involvement of autophagy in lipolysis of LDs in hepatocytes has been reported by the groups of Czaja and Cuervo, who showed that loss of Atg7 (Atg7Flox/Flox:albumin-Cre mice) results in accumulation of LDs in hepatocytes [18, 23-25]. Lipophagy, which is a form of autophagy that enwraps LDs by the isolation membrane has recently been considered important for the production of FFAs by degrading TAGs under acidic milieu and the FFAs produced fuel cellular rates of mitochondrial -oxidation [18, 23-25]. This process, called lipophagy has recently been

#### 156 Lipid Metabolism

thought to function to regulate intracellular lipid stores, cellular levels of free lipids such as fatty acids and energy homeostasis [25]. On the contrary, as described in this chapter, it has been shown that loss of Atg7 (Atg7Flox/Flox:albumin-Cre mice) largely suppresses LD formation in hepatocytes and cardiac myocytes 24 hours after the start of starvation, although numerous LDs accumulate in normal hepatocytes and cardiac myocytes under the same conditions [9] (Fig. 1).

Autophagy Regulates Lipid Droplet Formation and Adipogenesis 157

In adipose tissue, knock out of either Atg7 [9] or Atg5 [17] leads to reduced accumulation of lipid and impaired differentiation of adipocytes. The mutant white adipocytes are smaller with multiple LDs. It has been suggested that Atg5 deletion causes adipogenesis arrest at the later stages of mouse embryos. The possibility that a defect of Atg5 dependent Atg8 (LC3) lipidation and translocation to LDs of adipocytes may result in inefficient droplet fusion, which contributes to the defect of adipogenesis cannot be ruled out. The same authors observed reduced differentiation in Atg 5-deficient mouse embryonic fibroblasts (MEFs) model. An essential role of autophagy in lipid storage is also suggested in fat body cells of Drosophila. Knockdown of Atg1 or Atg6 led to small lipid droplets [27]. The paper has also indicated that Rab32 may regulate lipid storage by affecting autophagy [27]. Rab32 and several other Rabs have been found to affect the size of lipid droplets [27] and the mechanism of droplet fusion with atg8 (LC3) and Rab families awaits further studies. Thus, in adipocytes autophagy may contribute to LD

For hydrolysis of TAG in LDs of adipocytes, the molecular processes of lipolysis are becoming clear. Adipose tissue TAG lipase first acts on TAG to hydrolyze a fatty acyl chain [28]. Hormone sensitive lipase mediates the second step of lipolysis, diacylglycerol cleavage to monoacylglycerol. Finally monoacylglycerol lipase hydrolyzes the last side

Lipid droplets (LDs) are key cellular organelles involved in lipid storage and mobilization. In non-adipocytes, LDs are small, mobile and interact with other cellular compartments. In contrast, adipocytes primarily contain very large, immotile LDs. The marked morphological differences between LDs in adipocytes and non-adipocytes suggest that key differences must exist in the manner in which LDs in different cell types interact with other organelles and undergo fusion and fission with other droplets. It has suggested that droplet fusion is dependent on microtubules, the motor protein dynein [29], Rab proteins [27] and the

PAT proteins also target to the surface of LDs likely through different mechanisms. In adipocytes, TIP47 is found on smaller droplets and perilipin is found on larger LDs [31]. They may bind to different droplets associated proteins. Cleaved (and lipidated) LC3 demonstrated on isolated LDs from non-adipocytes [9] may be involved in LDs biology as well as PAT proteins (Figs. 2 and 3). Nakatogawa et al. [32] have shown that lipidated ATG8 (yeast homologue of LC3) mediates tethering between adjacent membranes of liposomes and stimulates membrane hemifusion, an event that may mimic expansion of the autophagosomal membrane during autophagy (Fig. 4). In LDs, homotropic interactions in adipocytes and non-adipocytes may involve a hemifusion type mechanism to facilitate lipid transfer (Fig.4). In adipocytes lipidated LC3 may also involved in growth of LDs (Fig. 4), as shown by morphological evidence that the mutant white adipocytes are

formation and not significantly to lipolysis.

**6. Concluding remarks** 

SNARE fusion machinery [31].

smaller with multiple LDs [17].

chain.

Electron microscopic analysis of wild-type mice by the former group shows LD-containing autophagosomes (lipophagosomes) under starvation conditions, although the latter group indicates that it is hard to see the presence of LDs enwrapped by double membranes even under starvation conditions, and that different from lipophagy, mitophagy can easily be found in hepatocytes if the mice are starved for 48 hours.

Two groups used different experimental approaches in their studies, which may underlie different conclusions. One critical point is to consider that Atg7Flox/Flox: albumin-Cre mice cause hepatomegaly and hepatitis with accumulation of abnormal organelles in hepatic cells [26].

Recently, metabolic contributions of amino acids released from liver by starvation-induced autophagy in adult animals using liver-specific Atg7-deficient mice have also been studied systematically [8]. That is, liver specific conditional Atg7-knockout mice (Atg7Flox/Flox:Mx1 mice) are generated by the different method from Atg7Flox/Flox:albumin-Cre mice [25]. To delete Atg7 from the liver, Cre expression in the liver was induced by intraperitoneal injection of polyinosinic acid-polycytidilic acid (pIpC), while complete deletion of the Atg7 protein in the liver was verified using immunoblotting analyses. Atg7-knockout mice are used 10 days after the injection of pIpC. No sign of hepatomegaly and hepatitis is observed within 2 weeks after the injection of pIpC. For synchronous induction of autophagy in the liver, mice previously fasted for 24 hours and preserve numerous LDs in hepatocytes, are fed by a pelleted laboratory diet for 2 hour (20:00–22:00) in the dark to suppress autophagic activity to a minimum [8]. The diet is then withdrawn and the mice are again starved. In separate experiments, it has been confirmed that the stomach and intestine are filled with a digested diet at the end of the 2 hour-feeding period. In this situation, hepatocytes become to change from the stage of lipogenesis and glycolysis to that of lipolysis and glycogenesis in hepatocytes. Electron microscopic examinations clearly show that in the control mice numerous LDs are continuously present in hepatocytes after 2 hour-feeding, and decrease during starvation [8]. Most LDs disappear in the liver after 24 hours of starvation, while glycogen granules increase in hepatocytes. In contrast, the number of autophagic vacuoles in the liver after 24 hour of starvation is increased dramatically. Such vacuoles and LDs are not observed in liver-specific Atg7-deficient mice [8].

These findings seem to indicate that LDs that accumulate in the liver during 24 hours fasting decrease in parallel with the following activation of autophagy after a 2 hour-feeding , but that loss of autophagy does not inhibit the disappearance of LDs in the liver. Thus another mechanism may operate in the removal of LDs from the liver that accumulated by starvation.

In adipose tissue, knock out of either Atg7 [9] or Atg5 [17] leads to reduced accumulation of lipid and impaired differentiation of adipocytes. The mutant white adipocytes are smaller with multiple LDs. It has been suggested that Atg5 deletion causes adipogenesis arrest at the later stages of mouse embryos. The possibility that a defect of Atg5 dependent Atg8 (LC3) lipidation and translocation to LDs of adipocytes may result in inefficient droplet fusion, which contributes to the defect of adipogenesis cannot be ruled out. The same authors observed reduced differentiation in Atg 5-deficient mouse embryonic fibroblasts (MEFs) model. An essential role of autophagy in lipid storage is also suggested in fat body cells of Drosophila. Knockdown of Atg1 or Atg6 led to small lipid droplets [27]. The paper has also indicated that Rab32 may regulate lipid storage by affecting autophagy [27]. Rab32 and several other Rabs have been found to affect the size of lipid droplets [27] and the mechanism of droplet fusion with atg8 (LC3) and Rab families awaits further studies. Thus, in adipocytes autophagy may contribute to LD formation and not significantly to lipolysis.

For hydrolysis of TAG in LDs of adipocytes, the molecular processes of lipolysis are becoming clear. Adipose tissue TAG lipase first acts on TAG to hydrolyze a fatty acyl chain [28]. Hormone sensitive lipase mediates the second step of lipolysis, diacylglycerol cleavage to monoacylglycerol. Finally monoacylglycerol lipase hydrolyzes the last side chain.
