**6. Concluding remarks**

156 Lipid Metabolism

the same conditions [9] (Fig. 1).

found in hepatocytes if the mice are starved for 48 hours.

not observed in liver-specific Atg7-deficient mice [8].

starvation.

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

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

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 The diet is then withdrawn and the mice are again starved.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

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 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 SNARE fusion machinery [31].

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 smaller with multiple LDs [17].

Autophagy Regulates Lipid Droplet Formation and Adipogenesis 159

It must be readdressed whether autophagy may affect lipid metabolism in a tissue- or cellspecific manner. If autophagy functions and regulates lipogenesis or lipolysis in a different way in non-adipocytes, it must be answered what molecular mechanisms work by which the autophagic machinery recognize LDs to enwrap for degradation or fuse with each other,

This paper was supported by Grants-in-Aid for Scientific Research (B), (23390041) and partly for Scientific Research on Innovative Areas (23111004), and by MEXT-supported

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*Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan* 

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**Figure 4.** As shown by Murphy [16], a large LD3 that is attached to the endoplasmic reticulum may be degraded by ER-associated lipases and TAG may be re-synthesized. Newly formed LD1 would re-grow as LD2 in the cytoplasm. As has been shown by Nakatogawa et al. [32], bilayered liposomes with Atg8 undergo hemifusion (left upper), since Atg8 molecules are oligomerized with each other. If LC3 molecules are oligomelized on the surface of LDs (right upper), LDs would undergo fusion and grow in size.

It must be readdressed whether autophagy may affect lipid metabolism in a tissue- or cellspecific manner. If autophagy functions and regulates lipogenesis or lipolysis in a different way in non-adipocytes, it must be answered what molecular mechanisms work by which the autophagic machinery recognize LDs to enwrap for degradation or fuse with each other, and how they are regulated.

### **Author details**

158 Lipid Metabolism

size.

**Figure 4.** As shown by Murphy [16], a large LD3 that is attached to the endoplasmic reticulum may be degraded by ER-associated lipases and TAG may be re-synthesized. Newly formed LD1 would re-grow as LD2 in the cytoplasm. As has been shown by Nakatogawa et al. [32], bilayered liposomes with Atg8 undergo hemifusion (left upper), since Atg8 molecules are oligomerized with each other. If LC3 molecules are oligomelized on the surface of LDs (right upper), LDs would undergo fusion and grow in Yasuo Uchiyama and Eiki Kominami *Departments of Cell Biology and Neurosciences, and Biochemistry, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan* 
