**3. Inhibition of LD formation in hepatocytes deficient in Atg7 after deprivation**

It has been shown that autophagy may play an important role in normal adipogenesis and that inhibition of autophagy by disrupting the *atg7* gene has a unique anti-obesity and insulin sensitization effect [17]. LDs are ubiquitous in eukaryotic cells, while excess free fatty acids and glucose in plasma are converted to TAG and stored as LDs (Fig. 1). However, the mechanism for the generation and growth of LDs in cells is largely unknown. As stated above, Atg7 that mediates LC3 lipidation and is essential for autophagy is involved in LD formation [4, 9]. LD formation accompanied by accumulation of TAG induced by starvation is largely suppressed in hepatocytes that cannot execute autophagy.

It is well known that LC3, microtubule-associated protein A/B light chain 3, is localized on the surface of the isolation membrane when starvation is induced [9]. Using GFP-LC3 transgenic mice, GFP-LC3 becomes dot-shaped, cap-shaped and ring-shaped in hepatocytes and cardiac myocytes under starvation conditions [21]. When immunostained for LC3 under starvation conditions, positive staining is, as shown by GFP-LC3 in TG mice, dot-shaped, cap-shaped, and ring-shaped in hepatocytes and cardiac myocytes (Figs. 2 and 3, Tables 1 and 2). Interestingly, positive staining of LC3 in cardiac myocytes is longitudinally arrayed in parallel to myofilaments. In both hepatocytes and cardiac myocytes, LDs are abundant 24 hours after the onset of starvation [9]. In particular, electron microscopic observations show that no clear-cut autophagosomes are detected in cardiac myocytes, although many large LDs are arranged longitudinally in parallel to the array of myofilaments together with mitochondria. This arrangement of LDs in cardiac myocytes is very similar to that of LC3-positive granules. To examine the relationship between staining patterns of LC3 and LDs in both hepatocytes and cardiac myocytes, double staining for perilipin with LC3 was performed. The results indicate that perilipinpositive LDs are also immunopositive for LC3 on the surface of LDs in both hepatocytes and cardiac myocytes [9]. In hepatocytes, there is also dotted staining of LC3 that is free of LD staining, whereas most LD-positive staining is co-localized with LC3 in cardiac myocytes (Figs. 2 and 3, Tables 1 and 2).

Autophagy Regulates Lipid Droplet Formation and Adipogenesis 153

**Figure 3.** Schematic demonstrations of LC3 staining in cells from mice under fed (left panel) and starved (right panel) conditions. When mice are fed, cells start to produce and store glycogen granules, whereas under starved conditions, lipid droplets are increased and many autophagic granules with double membranes increase in the cytoplasm. In this situation, the lipidated form of LC3 is attached to the isolation membrane of autophagosomes (blue color) and the surface membrane of LDs (yellow). Starvation is further continued, and some mitochondria are enwrapped by the isolation membrane (mitophagy).

**Figure 2.** LC3 localizes not only to autophagosomes but also LDs in liver and heart tissues of mice under starvation conditions for 24 hours. (A-L) Double staining of LC3 (green) (A, D, G, J) and perilipin (B, E) or BODIPY (H, K) (red) in liver (A-C, G-I) and heart (D-F, J-L) tissues. Ring-shaped structures that were costained for LC3 and perilipin in boxed areas (C, F) are enlarged in insets. One of the ring-shaped structures that were costained for LC3 and BODIPY in a boxed area (L) was enlarged in an inset. Bar indicates 5 µm (1 µm in inset). This figure is referred from Biochemical and Biophysical Research Communications (Shibata et al., 382 (2009) 419–423).

152 Lipid Metabolism

myocytes (Figs. 2 and 3, Tables 1 and 2).

Communications (Shibata et al., 382 (2009) 419–423).

hepatocytes and cardiac myocytes under starvation conditions [21]. When immunostained for LC3 under starvation conditions, positive staining is, as shown by GFP-LC3 in TG mice, dot-shaped, cap-shaped, and ring-shaped in hepatocytes and cardiac myocytes (Figs. 2 and 3, Tables 1 and 2). Interestingly, positive staining of LC3 in cardiac myocytes is longitudinally arrayed in parallel to myofilaments. In both hepatocytes and cardiac myocytes, LDs are abundant 24 hours after the onset of starvation [9]. In particular, electron microscopic observations show that no clear-cut autophagosomes are detected in cardiac myocytes, although many large LDs are arranged longitudinally in parallel to the array of myofilaments together with mitochondria. This arrangement of LDs in cardiac myocytes is very similar to that of LC3-positive granules. To examine the relationship between staining patterns of LC3 and LDs in both hepatocytes and cardiac myocytes, double staining for perilipin with LC3 was performed. The results indicate that perilipinpositive LDs are also immunopositive for LC3 on the surface of LDs in both hepatocytes and cardiac myocytes [9]. In hepatocytes, there is also dotted staining of LC3 that is free of LD staining, whereas most LD-positive staining is co-localized with LC3 in cardiac

**Figure 2.** LC3 localizes not only to autophagosomes but also LDs in liver and heart tissues of mice under starvation conditions for 24 hours. (A-L) Double staining of LC3 (green) (A, D, G, J) and perilipin (B, E) or BODIPY (H, K) (red) in liver (A-C, G-I) and heart (D-F, J-L) tissues. Ring-shaped structures that were costained for LC3 and perilipin in boxed areas (C, F) are enlarged in insets. One of the ring-shaped structures that were costained for LC3 and BODIPY in a boxed area (L) was enlarged in an inset. Bar indicates 5 µm (1 µm in inset). This figure is referred from Biochemical and Biophysical Research

**Figure 3.** Schematic demonstrations of LC3 staining in cells from mice under fed (left panel) and starved (right panel) conditions. When mice are fed, cells start to produce and store glycogen granules, whereas under starved conditions, lipid droplets are increased and many autophagic granules with double membranes increase in the cytoplasm. In this situation, the lipidated form of LC3 is attached to the isolation membrane of autophagosomes (blue color) and the surface membrane of LDs (yellow). Starvation is further continued, and some mitochondria are enwrapped by the isolation membrane (mitophagy).

#### 154 Lipid Metabolism


Autophagy Regulates Lipid Droplet Formation and Adipogenesis 155

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

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 knockdown-

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, BODIPY-

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

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

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

production fat bodies in the tissue [17].

cells is also evident in the control cells.

positive LDs largely disappear.

autophagy and lipid metabolism.

Apg, autophagosome; EM, electron microscopy; h, hours; ICH, immunocytochemistry; ++, highly positive; +, positive; +/-, rare; WB, Western blot

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


++, highly positive; +, positive

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

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 metabolism via LD formation.
