**2. Microtubule-associated protein 1A/1B light chain 3 (LC3) localizes not only to autophagosomal membranes but also on the surface membrane of LDs in hepatic and cardiac tissues under starvation conditions**

It is well known that hepatocytes are morphologically and functionally different between the periportal and perivenous regions; periportal hepatocytes are glycogenic and lipolytic, while perivenous hepatocytes are glycolytic and lipogenic [19]. Lysosomes in hepatocytes are also more abundant in the perivenous region than in the periportal region [19, 20].

Such a morphological difference is largely altered when mice are starved for 24 or 48 hours; numerous autophagosomes that contain part of the cytoplasm and possess the cisternal or double isolation membranes and autolysosomes appear near bile canaliculi in hepatocytes 24 hours after the commencement of starvation and those that in some cases contain mitochondria are seen 48 hours later [4, 9]. In addition to autophagosomes and autolysosomes, LDs accumulate abundantly in the cytoplasm of hepatocytes. When livers of conditional Atg7-deficient mice (Atg7Flox/Flox: Albumin-Cre) at 22 days, or 6 or 8 weeks of age were examined, positive staining for LDs is sparsely detected in the hepatocytes under starvation conditions for 12 or 24 hours, although they are abundant in the cytoplasm of the hepatocytes of the control littermate mouse liver (Fig. 1). Size and amount of stained LDs were much smaller in hepatocytes deficient in Atg7 than in the control hepatocytes. When examined by electron microscopy, the diameter of LDs in the control hepatocytes is various and ranged up to 2.61 m (the mean diameter (± SD)) is 1.12 ± 0.17 m). Different from the control, mutant hepatocytes mainly possess smaller LDs whose diameter ranged up to 0.83 m (the average diameter is 0.19 ± 0.17 m). Moreover, small LD-like bodies were observed in the luminal space of rough surfaced endoplasmic reticulum (rER), Golgi cisternal ends, and vesicles near the Golgi apparatus of the cells, indicating that the lumenally-sorted LDs are normally produced in the rER of both mutant and control hepatocytes. Importantly, the total TAG amount in Atg7-deficient liver and control livers is half of that in the control liver. These data indicate that the conjugation system of LC3 by Atg7 is required for the formation of LDs.

150 Lipid Metabolism

cells.

via LD formation..

FFAs are also converted to triacylglycerol (TAG) and stored as lipid droplets (LDs) that are

It has been shown that loss of Atg7 largely suppresses LD formations 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 [11]. Moreover, a mouse model with a targeted deletion of *atg7* in adipose tissue has been generated; the mutant mice were slim and contained only 20% of the mass of white adipose tissue (WAT) found in wild-type mice [17]. These mutant mice exhibit a high sensitivity to insulin that reduces low fed plasma concentrations of FFAs, and also exhibit a marked decrease in plasma concentrations of leptin but not adiponectin, and lower plasma concentrations of TAG. LDs, initially considered inert lipid deposits, have gained the classification of cytosolic organelles during the last decade due to their defined composition and the multiplicity of specific cellular functions in which they are involved [18]. At present, it remains largely unknown how autophagy is involved in LD metabolism, although lipophagy may occur in

One thing that we have found is that a lipidated form of LC3, representing the Atg8 family of proteins, is localized on the surface of LDs and also in LD fractions, in addition to ADRP and perilipin, representing the PAT family of proteins that cover the surface of LDs. In this review we will introduce the LC3 conjugation system that is involved in lipid metabolism

**2. Microtubule-associated protein 1A/1B light chain 3 (LC3) localizes not only to autophagosomal membranes but also on the surface membrane of** 

It is well known that hepatocytes are morphologically and functionally different between the periportal and perivenous regions; periportal hepatocytes are glycogenic and lipolytic, while perivenous hepatocytes are glycolytic and lipogenic [19]. Lysosomes in hepatocytes are also more abundant in the perivenous region than in the periportal region [19, 20].

Such a morphological difference is largely altered when mice are starved for 24 or 48 hours; numerous autophagosomes that contain part of the cytoplasm and possess the cisternal or double isolation membranes and autolysosomes appear near bile canaliculi in hepatocytes 24 hours after the commencement of starvation and those that in some cases contain mitochondria are seen 48 hours later [4, 9]. In addition to autophagosomes and autolysosomes, LDs accumulate abundantly in the cytoplasm of hepatocytes. When livers of conditional Atg7-deficient mice (Atg7Flox/Flox: Albumin-Cre) at 22 days, or 6 or 8 weeks of age were examined, positive staining for LDs is sparsely detected in the hepatocytes under starvation conditions for 12 or 24 hours, although they are abundant in the cytoplasm of the hepatocytes of the control littermate mouse liver (Fig. 1). Size and amount of stained LDs were much smaller in hepatocytes deficient in Atg7 than in the control hepatocytes. When examined by electron microscopy, the diameter of LDs in the control hepatocytes is various

**LDs in hepatic and cardiac tissues under starvation conditions** 

used for an energy source when starvation continues.

**Figure 1.** BODIPY staining (green) of hepatocytes obtained from control littermate (left) and Atg7 deficient (right) mice at the age of 6 weeks. Mice were housed under starvation conditions for 24 hours. BODIPY-positive LDs are abundant in hepatocytes from control littermate mouse, while positive LDs largely disappear from the Atg7-deficient hepatocytes. Bar= 50 m
