**1. Introduction**

148 Lipid Metabolism

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Proteolysis in eukaryotic cells can be separated into two major pathways: one is mediated by the ubiquitin-proteasome system and by the autophagy-lysosome system. Substrates of lysosomes may be taken through heterophagocytosis, endocytosis, or autophagy into heterophagosomes, early endosomes, or autophagosomes, which receive lysosomal enzymes via transporting vesicles from the trans-Golgi network or lysosomes and become heterophagolysosomes, late endosomes, or autolysosomes [1-4]. Different from endocytosis and heterophagocytosis, substrates of autophagy are limited to intracellular constituents and include various membranous organelles together with a part of the cytoplasm [3]. Until recently, 18 autophagy-related proteins (Atgs) have been shown to be involved in autophagosome formation, although more than 30 Atgs have been uncovered to regulate autophagy [2, 5]. Such autophagy contributes to the maintenance of cellular homeostasis [6]. Impairment of autophagy, therefore, causes severe degenerative alterations in various tissue cells [1, 4, 7]. In addition to the maintenance of basal cellular metabolism, autophagy is induced in response to various stresses such as starvation and diseases [8-13].

Since one of the major sources in living organisms is lipid, the metabolism of lipid is finely regulated. Besides adipose tissues, neutral lipids, most of which are mainly triacylglycerol (TAG) and cholesterol ester (CE) in cells, are stored in a sort of inclusion body, called the lipid droplet or lipid body [14-16]. Under nutrient-rich situations, excess fatty acids may be converted to TAG through lipogenesis and stored in lipid droplets, whereas lipid droplets under starvation conditions may be a source to produce lipids by lipolysis for cell usage [16]. Under stress or starvation states, proteins are also degraded to produce an amino acid pool that is used in part for energy metabolism through glyconeogenesis, while free fatty acids (FFAs) from adipocytes are delivered to hepatocytes and cardiac myocytes where they are used as an energy source via -oxidation [15, 16]. At the initial stage during fasting,

© 2013 Uchiyama and Kominami, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### 150 Lipid Metabolism

FFAs are also converted to triacylglycerol (TAG) and stored as lipid droplets (LDs) that are used for an energy source when starvation continues.

Autophagy Regulates Lipid Droplet Formation and Adipogenesis 151

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

**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

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

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

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

largely disappear from the Atg7-deficient hepatocytes. Bar= 50 m

is largely suppressed in hepatocytes that cannot execute autophagy.

of LDs.

**deprivation** 

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 cells.

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 via LD formation..
