Lipid Droplet Formation and Autophagy

Objective: Lipid droplets (LDs) are cytoplasmic inclusions that have a neutral lipid core with a single phospholipid layer, while excess free fatty acids and glucose in plasma are converted to triacylglycerol (TAG) and stored as LDs. However, the mechanism for the generation and growth of LDs in cells is largely unknown. This review will explain the relationship between LD formation and autophagy. Results: LD formation accompanied by accumulation of TAG induced by starvation was largely suppressed in the hepatocytes and cardiac myocytes that cannot execute autophagy. Under starvation conditions, LDs in addition to autophagosomes were abundantly formed in the cytoplasm of these tissue cells, although autophagosomes did not appear in wild-type and autophagy-unable cardiac myocytes. Moreover, LC3 was localized on the surface of LDs and LC3-II (lipidation form) was fractionated to a perilipin-positive lipid fraction from the starved liver. Conclusion: Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid metabolism via LD formation.


Introduction
It is well known that there are two major pathways for proteolysis in eukaryotic cells: the proteasome and lysosome.Substrates transferred to lysosomes may be taken into heterophagocytosis, endocytosis, or autophagy into heterophagosomes, early endosomes, or autophagosomes.These phagosomes receive lysosomal enzymes via transporting vesicles from the trans-Golgi network or lysosomes and become heterophagolysosomes, late endosomes, or autolysosomes 1) -6) .Old and unneeded intracellular components containing membranous organelles and part of the cytoplasm are substrates of autophagosomes.Autophagy is now believed to play an essential role in the maintenance of cellular metabolism.In fact, impairment of autophagy causes severe degenerative alterations in various tissue cells 4) 7) -10) .
Neutral lipids, mainly triacylglycerol (TAG) and cholesterol ester (CE), are stored in lipid droplets or lipid bodies 11) 12) .Excess fatty acids are converted to TAG through lipogenesis under nutrient-rich conditions and stored in lipid droplets, while lipid droplets are degraded to lipids by lypolysis under fasting conditions.Proteins are degraded to produce an amino acid pool that is used in part for energy metabolism through glyconeogenesis under starvation conditions, while free fatty acids (FFAs) from adipocytes are delivered to hepatocytes and cardiac myocytes where they are used as an energy source via β-oxidation 12) 13) .
The microtubule-associated protein 1A/1B light chain 3 (LC3) is analogous to Atg8 in yeast and conjugated with phosphatidylethanolamine (PE).LC3 is synthesized as a precursor form, is processed to expose a glycine residue at its C-terminus and becomes cytosolic LC3-I 14) .In response to starvation, the cytosolic form of LC3 is converted to the membrane-bound form by covalent conjugation to an amino group of PE through an enzymatic cascade consisting of Atg7 (E1) and Atg3 (E2) 5) .
We have 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 hepatocytes and cardiac myocytes of wild-type mice under the same conditions 15) .Moreover, Atg7-deficient mice specifically in adipose tissue are slim and contained only 20% of the mass of white adipose tissue (WAT) found in wild-type mice 16) .It is interesting that these mutant mice are highly sensitive to insulin that reduces low fed plasma concentrations of FFAs, while they exhibit markedly decreased plasma concentrations of leptin but not adiponectin, and lower plasma concentrations of TAG.LDs, initially considered inert lipid deposits, have gained during the last decade the classification of cytosolic organelles due to their defined composition and the multiplicity of specific cellular functions in which they are involved 17) .
Since ubiquitin aggregates have been shown to accumulate in cells and neurons deficient in Atg7 18) 19) , selective autophagy plays an important role in the quality control of cellular components through p62 and NBR1 that have specific domains to bind LC3 and ubiquitin and work as adapter proteins 20) 21) .Recent studies show that selective autophagy is required to eliminate damaged cellular components including proteins and cytoplasmic organelles such as mitophagy for mitochondria, pexophagy for peroxisomes, lipophagy for lipid droplets, ribophagy for ribosomes, zymophagy for zymogen granules, reticulophagy for endoplasmic reticulum, glycophagy for glycogen granules, and aggrephagy for protein aggregates 22) .At present, it is largely elusive whether autophagy is involved in LD metabolism, although lipophagy may happen to occur in cells.

Loss of Atg7 suppresses LD formation
It is well know that morphofunctional aspects of hepatocytes differ depending on the location within the liver acinus; Periportal hepatocytes are glycogenic and lypolitic, while perivenous hepatocytes are glycolytic and lipogenic 23) 24) .Although quantities of cytoplasmic organelles in hepatocytes significantly vary within 24 hours, lysosomes and lipid droplets are more abundant in perivenous hepatocytes than in periportal hepatocytes.Such organization of cytoplasmic organelles is drastically altered in hepatocytes, when animals are starved for 24 hours 25) 26) ; 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 start of starvation and those that in some cases contain mitochondria are detected when starvation is extended to 48 hours.In addition to autophagosomes and autolysosomes, LDs accumulate abundantly in the cytoplasm of hepatocytes located in both periportal and perivenous regions of liver acini.By using Atg7-deficient mice specifically in the liver (Atg7 flox/flox : Albumin-Cre mice), LD formation is confirmed to be normal in hepatocytes at 22 days, or 6 or 8 weeks of age.Positive staining for LDs is sparsely detected in Atg7-deficient 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.LDs stained by BODIPY were smaller in size and much less in number in hepatocytes deficient in Atg7 than those in control.By electron microscopy, the size of LDs in the control hepatocytes was 1.12 ± 0.17 μm (the mean diameter (± SD)) and range to 2.61 μm (diameter).In mutant hepatocytes, however, smaller LDs are mainly detected; the size of smaller LDs ranges to 0.83 μm and the average diameter is 0.19 ± 0.17 μm.
Importantly, the total TAG amount in Atg7deficient 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 25) .

LC3 anchors the surface membrane of LDs
Autophagy has been shown to play an important role in normal adipogenesis; inhibition of autophagy by disrupting the atg7 gene has a unique antiobesity and insulin sensitization effect 16) .LDs are ubiquitous in eukaryotic cells, while excess free fatty acids and glucose in plasma are converted to TAG and stored as LDs.However, the mechanism for the generation and growth of LDs in cells remains largely elusive.As stated above, Atg7 that mediates the LC3 lipidation and is essential for autophagy is involved in LD formation.
LC3 is known to be localized on the surface of the isolation membrane when starvation is induced (Figure -1).Using transgenic (TG) mice of GFP-LC3, GFP-LC3 becomes dot-shaped, cap-shaped and ring-shaped in hepatocytes and cardiac myocytes under starvation conditions 27) .Like GFP-LC3 TG-mice, positive staining for LC3 appears dotshaped, cap-shaped, and ring-shaped in hepatocytes and cardiac myocytes under starvation conditions 25) (Figure -1).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.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 the 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 is performed.The results indicate that perilipin-positive LDs are also immunopositive for LC3 on the surface of LDs in both hepatocytes and cardiac myocytes.In hepatocytes, dotted staining of LC3 is free of staining of LD, but most LD-positive staining is colocalized with LC3 in cardiac myocytes (Figure -1).
In fact, cytosolic LC3 is converted to membranebound LC3 (LC3-II) in both hepatocytes and cardiac myocytes 24 hour after the start of starvation.By electron microscopic morphometry, the volume densities of autophagosomes/autolysosomes increase from 0.11% to 0.58 ± 0.10 (mean ± SD, %) at 24 hours and 0.61 ± 0.19 at 48 hours after the onset of starvation, while those of LDs increases from 0.2 ± 0.44 to 18.67 ± 2.44 at 24 hours and 10.89 ± 3.11 at 48 hours.Autophagosomes and autolysosomes, however, are rarely found in cardiac myocytes.The volume densities of LDs are only counted in cardiac myocytes and significantly increase from 0.85 ± 0.19 (mean ± SD, %) to 3.38 ± 0.58 at 24 hours and 2.31 ± 0.35 at 48 hours after the start of starvation.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 When mice are fed, cells start to produce and store glycogen granules, whereas under starved conditions, lipid droplets increase 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 and the surface membrane of LDs.Starvation is further continued, and mitochondria are enwrapped by the isolation membrane (mitophagy).24 hours.Moreover, LC3 is localized on the surface membrane of LDs and the lipidated form of LC3 is fractionated to a perilipin and ADRP (LD marker)positive lipid fraction from the starved liver and cardiac myocytes, respectively.In fact, such LDs obtained from the LD fraction are labeled by gold particles showing the antigenicity of LC3 25) .Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid metabolism via LD formation.

Conclusions
It has been proposed that lipophagy and lypolysis under acidic conditions contribute the breakdown of TAG into FFAs that are used for mitochondrial β-oxidation 28) .Electron microscopic observations, however, show that it would be hard to see the presence of LDs that are enwrapped by double membranes even under starvation conditions.Different from lipophagy, mitophagy can easily be found in hepatocytes if the mice are starved for 48 hours.Hormone-sensitive lipase (HSL) plays a key role in the metabolism of neutral lipids and FFA products, to which other lipases take part in, are used for energy metabolism 29) .In this meaning, it may be important to understand phenotypes of targeted animals of lipases and phospholipases in lysosomes.
According to a model of the trafficking of cytosolic LDs and secreted lipoproteins in mammalian cells including hepatocytes, there is cytosolic LD cycling that may have a constitutive function in cells whereby imported or de novo synthesized fatty acids are buffered in a cytosolic pool of TAG 30) .During this cycling, cytosolic LDs are closely associated with the membrane of ER and some are re-fused with the ER membrane from which ADRP-bound LDs are released into the cytosol.Our data simply show that 1) loss of Atg7 inhibits LD formations in hepatocytes and cardiac myocytes under starvation conditions, and 2) LC3 is localized on the surface of LDs and fractionated into the fraction of ADRP and perilipin.It may be that released LDs repeat fusion and increase in size, but this fusion is a regulated cell-specific process.Although the SNARE system has been reported to be in part involved in this LD fusion, the regulatory mechanism of LD formation remains largely unknown.The surface of LDs is either a hemi-membrane or a phospholipids monolayer containing cholesterol, while oligomelized Atg8 is suggested to be involved in hemifusion of liposomes 31) .When considering that LC3 is localized on the surface of LDs under starvation conditions, oligomerization of LC3 may contribute to the fusion and growth of LDs.

Figure- 1
Figure-1 Localization of LC3 in cells from mice under fed (left panel) and starved (right panel) conditions