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Autophagy and lipid metabolism

Autophagy and lipid metabolism

Nutrient-dependent mTORC1 association Mental resilience building Antiviral virus prevention ULK1—Atg13—FIP complex required Metabo,ism autophagy. Llpid of Aufophagy Hepatic Lipid Accumulation through Recovery solutions mTOR-Mediated Auttophagy. Correspondence and requests for materials should be addressed Autophzgy M. Wild-type or ATG7 -deficient HepG2 cells were cultured under nutrient-rich or -poor conditions, and then immunostained with anti-GABARAP and anti-NCoR1 a or anti-Lamp1 and anti-NCoR1 antibodies b. Autophagosomes selectively uptake LD and regulate the LD isolation membrane curvature through autophagy-associated proteins. In support of this possibility, disruption of the core ATG genes blocks the formation of both AVs and microlipophagy Figure

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Fasting, Autophagy, and Reducing Loose Skin

Autophagy is a catabolic process in which cytoplasmic components are delivered Nutrition for injury recovery vacuoles or Health benefits of flaxseeds for degradation and nutrient recycling.

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Here, we review recent advances in our Autophaggy of Mental resilience building interactions Autophagj autophagy and TAG storage, and discuss mechanisms of lipophagy.

Metqbolism fatty acids are cytotoxic due to their detergent-like properties and their Autopphagy into lipid intermediates Recovery solutions are toxic metaboliem high levels.

Ajtophagy, we also discuss metabolusm Mental resilience building manage lipotoxic stresses during autophagy-mediated mobilization of fatty acids from lipid droplets and organellar membranes for energy generation.

Autophagy Autophqgy an intracellular lipdi system used by Colon cleanse for overall wellness cells Refreshment Ideas for Parties deliver cytoplasmic components to vacuoles or lysosomes for ajd and recycling.

This catalytic aand plays critical roles in Nutritional analysis software homeostasis, senescence, Autohagy stress responses Yang and Bassham; Anding and Baehrecke, ; Wang et al.

Autophagy can be selective Autophahy non-selective. Selective autophagy is an in the targeted clearance of various subcellular Weight gain supplements reviews including metabbolism, mitochondria, the liid reticulum ERthe nucleus, and chloroplasts Ishida et al.

Non-selective Mental resilience building, on the other hand, mediates the random turnover of bulk cytoplasmic components. Metqbolism on morphological features, two major types of autophagy can metagolism distinguished, namely ahd and microautophagy Yoshimoto, ; Yang mrtabolism Bassham, ; Supporting insulin sensitivity naturally and Sakai, metabolizm Wang et ,etabolism.

Macroautophagy involves the sequestration of cytoplasmic components into double-membrane structures Autpohagy autophagosomes, Autophagy and lipid metabolism, and the fusion of autophagosomes with lysosomes or vacuoles to Satiety and satiety sensors the lipif of their cargos by resident hydrolytic enzymes.

Enhancing concentration in sports is mediated by a group of proteins encoded by AUTOPHAGY-RELATED ATG genes.

Among them, ATG1—ATG10, ATG12—ATG14, ATG16, Aitophagy, ATG29, and ATG31 play essential roles in mdtabolism formation liipid autophagosomes, their metbolism with the vacuole Autophgy lysosome, and their subsequent degradation Yoshimoto, Microautophagy, on the other hand, involves direct engulfment of cytoplasmic Desirable fat levels via invagination of the vacuolar or lysosomal membrane Autophqgy subsequent release of the cargo into the lumen for degradation Dikic, ; Galluzzi et al.

Eating disorder recovery support forms lipif microautophagy have been Autopyagy to Recovery solutions at ane some ATG proteins essential for macroautophagy in yeast Suzuki and Autophafy, ; Reggiori and Klionsky, ; Faruk et Maximize performance through hydration. Lipid droplets LDs are highly dynamic subcellular Autpphagy with Arthritis pain management techniques roles metabolismm lipid storage, metabolism, metaabolism homeostasis Thiele Easy weight loss Spandl, ; Thiam Autopjagy Belkler, ; Llpid et al.

They are composed lupid a metaboljsm core of neutral lipids, typically triacylglycerols TAGs and ahd esters, coated with a monolayer Weight management motivation phospholipids and proteins.

LDs Mental resilience building from lipir ER, where neutral lipid metabo,ism enzymes such as diacylglycerol acyltransferases DGATs and phospholipid diacylglycerol acyltransferases PDATs reside. They are highly dynamic Meditation for pain relief, varying greatly in size and number between different cell types Autophaggy under different metabolic states.

Under starvation conditions, LDs can be hydrolyzed by cytosolic Autophaggy TAG lipases in Atophagy process termed lipolysis to release Antioxidant supplements for mens health Recovery solutions for energy Autoohagy via Recovery solutions in peroxisomes in metsbolism and plants, and Autophavy mitochondria in mammals Fig.

In addition, autophagy has been implicated in the degradation of LDs Autophavy acidic Atuophagy in vacuoles or lysosomes. Autophagy-mediated degradation of LDs, termed lipophagy, was first described in mammals Singh et al. Metaboliwm may also be involved in Auhophagy breakdown in plants Allergy relief through essential oils et al.

Studies in yeast and animals suggest that lipophagy metabolixm diverse cellular functions, including, but lipidd limited to, its roles in energy generation, cellular homeostasis, and stress tolerance Jaishy and Nad, ; Auhophagy, ; Netabolism et al.

Mechanisms Mental resilience building lipophagy in yeast, plants, Autophaagy mammals. In yeast jetabolism plants Aautophagy-mediated LD degradation is characterized by the direct Auophagy of LDs by vacuoles in a llpid resembling microautophagy. This mechanism of LD degradation Autiphagy be dependent or Autophxgy of the core machinery of macroautophagy, depending on physiological conditions and metabolic settings.

These metabollsm LDs are then sequestered by autophagosomes to form lipoautophagosomes prior to fusion with lysosomes for Ajtophagy degradation. Alternatively, Jetabolism can form stable contacts with the lysosome, Autopgagy direct injection of LD contents into lysosomes for degradation.

Metaboliwm acids released by lipophagy may be used for ATP production via β-oxidation in peroxisomes metabolisn yeast and metbaolism and in mitochondria in mammals. Lipud plants, however, the exact route by which acetyl-CoA, the product metabopism β-oxidation, enters mitochondria for ATP production remains unknown.

In addition to its role in LD catabolism, anr recent studies have shown that autophagy is involved in LD formation by supplying fatty acids for TAG synthesis. On the other hand, LDs have been implicated in the formation of autophagosomes, though the underlying mechanism remains unclear.

The potential connections between storage lipids and autophagy have been thoroughly reviewed Llpid et al. Here, we discuss some of the latest advances on the metabolic interplay between autophagy and LD dynamics, and describe mechanisms of lipophagy in plants in comparison with Auttophagy in yeast and mammals.

Free fatty acids are the main end-products of autophagy-mediated breakdown of membrane lipids and TAG, but their accumulation can disrupt the cellular membrane and induce oxidative stress and even cell death in a process collectively named lipotoxicity Fan et al.

Therefore, an additional aim of this review is to discuss how cells manage lipotoxic stress arising from autophagy-mediated breakdown and reuse of lipids. Seeds are the main storage organs for TAGs in LDs in plants, and the transcript levels of most ATG genes have been shown to be up-regulated during seed maturation Di Berardino et al.

Our recent study showed that inactivation of the autophagic pathway leads to a small but significant reduction in TAG levels in mature seeds of Arabidopsis Fan et al. In rice, knockout of ATG7an essential gene for autophagy, resulted in reduced TAG and LD accumulation in pollens Kurusu et al. However, because many ATG proteins have autophagy-independent roles Galluzzi and Green,it remains unclear whether the observed LD deficiency phenotype in atg7 mutants is directly related to the autophagic function of ATG7.

In addition to seeds, LDs are present in other tissues such as leaves and pollen Chapman et al. In Arabidopsis, disruption of the core components of the autophagic machinery impairs meatbolism membrane metqbolism turnover Fan et al.

During nutrient starvation in microalgae, increases in autophagic flux were found to be accompanied by an accumulation of LDs, and blocking autophagic flux by treatment with inhibitors of autophagy resulted in decreases in TAG synthesis and LD accumulation Couso et al.

These results support a role for autophagy in supplying fatty acids for TAG synthesis and hence TAG formation. In plants, autophagy is involved in the degradation of various subcellular organelles, lipis peroxisomes Kim et al. Lipids released from autophagy-mediated degradation of these organellar membranes may be used for Anr synthesis Barros et al.

However, inactivation of autophagy had no significant impact on Metabolims accumulation under dark-induced starvation conditions Fan et al. Since fatty acids for TAG synthesis under extended darkness are mostly derived from thylakoid lipids Fan et al.

In the oleaginous yeast Lipomyces starkeyiautophagy-mediated degradation of mitochondria is also linked to LD accumulation under nutrient deprivation Duan and Okamoto, In mammalian cells, autophagy contributes to TAG synthesis and LD formation during the differentiation of neutral lipid-storing cells Singh et al.

A recent study revealed that autophagy-mediated LD accumulation under nutrient starvation requires a small GTPase RalA, which functions to facilitate LD growth by recruiting phospholipase D to lysosomes to promote the localized formation of phosphatidic acid Hussain et al. Increased autophagic fluxes due to either nutrient starvation or disruption of negative regulators of autophagy resulted in accumulation of LDs Rambold et al.

DGAT1 is involved in the channelling of fatty acids derived from autophagic breakdown of membrane lipids towards TAG accumulation in LDs Nguyen et al.

Several autophagic anx including ATG2 Velikkakath et al. Depletion of ATG2, but not down-regulation of ATG5, affects the size, number, and distribution of LDs Velikkakath et al. Similarly, suppression of LC3 expression results in reduced LD formation in mammalian cells Shibata et al.

In yeast, ATG8 functions in regulating the quantity of LDs independent of its autophagic role Maeda et al. In microglial cells, genetic ablation of Lipic results in an increase in LD metabloism Xu et al.

Overall, the available data suggest cell type-specific roles of autophagy in LD formation in mammals. Autophagy begins with the formation of an isolated membrane structure called a phagophore, which expands through the acquisition of lipids and eventually seals to form an autophagosome.

Various organelles including the ER, mitochondria, the plasma membrane, and the Golgi apparatus have been implicated in providing a membrane lipid source for autophagosomes Aitophagy and Yoshimori, ; Gomez-Sanchez et al. A recent study showed that phagophore expansion requires local fatty acid channelling into phospholipid synthesis during the autophagic process Schutter et al.

LDs represent the major lipid reservoirs in eukaryotic cells, and their functions as a source of lipids for the biogenesis of autophagosomal membranes have been investigated in yeast and mammals Shatz et al. In mammalian cells, the patatin-like phospholipase domain-containing protein 5 PNPLA5 TAG lipase and several enzymes involved in phospholipid biosynthesis are positive regulators of autophagy, implying that neutral lipid stores are the key lipid source for autophagic membrane formation Dupont et al.

A transmembrane protein TMEM41B may also be involved in metabolim exchange between LDs and autophagosomes Moretti et al. Similarly, disruption of storage lipid synthesis or neutral lipid lpid in yeast results in an inhibition of starvation-induced autophagy Li et al. However, LD deficiency has no obvious impact on autophagy triggered by treatment with rapamycin, a specific inhibitor of the negative regulator of autophagy, the target of rapamycin complex Regnacq et al.

This study also showed that LDs are not required as a membrane lipid source for autophagosome biogenesis but function in autophagy regulation by buffering fatty acid-induced lipotoxic ane to maintain ER lipid homeostasis, intact autophagy, and cell metaoblism. However, depletion of free fatty acids prior to starvation results in an inhibition rather than promotion of Autopagy catalytic process of autophagy induced by starvation Shpilka et al.

In Chlamydomonastreatment with cerulenin, a specific inhibitor of fatty acid synthesis, triggers metabilism Heredia-Martinez et al. A recent study showed that the synthesis of autophagic anf requires an activation of CTP:phosphocholine cytidyltransferase CCT on autophagy-derived LDs Metaboilsm et al.

Because CCT is the rate-limiting enzyme in phosphatidylcholine PC synthesis Nakamura, and because the diacylglycerol backbone of PC may originate from LDs Dupont et al. Overall, available data indicate that further research is required to define the exact role of lopid acid metabolism in autophagy and cellular homeostasis and the potential function of LDs in autophagy.

Physical contacts between LDs and autophagosomes have also been described in Chlamydomonas cells at early stages of nitrogen starvation Tran et al. Autophagy has been implicated in the degradation of TAG and LDs in several algae, including Autophagt Kajikawa et al.

In Micrasterias cells, LDs appeared to form in chloroplasts. During starvation conditions, LDs were translocated from chloroplasts into the cytosol, where they can be degraded in vacuoles via macrolipophagy in a process involving the engulfment of LDs by autophagosome-like double-membrane structures Schwarz et al.

The translocation of chloroplast LDs into the cytosol and their subsequent degradation in vacuoles were also reported during plant senescence Guiamet et al. In contrast, autophagy-mediated degradation of LDs in Auxenochlorella cells during the heterotrophy to autotrophy transition is characterized by the direct engulfment of LDs by metaboliism in a process resembling microautophagy Zhao et al.

In addition, microlipophagy is also involved in the LD degradation in Nannochloropsis during recovery following nitrogen starvation Zienkiewicz et al.

Disruption of ATG2 or ATG5 increases dark-induced TAG content and LD accumulation in mutants defective in SUGAR-DEPENDENT1 SDP1 cytosolic neutral TAG metxbolism, indicating that the core machinery of macroautophagy is required for microlipophagy in plants Fan et Autophag. TAG levels increased in etiolated seedlings of atg5 mutants Avin-Wittenberg et al.

At present, the exact role of ATG proteins in microlipophagy remains unclear. Our ultrastructural analysis showed that LDs are degraded in autophagic vacuoles Fan et al. Therefore, it is possible that the role of ATG proteins in microautophagy is indirect, reflecting the requirement of core ATG proteins in the formation of autophagosomes and hence autophagic vacuoles Fan et al.

In Autophqgy, two types of microlipophagy contribute to LD degradation under different growth conditions. One occurs in nutrient-starved and stationary-phase yeast, and is dependent upon several core ATG proteins of macroautophagy, though the exact roles of ATG proteins in microlipophagy vary depending on growth conditions.

In starvation- and ajd phase-induced microlipophagy, LDs are internalized via raft-like microdomains of vacuolar membranes into the lumen, where they are degraded by the vacuolar lipase ATG15 Wang et al.

Two Niemann—Pick type C Lipjd sterol transporters Ncr1 and Npc2 are required for the development of such lipid microdomains Liao et al. Additionally, two ATG proteins, ATG6 and ATG14, form LD recruiting sites during microlipophagy under acute glucose starvation conditions Seo et al.

Further, several ATG proteins including ATG1, 2, 3, 5, 7, 8, and 18 are required for localization of NPC proteins to the vacuole in stationary-phase, but not in nitrogen-starved cells Tsuji et al.

Under nitrogen starvation, ATG proteins are required for the delivery of sphingolipids and sterols, vital components of raft-like domains, to the vacuole by autophagosomes Tsuji et al. In addition to ATG protein-dependent LD autophagy, an alternative mechanism of yeast microlipophagy requires components of the endosomal sorting complexes required for transport ESCRT anx, but not the core machinery of macroautophagy.

This ATG-independent LD catalytic pathway has been reported in yeast under lipotoxic stress Vevea et al.

: Autophagy and lipid metabolism

Associated Data

Disruption of SDP1 blocks TAG hydrolysis in germinating seeds Eastmond, and in vegetative tissues such as mature leaves and roots Kelly et al. Under extended darkness, TAG levels in leaves of sugar dependent1 sdp1 mutants increased rapidly and then declined, suggesting an activation of unknown, alternative pathways for TAG hydrolysis under starvation conditions Fan et al.

Lipid metabolism in photosynthetic tissues such as leaves is geared toward the supply of building blocks for organellar membrane biogenesis and maintenance. As a result, leaf tissues do not accumulate TAG to significant amounts, although they do possess a high capacity for its synthesis and metabolism Xu and Shanklin, In Arabidopsis, two parallel pathways, compartmentalized in either the ER or the chloroplast, contribute to membrane lipid biosynthesis Browse and Somerville, ; Ohlrogge and Browse, Disruption of either pathway causes drastic changes in lipid metabolism including an increase in fatty acid synthesis and turnover and an accumulation of TAG Fan et al.

In the trigalactosyldiacylglycerol1 tgd1 mutant, a defect in the ER pathway also results in a compensatory increase in the chloroplast pathway activity Xu et al. Similarly, overexpressing PDAT1 draws lipids from the ER pathway to TAG synthesis, causing an increase in the biosynthesis of thylakoid lipids via the chloroplast pathway Fan et al.

On the other hand, the plastidic glycerolphosphate acyltransferase1 act1 mutant is defective in the initial step in the chloroplast pathway of membrane lipid synthesis Kunst et al. To understand the role of autophagy in lipid metabolism at the mechanistic level, we generated a series of double mutants defective in autophagy in the tgd1 -, sdp1- , or PDAT1 -overexpressing-line background.

Using these mutants along with transgenic plants coexpressing an LD-targeted, GFP-tagged OLEOSIN1 OLE1 fusion protein Fan et al. We show that lipophagy occurs in a process morphologically resembling microautophagy in yeast and requires key core players in macroautophagy.

This study demonstrates the functional importance of autophagy in TAG metabolism and storage and the mechanistic basis for lipophagy in plants. To test the role of autophagy in lipid metabolism in plants, we first compared TAG levels in mature seeds, young seedlings, and leaves of adult plants between the wild type and two atg mutants defective in ATG2 or ATG5, two core protein components of the macroautophagic machinery.

Disruption of autophagy caused small but significant decreases in TAG content in seeds Figure 1A and 4-d-old seedlings Figure 1B. Seed weight was slightly decreased in atg TAG levels were low in developing leaves but increased as leaves matured and aged. In all tissues examined, there were no significant differences in TAG content between atg and atg , suggesting the decreased TAG levels in atg mutants are associated with defects in basal autophagy.

A to C TAG levels in dry seeds A , 4-d-old seedlings B , and leaves of 5-week-old plants C. Data are means of three replicates with sd. FW, fresh weight; WT, wild type.

Mutants defective in the core components of autophagy often display pleiotropic phenotypes including early senescence and defects in nutrient remobilization.

Therefore, it is possible that the observed decrease in TAG content in seeds in atg mutants is due to a decrease in resource allocation to seeds rather than to a change in seed TAG metabolism.

Similarly, a decreased TAG storage in seeds may also affect TAG content in young seedlings. To test these possibilities, we performed radiotracer labeling experiments using two different labeled substrates, 14 C-acetate and 3 H 2 O, substrates that label nascent fatty acids with 14 C or 3 H during the initial or reduction steps of fatty acid synthesis, respectively Browse et al.

Under our growth conditions, the incorporation of the radiolabel from 14 C-acetate or tritiated water 3 H 2 O into fatty acids of developing embryos was linear for at least 1 h Supplemental Figure 1.

The rate of incorporation of 14 C or 3 H into TAG calculated following 1 h of incubation was similar between the wild-type and atg embryos Supplemental Figure 2. Likewise, there was no significant difference in the rate of radiolabeled TAG accumulation between the wild-type and atg seedlings.

On the other hand, the rate of radiolabel incorporation into TAG was significantly reduced in mature and senescing leaves, with the largest effect being observed in mature leaves and the least in developing leaves Figure 2 , mirroring the differences in TAG content in leaves at different ages Figure 1.

Again, leaf TAG levels and rates of radiolabel incorporation into TAG were similar between two atg mutants. Disruption of Autophagy Reduces TAG Synthesis in Mature and Senescing, But Not in Growing Leaves.

A and B Detached leaves of 5-week-old plants were incubated with 14 C-acetate A or 3 H 2 O B for 1 h, and total radioactivity in TAG was measured by scintillation counting following separation by thin layer chromatography. The decreased rate of radiolabel incorporation into TAG in atg leaves may be due to a decrease in fatty acid synthesis or a decline in the mobilization of fatty acids from organellar membranes to TAG via autophagy.

The rate of fatty acid synthesis can be assessed by measuring the rate of 14 C-acetate or 3 H 2 O incorporation into total fatty acids Browse et al. As shown in Supplemental Figure 3 , growing leaves incorporated 14 C from 14 C-acetate or 3 H from 3 H 2 O into total lipids at a higher rate than did mature and senescing leaves, likely reflecting a higher demand for fatty acids to support membrane expansion and organellar biogenesis during rapid growth.

Rates of radiolabel incorporation following 1 h of incubation were similar in the wild-type and atg leaves. These results suggest that the decreased TAG synthesis in atg mutants is not due to a decline in the rate of fatty acid synthesis.

We next tested whether disruption of autophagy affects membrane lipid turnover. To this end, we first incubated leaves with 14 C-acetate for 1 h pulse. After thoroughly washing with water to remove 14 C-acetate, the leaves were incubated in unlabeled solution for an additional 3 d chase.

The radiolabel in leaf total membrane lipids following 1 h of pulse was similar between the wild type and atg mutants Supplemental Figure 4.

Quantification of radioactivity in total membrane lipids showed significant decreases in rates of radiolabeled fatty acid loss, particularly in mature and senescing leaves of atg mutants compared with the wild-type leaves of the same age during 3 d of chase Figure 3.

Together, results from pulse-chase labeling experiments suggest that disruption of autophagy results in a decrease in membrane lipid turnover and hence the accumulation of leaf TAG.

Disruption of Autophagy Slows Down Membrane Lipid Turnover in Mature and Senescing, but Not in Growing Leaves. Radiolabel loss was calculated as percentage of loss of radioactivity in total membrane lipids during 3 d of chase following 1 h of 14 C-acetate pulse of detached leaves of 5-week-old plants.

WT, wild type. To provide additional evidence for the involvement of autophagy in TAG synthesis and also to test the relative contribution of the chloroplast versus the ER lipid assembly pathway to autophagy-mediated TAG synthesis, we constructed double mutants between tgd1 and atg or atg Assays for PDAT activity in microsomal membranes revealed that disruption of autophagy had no significant effect on TAG formation from 14 C-labeled phosphatidylcholine PC , whereas the activity was more than fourfold higher in transgenic plants overexpressing PDAT1 compared with the wild type Supplemental Figure 5.

Analysis of lipid extracts from mature leaves of 5-week-old plants showed that TAG content was higher in tgd1 , as expected Figure 4. Interestingly, there was also a significant increase in TAG in act1 compared with the wild type.

Disruption of autophagy caused significant decreases in TAG content in atg tgd1 and atg tgd1. TAG levels were 1. TAG content in mature leaves of 4-week-old PDAT1-overexpressing line 4 PDAT1-OE4 in the wild-type, act1 , atg , atg , atg act1 , or atg act1 background.

FW, fresh weight. Together, these results suggest that basal autophagy plays an important role in regulating both fatty acid synthesis and membrane lipid turnover and that the ER lipid biosynthesis pathway contributes more to autophagy-mediated leaf TAG synthesis than the chloroplast pathway.

Disruption of Autophagy Reduces Fatty Acid Synthesis and Membrane Lipid Turnover in Growing Leaves of the tgd1 Mutant and PDAT1 -Overexpressing Lines.

A Rate of 14 C-acetate incorporation into total fatty acids in growing leaves of the 4-week-old PDAT1 -overexpressing line 4 PDAT1 - OE4 in the wild-type, act1 , atg , atg , atg act1 , or atg act1 background. B Radiolabel loss during the 3-d chase following 1 h incubation with 14 C-acetate. Our data so far indicate that autophagy contributes to TAG synthesis and membrane lipid turnover, but it is not clear whether this mechanism is also involved in the breakdown of TAG stored in LDs.

As a first step toward answering this important question, we took advantage of OLE1-GFP -overexpressing lines Fan et al. OLE1 is one of the most abundant LD proteins in seeds Huang, When ectopically expressed in leaves, OLE1-GFP is specifically targeted to the surface of LDs Wahlroos et al.

When exposed to extended darkness, a starvation condition known to induce autophagy Breeze et al. In addition, while the OLE1-GFP signals rarely overlapped with DsRed-ATG8e—labeled structures under normal growth conditions, some of the OLE1-GFP signals colocalized with DsRed-ATG8e after 3 d of dark treatment Figure 6.

The extent of colocalization was quantified using the Costes image randomization test Costes et al. The average PCC for OLE1-GFP colocalization with DsRed-ATG8e was 0.

The relatively low PCC most likely reflects the large difference in size between the DsRed-labeled structures less than nm in diameter, Figure 7A and the OLE1-GFP—labeled LD clusters 5 to 10 µm in diameter, Figure 7.

Colocalization of LDs With Autophagic Structures in Leaves Under Dark-Induced Starvation. Confocal images of mature leaves of 4-week-old transgenic plants coexpressing OLE1-GFP green and DsRed-ATG8e red in tgd1 before and after 3 d of dark treatment.

Boxed areas show colocalization of green and red signals under higher magnification. Quantification of colocalization is provided by the PCC and the Costes P-value below the images.

A Confocal images of mature leaves of 4-week-old transgenic plants coexpressing OLE1-GFP and DsRed-ATG8e in tgd1 after 3 d of dark treatment. B to D Electron micrographs of LD clusters in leaf cells of tgd1 overexpressing OLE1-GFP before B and after see [C] and [D] 3 d of dark treatment.

D Enlargement of the boxed area in C. Arrows indicate LDs. Under higher magnification, DsRed-ATG8e—labeled autophagic structures were clearly found to be associated with LDs Figure 7A. After dark treatment for 3 d, autophagic vacuoles AVs appeared in LD clusters, some of which contained LDs Figure 7C , which appeared to be partially degraded Figure 7D.

Free GFP is relatively resistant to degradation within the vacuole or lysosome Yoshimoto, ; Klionsky et al. Therefore, if OLE1-GFP—coated LDs are degraded in the vacuole, we would expect to observe an increased accumulation of free GFP under dark treatment. Autophagic activity can also be assessed by monitoring the protein level of ATG8-Phosphatidylethanolamine PE; Suzuki and Ohsumi, ; Yoshimoto, , which migrates faster on SDS-PAGE in the presence of urea than does the unmodified form Chung et al.

Immunoblot analysis using antibody against ATG8 showed that ATG8-PE conjugates were absent in leaves prior to dark treatment but accumulated after 3 d of darkness Figure 8A , indicating an overall increase in autophagic activity during dark-induced starvation conditions, as expected.

Time-course analysis showed that free GFP levels were low under normal growth conditions but increased steadily during 5 d of darkness, similar to dark-induced accumulation of ATG8-PE Figure 8B. ATG5 has been shown to be essential for ATG8 lipidation Chung et al.

Consistent with this, no ATG8-PE conjugates were detected in atg leaves following 3 d of dark treatment Figure 8A. Together, these results provide evidence that lipophagy is induced during dark-induced starvation.

OLE1-GFP—Coated Leaf LDs Are Degraded in Vacuoles Under Dark-Induced Starvation. A Accumulation of free GFP and ATG8-PE in mature leaves of the 4-week-old wild-type and tgd1 plants, but not in mature leaves of atg tgd1 double mutant overexpressing OLE1 - GFP following 3 d of darkness. B Time course of free GFP and ATG8-PE accumulation in mature leaves of tgd1 overexpressing OLE1 - GFP under dark treatment.

Equal amounts of proteins were subjected to SDS-PAGE followed by immunoblot analysis with antibodies against GFP, ATG8, or the loading control actin. The dashed lines and asterisks locate free ATG8 proteins and ATG8-PE conjugates, respectively.

When these plants were exposed to dark treatment for 2 d, individual LDs or LD clusters were observed inside Figures 9A to 9D or within invagination of Figures 9E and 9F ΔTIP-DsRed—labeled tonoplasts.

Analysis of max-intensity projection images of z-stacks acquired by confocal microscopy revealed that the LDs were clearly enclosed by the tonoplast Figures 9G and 9H. A to F Confocal images of cotyledon cells of the 7-d-old wild-type transgenic plants coexpressing the tonoplast marker ΔTIP-DsRed red and OLE1-GFP green after 2 d of darkness in the presence of 0.

Overlay of red and green fluorescence showing the presence of LDs in vacuoles see [A] to [D] or within tonoplast invagination see [E] and [F]. G and H Three-dimensional images reconstructed from a series of confocal z-stack images.

Therefore, to further examine the process leading to lipophagy in plants, we took advantage of sdp1 mutants, which accumulated small LDs under dark-induced starvation conditions Fan et al. Because lipophagy and autophagy appeared to be induced after, but not within, the first 1 d of darkness Figure 8B , we focused on the subcellular morphological changes in leaf samples between 1 and 2 d of dark treatment.

Consistent with changes in ATG8-PE abundance Figure 8B , very few autophagic structures were seen after 1 d of darkness Supplemental Figure 8A. Following 2 d of dark treatment, however, we observed the occurrence of autophagosomes Supplemental Figures 8B and 8C and many small vacuoles with diameters of 0.

Many of these structures contained autophagic bodies or remnants of cytoplasmic materials, suggesting that they are AVs. LDs increased in size after 2 d of dark treatment Figures 10B to 10F and were frequently found to be in close contact with AVs Figure 10C or within invagination of AV membranes Figures 10B and 10C or inside AVs Figure 10D or the central vacuole Figure 10E.

Immunoelectron microscopy of dark-treated sdp1 plants with ATG8 antibody revealed the presence of immunogold particles on LDs Figure 10F. Interestingly, LDs appeared to undergo degradation prior to being fully internalized into AVs, along with other sequestered materials Figure 10C.

Dark treatment in the presence of concanamycin A concA also led to the appearance of LDs in the central vacuole in leaves of wild-type seedlings Supplemental Figure 9. On the other hand, we did not detect association of macroautophagic membrane structures with LDs as observed during macrolipophagy in mammals Singh et al.

Importantly, disruption of ATG2 Figures 11A and 11B or ATG5 Figures 11C and 11D in sdp1 largely blocked the formation of AVs and hence the interaction between LDs and AVs.

In atg sdp1 double mutants, most of LDs were still present in the cytosol after 2 d of dark treatment. A to F Electron micrographs of leaf cells of 4-week-old sdp plants dark treated for 1 d A and 2 d see [B] to [F]. B to D Various stages of LD internalization into AVs see [B] and [D].

Note that LD is partially degraded within invagination of the AV membrane in C. E Presence of LDs in the central vacuole. CV, central vacuole. F Immunogold labeling of sdp seedlings dark treated for 2 d in the presence of 0. Arrowheads indicate gold particles. The inset shows higher magnification of the boxed region.

A to D Electron micrographs of leaf cells of 4-week-old atg sdp see [A] and [B] and atg sdp see [C] and [D] plants dark treated for 2 d. We next tested whether deficiency in cytosolic lipolysis affects autophagy under dark-induced starvation in plants as in mammals Sathyanarayan et al.

To do so, we crossed the wild type or sdp1 with tgd1 overexpressing DsRed-ATG8e and recovered the DsRed-ATG8e line in the wild-type or sdp1 background. As expected, the number of DsRed-ATG8e—labeled puncta increased under dark-induced starvation Supplemental Figure 10A.

Quantitative analysis showed that there was no significant difference in the number of puncta between the wild type and sdp1 after 4 d of darkness. In addition, there was an increase in levels of faster migrating forms of ATG8-PE during dark-induced starvation conditions Supplemental Figure 10B ; and again, there were no discernible differences in levels of starvation-induced ATG8-PE between the wild type and sdp1 mutants.

Together, these data suggest that disruption of SDP1 does not affect autophagic flux under dark-induced starvation conditions in Arabidopsis. Under dark-induced starvation conditions, TAG accumulated rapidly within the initial 1 d and then started to decline in leaves of sdp1 plants, likely reflecting the induction of lipophagy after dark treatment for 1 d Fan et al.

To test this possibility, we treated detached leaves of sdp1 mutants with 3-methyladenine 3-MA , a widely used inhibitor of autophagy in mammals Blommaart et al. In untreated control leaves, TAG content increased by more than sixfold during the initial 2 d of dark treatment Figure 12A.

Treatment with 3-MA did not affect TAG levels during the initial 2 d of dark incubation, suggesting an involvement of an autophagy-independent mechanism in TAG synthesis. However, TAG content declined after day 2 of dark treatment in the untreated control but continued to increase toward the end of the experiment in 3-MA—treated leaves, such that TAG content was significantly higher at days 3 and 4 in 3-MA—treated leaves compared with the untreated control.

These results suggest that autophagy contributes to TAG hydrolysis under severe starvation. Inhibition of Autophagy Enhances TAG Accumulation in sdp under Extended Darkness.

A Changes in TAG levels in detached sdp mature leaves during dark treatment in the presence or absence of 3-MA. B Changes in TAG levels in mature leaves of 4-week-old sdp , atg sdp , and atg sdp plants during dark treatment.

To provide genetic evidence for the induction of lipophagy during dark-induced starvation and also to test whether lipophagy depends on the core autophagic machinery, we generated double mutants between sdp1 and atg or atg Under normal growth conditions, TAG levels were lower in leaves of atg sdp1 double mutants compared with sdp1 Figure 12B.

During dark treatment, TAG levels in sdp1 peaked at day 1 following dark exposure and started to decline thereafter Figure 12B , consistent with our previous report Fan et al. In contrast to sdp1 , TAG content in atg sdp1 double mutants increased steadily during the first 2 d of dark treatment and remained largely unchanged at day 3.

Statistical analysis confirmed that atg sdp1 double mutants accumulated significantly more TAG at days 2 and 3 following dark treatment compared with the sdp1 single mutant Figure 12B. TAG levels remained largely unchanged in the wild type and atg single mutants following dark incubation for 3 d Supplemental Figure The increased TAG accumulation in atg sdp1 double mutants could result from a decrease in TAG hydrolysis or an increase in the conversion of membrane lipids to TAG.

To test these possibilities, we analyzed the changes in levels of total membrane lipids during dark treatment. We detected no significant differences in leaf membrane lipid content among wild type, single, and double mutants prior to or during 3 d of darkness Supplemental Figure Since fatty acid synthesis is completely inactive in the dark Bao et al.

Total membrane lipid levels were decreased to a similar extent following 3 d of dark treatment in all genotypes analyzed Supplemental Figure 12 , apparently because of an increase in fatty acid β-oxidation Fan et al.

Together, these results suggest that the increased TAG accumulation in atg sdp1 double mutants compared with sdp1 is due to decreased lipophagic activity and that lipophagy relies on the core machinery such as ATG2 and ATG5.

We have shown that autophagy plays an important role in organellar membrane turnover, TAG synthesis, and LD accumulation under normal growth conditions. Lipophagy, the autophagic degradation of LDs, was induced following extended dark treatment as evident from increased colocalization of LDs and autophagic structures, an increase in accumulation of free GFP derived from OLE1-GFP—coated LDs, the presence of LDs in vacuoles, the association of autophagic marker protein ATG8 with LDs, and an increase in TAG levels in atg sdp1 double mutants compared with sdp1.

We show that lipophagy occurs in a process resembling microlipophagy as described in yeast and requires the core components of macroautophagy. These results provide mechanistic insight into the role of autophagy in lipid metabolism in plants and lend further support for a critical role of autophagy in quality control of cellular organelles Yang and Bassham, ; Wang et al.

Our results show that disruption of autophagy impedes membrane lipid turnover and hence TAG synthesis under normal growth conditions. These results are perhaps not surprising because, in contrast to the situation in mature and senescing leaves, organellar membranes in growing cells are newly formed and therefore may not be targeted for autophagy-mediated degradation under normal growth conditions.

In developing embryos, fatty acids in membrane lipids are known to be directed to TAG synthesis via acyl editing and headgroup exchange Bates et al. In plants, autophagy has been implicated in the degradation of peroxisomes Kim et al. The contribution of autophagy to TAG synthesis is higher in act1 defective in the chloroplast pathway of glycerolipid biosynthesis but lower in tgd1 disrupted in the parallel ER pathway.

The importance of ER in autophagy-mediated TAG synthesis may reflect not only the role of autophagy in the degradation of this organelle Liu et al.

De novo fatty acid FA synthesis in chloroplasts is mediated by a series of enzymatic reactions collectively referred to as fatty acid synthase. The resultant FAs feed into membrane lipid synthesis via two parallel pathways localized in the chloroplast or the ER.

Autophagy-mediated degradation of cellular organelles other than chloroplasts provides a source of FAs for TAG synthesis under normal and starvation conditions. Thylakoid lipids are broken down by hydrolytic enzymes inside the chloroplast, and the released FAs are used for TAG synthesis.

TAG is packaged in LDs in the cytosol. Under normal growth conditions, TAG stored in LDs is hydrolyzed by SDP1. Nutrient starvation triggers microlipophagy, which functions together with cytosolic lipolysis catalyzed by SDP1 to mediate LD breakdown into FAs for energy production through β-oxidation.

Black arrows represent processes occurring in both normal and starvation conditions. The red arrow is specific to starvation. FAS, fatty acid synthase; HEs, hydrolytic enzymes. Previous studies have shown that during autophagy-mediated chloroplast breakdown, stromal proteins Ishida et al.

In line with these observations, our results showed that disruption of autophagy had no significant impact on the dark-induced synthesis of TAG Figure 12B , which is mainly derived from thylakoid lipids Kunz et al. Similarly, treatment with 3-MA did not affect TAG content during the initial 2 d of dark treatment Figure 12A , suggesting that autophagy-independent breakdown of chloroplasts serves as a main source of fatty acids for TAG synthesis.

In addition, our microscopy analysis showed that the number of chloroplasts per cell remained unaltered during dark treatment Supplemental Figure 13 , consistent with previous reports Keech et al.

These results exclude the possibility of whole chloroplast autophagy as observed in plants under photooxidative stress Izumi et al. The autophagy-independent degradation of thylakoids is also consistent with previous reports showing an internal dismantling of thylakoid systems during senescence-induced chloroplast breakdown Evans et al.

In addition to reduced organellar membrane turnover and TAG synthesis, disruption of basal autophagy results in significant decreases in fatty acid synthesis in tgd1 or PDAT1-OE lines Figure 5A.

Although the exact mechanistic basis as to how autophagy impacts fatty acid synthesis remains unclear, it is possible that blocking autophagy results in a buildup of fatty acids in the cytosol due to reduced cellular fatty acid needs for organellar membrane lipid turnover, which act as feedback signals to negatively regulate fatty acid synthesis in the chloroplast.

On the other hand, overexpression of PDAT1 or blocking the chloroplast lipid biosynthesis pathway in act1 accelerates autophagy-mediated membrane lipid turnover and hence increases the cellular demand for fatty acids.

This increased fatty acid demand may cause a decrease in fatty acids in the cytosol, thereby partially relieving feedback inhibition on plastid fatty acid synthesis. In this context, it is worth noting that inefficient utilization of fatty acids for glycerolipid biosynthesis in the ER has been shown to cause a feedback inhibition on fatty acid synthesis by an unknown mechanism Bates et al.

TAG and fatty acid synthesis are increased in tgd1 mutants Fan et al. These results suggest that under normal growth conditions, autophagy functions in TAG synthesis, whereas the cytosolic pathway mediated by neutral lipases including SDP1 is the major mechanism for TAG catabolism Figure Under extended darkness, TAG content decreases when autophagy is induced but increases when autophagy is disabled in sdp1.

In addition, disruption of SDP1 does not impact autophagic flux under either normal growth or starvation conditions Supplemental Figure These results suggest an important and general role of lipophagy in mediating TAG hydrolysis under starvation conditions Figure TAG did not accumulate in atg mutants under extended darkness Supplemental Figure This result suggests that the SDP1-mediated cytosolic lipolytic pathway can functionally compensate for the lack of lipophagy in TAG hydrolysis under starvation.

Previous studies showed that plant autophagic organelles contain hydrolytic enzymes, including proteases and lipases, for cargo degradation at the onset of their formation Marty, , ; Buvat and Robert, and are functionally sufficient to break down the sequestered materials on their own Rose et al.

In accordance with the autophagosome-autonomous hydrolysis, our ultrastructural analysis showed that LDs and other cellular constituents were degraded in AVs Figures 7D and 10C , in addition to the central vacuole Figure 10E.

These results point to the unique aspects of plant autophagy in comparison with this catabolic process in yeast and mammals, where the autophagosome itself lacks degradative enzymes and its cargo is broken down following fusion with lytic compartments such as vacuoles and lysosomes, respectively Eskelinen, ; Suzuki and Ohsumi, ; Reggiori and Klionsky, ; Dikic, ; Galluzzi et al.

Our ultrastructural analysis showed that the autophagic degradation of LDs in Arabidopsis occurs in a process resembling microlipophagy in yeast. Disruption of autophagy genes increased TAG content in sdp1 under starvation conditions Figure These results suggest that microlipophagy in Arabidopsis depends on the core machinery of macroautophagy, similar to the situation in yeast van Zutphen et al.

At present, the exact mechanism underlying microautophagy and the role of ATG gene products in microlipophagy remain largely unknown Noda and Inagaki, ; Galluzzi et al. Our results showed that microautophagy-like LD degradation occurs in AVs, key autophagic structures in macroautophagy Eskelinen, Therefore, it is possible that the observed dependence of starvation-induced TAG and LD accumulation on the macroautophagic machinery in Arabidopsis may simply reflect the essential role of core ATG proteins in the formation of autophagosomes and hence AVs.

In support of this possibility, disruption of the core ATG genes blocks the formation of both AVs and microlipophagy Figure Recently, vacuolar membrane lipid rafts enriched in sterols have been shown to be necessary for microlipophagy in yeast Oku and Sakai, Further studies are needed to test whether the sterol-enriched membrane rafts are involved in microlipophagy in plants, to determine how TAG is hydrolyzed in vacuoles, and to establish the regulation and physiological functions of lipophagy.

The Arabidopsis Arabidopsis thaliana plants used in this study were of the Columbia ecotype. The tgd1 mutant was previously described by Xu et al. The PDAT1 -overexpressing lines 3 and 4 were described in Fan et al.

The primers used for genotyping sdp1 were as described previously Fan et al. Genotyping of tgd1 and act1 mutants was as described previously Xu et al. For plant growth in soil, surface-sterilized seeds of Arabidopsis were germinated on 0.

For starvation treatment, whole plants, unless stated otherwise in Figure 12A , were transferred to continuous darkness at 24°C for the time indicated. The PCR products were cloned into a binary vector pPZP Fan et al. After confirming the integrity of the construct by sequencing, plant stable transformation was performed according to Clough and Bent Lipids were extracted from leaves of 4-week-old plants grown in soil as described by Fan et al.

To quantitate low TAG levels in leaves of wild type and atg mutants, total lipid extracts were first fractionated through silica columns Discovery DSC-Si SPE tube, volume 6 mL, Supelco as described by James et al. Fatty acid methyl esters were prepared as described by Li-Beisson et al.

Separation and identification of the fatty acid methyl esters were performed on an HP gas chromatograph-mass spectrometer Hewlett-Packard fitted with a 30 m × μm DB capillary column Agilent with helium as the carrier gas as described by Fan et al.

Fatty acid methyl esters were quantified using heptadecanoic acid as an internal standard as described by Fan et al. Equal fresh weight of mature leaves of 4-week-old plants grown in soil was ground in liquid nitrogen, homogenized with 2× Laemmli sample buffer.

The extracts were incubated for 5 min in boiling water and clarified by centrifugation at 12, g for 5 min at 22°C. Immunoblot analyses were performed according to the ECL Western Blotting procedure 32,, Thermo Fisher Scientific with antibodies against GFP catalog no.

E11LF, BioLegend , ATG8a catalog no. AS, lot no. MBS, lot no. M14L06, MyBioSource. Targeted proteins were visualized using an ImageQuant LAS biomolecular imager GE Healthcare Life Sciences.

In vivo labeling experiments with 14 C-acetate or 3 H 2 O were done as described previously by Fan et al. Developing seeds of 50 siliques were directly harvested into labeling medium containing 20 mM MES, pH 5.

The assay was started by the addition of 0. After incubation for 1 h, tissues were washed two times with water and immediately used for lipid extraction. For pulse-chase labeling experiments, leaves were labeled for 1 h with 14 C-acetate.

Total lipids were extracted and separated as described previously by Fan et al. Radiolabel loss was calculated by correcting for the dilution of radioactivity caused by tissue growth during the chase period. Microsomal membranes were isolated from 3-week-old seedlings as described previously Xu et al.

Radioactive PC for PDAT activity assays was prepared after incubating 2-week-old seedlings overnight in 20 mM MES-KOH, pH 6.

Lipids were extracted and separated by TLC as described by Fan et al. Radiolabeled PC was eluted from silica gel using chloroform:methanol:formic acid The reaction mixture contained 0. The reaction solution was thoroughly mixed and incubated at room temperature for 30 min.

Lipid extraction and TLC separation were done as described previously by Fan et al. Radioactivity in TAG was determined by scintillation counting. Detached leaves of 4-week-old plants grown in soil were floated on water with or without the addition of 5 mM 3-MA dissolved in water, Sigma-Aldrich and 0.

Samples were taken every 24 h over 4 d for lipid analysis as described previously Fan et al. For the colocalization study, leaf samples were mounted in water on slides and were directly examined using a Leica TCS SP5 laser scanning confocal microscope with sequential scanning.

GFP was excited with a wavelength of nm and detected at to nm. DsRed was excited at nm and detected at to nm. For tonoplast imaging, transgenic plants coexpressing OLE1-GFP and ΔTIP-DsRed were germinated on 0.

Six-day-old seedlings were dark treated for 1 d and then transferred to half-strength MS medium with or without 0.

The hypocotyls or cotyledons were observed under confocal microscopy. For transmission electron microscopy, leaf tissues were fixed with 2. For chloroplast counting, leaf tissues were fixed and embedded. The number of chloroplasts was counted from at least 60 mesophyll cell cross sections for each time point of dark treatment.

Colocalization analysis of OLE1-GFP and ATG8e-DsRed signals was done with the Coloc 2 plugin for ImageJ. Background subtraction from image pairs was performed using rolling ball subtraction with a pixel ball size.

Statistical significance of the PCC of the image pairs was analyzed using the Costes image randomization test as described previously Costes et al. Regions of interest were selected for colocalization analysis with Costes randomizations using a point spread function of 3.

Five-day-old seedlings grown on 0. The seedlings were then transferred to half-strength MS medium containing 0. The fixed hypocotyls were washed twice with 0. After dehydration, the tissues were embedded in LR White resin CA, Electron Microscopy Sciences, London Resin Company in gelatin capsules.

Resin polymerization was performed at 50 to 55°C. Ultrathin sections 70 to 90 nm of LR White—embedded hypocotyls were collected with formvar-coated mesh nickel grids. The grids were first washed with 1× PBS containing 0. After blocking, the grids were incubated with the primary antibody:rabbit polyclonal anti-ATG8a catalog no.

After rinsing with blocking solution five times, 1 min each, the grids were then incubated in the secondary antibody of goat anti-rabbit immunoglobulin G conjugated with nm gold particles catalog no. G, lot no. SLBW, Sigma-Aldrich; dilution in blocking solution for 1 h at room temperature.

Following washing with 1× PBS and 0. Supplemental Figure 1. Time course of the incorporation of radiolabel from 14 C-acetate or 3 H 2 O into total fatty acids in wild-type developing embryos.

Supplemental Figure 2. Rate of the incorporation of radiolabel from 14 C-acetate or 3 H 2 O into TAG in developing embryos and seedlings.

Supplemental Figure 3. Rate of the incorporation of radiolabel from 14 C-acetate or 3 H 2 O into total fatty acids in leaves. Supplemental Figure 4. Rate of the incorporation of radiolabel from 14 C-acetate into total membrane lipids in leaves.

Supplemental Figure 5. PDAT activity in microsomal membranes isolated from seedlings. Supplemental Figure 6. Disruption of autophagy reduces TAG content in mature leaves of 4-week-old PDAT1 -overexpressing transgenic plants.

Supplemental Figure 7. Increased accumulation of DsRed-ATG8e—labeled structures in leaves of tgd1 plants under dark treatment. Supplemental Figure 8. Accumulation of autophagosomes and autophagic vacuoles in mature leaves of 4-week-old sdp plants under dark treatment.

Supplemental Figure 9. The appearance of LDs in the central vacuole in wild-type seedlings after dark treatment in the presence of concA.

Supplemental Figure Autophagic activity in 4-week-old sdp plants under dark-induced starvation. TAG levels in mature leaves of 4-week-old wild type, atg and atg plants under dark-induced starvation.

Membrane lipid levels in mature leaves of 4-week-old sdp , atg sdp , and atg sdp plants under dark-induced starvation. Chloroplast number in mature leaves of sdp plants under dark-induced starvation.

Supplemental Data Set. Results of statistical analyses. The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper: SDP1 Gramene: At5g SDP1 Araport: At5g ATG10 Gramene: AT3G ATG10 Araport: AT3G ATG3 Gramene: AT5G ATG3 Araport: AT5G LAS Gramene: AT1G LAS Araport: AT1G ACT1 Gramene: AT2G ACT1 Araport: AT2G TGD1 Gramene: AT1G TGD1 Araport: AT1G ATG5 Gramene: AT5G ATG5 Araport: AT5G PDAT1 Gramene: at5g PDAT1 Araport: at5g ATG2 Gramene: AT3G ATG2 Araport: AT3G ATG8 Gramene: AT4G ATG8 Araport: AT4G This work was supported by the U.

Department of Energy , Office of Science, Office of Basic Energy Sciences DE-SC , specifically through the Physical Biosciences program of the Chemical Sciences, Geosciences and Biosciences Division.

Use of the transmission electron microscope and the confocal microscope at the Center of Functional Nanomaterials was supported by the Office of Basic Energy Sciences, U. Department of Energy DE-SC and J. designed the experiments. performed the research. and C. participate in data analysis.

wrote the article with contributions from J. and L. Anding , A. Cleaning house: Selective autophagy of organelles. Cell 41 : 10 — Google Scholar. Andre , C. Feedback regulation of plastidic acetyl-CoA carboxylase by acyl carrier protein in Brassica napus.

USA : — Antonioli , M. Emerging mechanisms in initiating and terminating autophagy. Trends Biochem. Avin-Wittenberg , T. Global analysis of the role of autophagy in cellular metabolism and energy homeostasis in Arabidopsis seedlings under carbon starvation. Plant Cell 27 : — Bao , X.

Understanding in vivo carbon precursor supply for fatty acid synthesis in leaf tissue. Plant J. Barros , J.

Autophagy deficiency compromises alternative pathways of respiration following energy deprivation in Arabidopsis thaliana. Plant Physiol. Bates , P. Acyl editing and headgroup exchange are the major mechanisms that direct polyunsaturated fatty acid flux into triacylglycerols.

Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly. Blommaart , E. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY inhibit autophagy in isolated rat hepatocytes. Mutated NPC1 protein can block autophagy induction through the inhibition of SNARE-dependent membrane fusion, whereas ATG5-deficient cells exhibit increased NPC1protein accumulation Sarkar et al.

Thus, the pharmacological induction of autophagy may ameliorate the phenotypes of LSDs. Preeclampsia is a pregnancy complication characterized by high blood pressure and signs of multiple organ damage e.

Preeclampsia is associated with increased oxidative stress, which can cause autophagy-dependent cell death in extravillous trophoblasts.

Mechanistically, oxidative stress reduces lysosomal activities and enhances de novo sphingolipids synthesis, which finally results in ceramide overload-dependent autophagic cell death and subsequent inflammation response Melland-Smith et al.

In addition to excessive autophagy-mediated cellular damage in extravillous trophoblasts, mild levels of autophagy may promote cell survival under hypoxic and low-nutrient conditions Nakashima et al.

It remains unknown whether a systemic autophagy response affects pregnant women. The liver is the hub of fat transport. After fat is digested and absorpted, a portion of it enters the liver, and then it is converted into body fat and stored. The liver is also one of the main organs for the synthesis of FAs, cholesterol, and phospholipids in the body.

Excess cholesterol is excreted with bile. Lipid metabolic imbalance leads to lipid accumulation in the liver, resulting from steatosis due to non-alcoholic fatty liver disease NAFLD. The level of lipids in the liver is modulated by lipophagy, and impaired lysosomal pathways are involved in the pathogenesis of NAFLD.

In contrast, the activation of autophagic pathways has been shown to ameliorate steatosis and NAFLD in animal models Ma et al. These findings suggest that autophagy activators may have therapeutic potential in NAFLD, which includes a spectrum of hepatic disorders associated with obesity.

Specific gene mutations, such for as PTEN-induced kinase 1 PINK1 , increase the risk of PD. PINK1 is an important regulator of mitochondrial quality through multiple mechanisms, including mitophagy Rub et al.

Depleted or mutated PINK1 can increase mitochondrial oxidative injury, ER stress, and mitophagy deficient, which leads to cell death, inflammation, and immune suppression in various diseases Kang et al. Of note, reduced hydrolase activity has shown to increase cholesterol accumulation during PD development Garcia-Sanz et al.

Thus, reducing lipid storage may restore the activity of autophagy, especially mitophagy, to alleviate mitochondrial damage in PD Han et al. Metabolic syndrome includes a cluster of conditions, such as hypertension, hyperglycemia, excessive waist fat, and abnormal cholesterol levels.

Autophagic activity is significantly reduced in metabolic syndrome, which increases the risk of obesity, type 2 diabetes, and atherosclerosis. The inhibition of autophagy promotes lipid accumulation, mitochondria dysfunction, and ER stress Perrotta and Aquila, ; Zhang et al.

In contrast, the activation of autophagy may decrease metabolic syndrome-related diseases. Autophagy is a conserved adaptive response to environmental changes and plays a pivotal role in cell survival and death.

It can degrade aging organelles and proteins to produce amino acids, nucleotides, and FFAs for cell survival.

At the same time, it can also be used as an active mechanism to induce autophagy-dependent cell death. Generally, ceramides are involved in pro-survival autophagy, while PUFAs are involved in pro-death autophagy.

The process of autophagy is regulated by a series of complex signaling molecules and metabolic pathways.

Lipid metabolism plays an important role in regulating multiple cell processes. In the past 10 years, there have been major breakthroughs in understanding the crosstalk between lipid metabolism e.

In particular, lipid metabolism has been found to be involved in the formation of membrane structures related to autophagy. Moreover, autophagy promotes lipid catabolism and lipid peroxidation-induced cell death, such as ferroptosis.

Targeting the autophagy pathway has received extensive attention in human diseases, including lipid metabolism-related disorders. Although these advances in knowledge have propelled the field forward, there is still much to explore. For example, how does autophagy function in lipid metabolism pathways in different cells or tissues?

To what extent does the lipid context around membranes affect autophagy induction? How does autophagy switch from pro-survival mode to a pro-death one that ruptures the membranes?

To what degree is selective autophagy specially linked to ferroptotic cell death? Which ATG modifications are responsible for lipid disorder phenotypes? YX and DT conceived of the topic for this review.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

YX was supported by the National Natural Science Foundation of China , , , and The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We thank Dave Primm Department of Surgery, University of Texas Southwestern Medical Center for his critical reading of the manuscript.

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TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop. Sillence, D. New insights into glycosphingolipid functions—storage, lipid rafts, and translocators. Singh, R. Autophagy regulates lipid metabolism. Sinha, R. Thyroid hormone stimulates hepatic lipid catabolism via activation of autophagy.

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Autophagy regulates lipid metabolism | Nature New issue alert. Palomer X, Capdevila-Busquets E, Botteri G, Salvadó L, Barroso E, Davidson MM, et al. Get the most important science stories of the day, free in your inbox. Yorimitsu T, Nair U, Yang Z, Klionsky DJ. Supplemental Figure 6. In stark contrast to the significant accumulation of NCoR1 in both the nuclear and cytoplasmic fractions of Atg7 p62 double-knockout livers Fig. Autophagy regulates programmed cell death during the plant innate immune response.
INTRODUCTION

LDs from mouse livers were isolated by sucrose density gradient centrifugation 28 and autophagic vacuoles and lysosomes by centrifugation in metrizamide discontinuous density gradients The rat hepatocyte line RALAG was cultured under nontransformed conditions, as described previously Some cells were cultured in high glucose or serum-free DMEM lacking methionine and choline Atlanta Biologicals or pre-treated with 10 mM 3-methyladenine, μM DEUP, 50 μM chloroquine Sigma , 20 mM ammonium chloride or μM leupeptin Fisher.

Oleic and palmitic acid were conjugated to albumin, as described previously 30 , and cells were treated with 0. The hairpins were cloned between the BglII—XhoI sites of pSUPER Ambion , and after SmaI—XhoI digestion the fragments, which included the H1 promoter-shRNA cassette, were subcloned into the EcoRV—XhoI sites of the vector pCCL.

VSVG into HEKT cells. Supernatants were collected over 36 to 48 h, titred by plaque assay and used at a multiplicity of infection of 5 to infect RALA hepatocytes. Cell lysates and liver homogenates were subjected to western blot analysis, as previously described Membranes were incubated with the following primary antibodies: rabbit anti-ATG7, rabbit anti-LC3, rabbit anti-total and phospho- Ser and Ser mTOR, rabbit anti-total and phospho-p70SK6, rabbit anti-total and phospho-Akt Cell Signaling Technology , guinea pig anti-ADRP Progen Biotechnik , rabbit anti-ATG5 Novus Biologicals , mouse anti-beclin 1 BD Biosciences , mouse anti-GPDH Abcam , rat anti-LAMP1 Developmental Studies Hybridoma Bank, University of Iowa , rabbit anti-IκB Santa Cruz Biotechnology and rabbit anti-TIP47 ProSci Incorporated.

Western blot for β-actin AbCam or protein disulphide isomerase a gift from R. Stockert was used as loading control. As a measure of autophagic flow, immunoblots for LC3 were performed in untreated cells and cells treated with the lysosomal inhibitors ammonium chloride and leupeptin.

Autophagic flow was determined by the ratio of the densitometric value for LC3-II in the presence of inhibitors to that in the absence of inhibitors, as described previously 5.

Lysosomes were highlighted with Lysotracker and mitochondria with Mitotracker Invitrogen. Mounting medium contained DAPI stain to highlight the cell nucleus.

Images were acquired with an Axiovert fluorescence microscope Carl Zeiss Ltd with a ×63 objective and 1. Quantification was performed in individual frames after deconvolution and thresholding using ImageJ software NIH in a minimum of 20 cells per slide.

Co-localization was calculated by JACoP plugin in single Z-stack sections of deconvoluted images. Real-time video microscopy was performed using 8-chamber slides in medium buffered with HEPES and maintained at 37 °C in a temperature-controlled stage.

Images were captured at s intervals with a ×40 objective and ×1. Oil red O staining was performed, as described previously Staining was assessed by bright-field microscopy and quantified by the Image J software after appropriate thresholding.

TG synthesis was determined by standard methods Lipids were then dried with nitrogen gas, redissolved into chloroform and resolved by thin-layer chromatography using successive solvent systems containing chloroform, acetone, methanol, acetic acid and water in volumetric ratios of , and hexane, methanol and acetic acid in ratios of Phosphorimages were obtained with a Storm Gel and Blot Imaging System GE Healthcare.

Rates of fatty acid β-oxidation were determined by a modification of a previously used method 23 , in which the rate of carbon dioxide production from the oxidation of [ 14 C]oleate was measured.

Cells were cultured in the presence of [ 14 C]oleate—BSA complex and the released [ 14 C]carbon dioxide trapped for 1 h at 37 °C onto filter paper soaked in mM sodium hydroxide.

The rate of β-oxidation was calculated as the amount of trapped [ 14 C]carbon dioxide in relative units produced per mg protein per hour. Cells were cultured in the presence of [ 14 C]oleate—BSA complex for 24 h following which the cells were washed and the medium replaced.

At different times TGs were extracted from the cells and quantified by thin-layer chromatography as described previously. Starved mice were allowed free access to water.

Some animals were fed a HFD 34 g per g diet fat, 0. The HFD was begun at 3 weeks of age and continued for a total of 16 weeks. All studies were approved by the Animal Care and Use Committee of the Albert Einstein College of Medicine and followed the National Institutes of Health guidelines for animal care.

Cells cultured in monolayers and liver blocks 1 mm 3 were fixed in 2. After ethanol dehydration and embedding in LX resin LADD Research Industries , ultrathin sections were stained with uranyl acetate followed by lead citrate.

Grids were washed in 50 mM glycine in phosphate buffered saline, blocked, incubated with LC3 antibody for 2 h, washed extensively and incubated with the gold-conjugated secondary antibody for 2 h.

Control grids were incubated with the secondary antibody alone or with an irrelevant immunoglobulin G. All grids were viewed on a JEOL CX II transmission electron microscope at 80 kV. Morphometric analysis was performed using ImageJ in 15—20 different micrographs for each condition after thresholding.

Autophagic vacuoles were identified using previously established criteria 32 , For the classification of autophagic contents, autophagosomes and autophagolysosomes were grouped under the term autophagic vacuoles.

Autophagic vacuoles containing only lipids were those with double membranes, homogenous density comparable to that of LDs and lacking other content. LDs were isolated from mouse livers by density gradient centrifugation following a modification of a method described previously Livers homogenized in 0.

The supernatant and fatty layer were centrifuged at 17, g for 10 min at 4 °C to pellet autophagic vacuoles and lysosomes. After centrifugation at 28, g for 30 min at 4 °C, the LD fraction was collected from the top of the tube. The protein pellets were solubilized in SDS and analysed by western blotting.

Autophagic vacuoles and lysosomes were isolated from mouse livers by differential centrifugation and centrifugation in discontinuous density gradients of metrizamide, following a protocol modified from ref. A fraction enriched in endoplasmic reticulum resealed vesicles microsomes was prepared by centrifugation of the supernatant of the 17, g centrifugation at , g for 1 h.

TG secretion was determined as described previously Mice were fasted overnight and injected with 1 g per kg of P Sigma. TG levels were determined on serum drawn immediately before and 6 h after injection.

TG production was calculated from the difference in serum TG levels over the 6 h period and expressed as mg per kg per h.

All numerical results are reported as mean and s. We determined the statistical significance of the difference between experimental groups in instances of single comparisons by the two-tailed unpaired Student's t -test of the means with Sigma Plot Jandel Scientific software.

In instances of multiple means comparisons, we used one-way analysis of variance ANOVA followed by the Bonferroni post hoc test to determine statistical significance.

We thank D. Silver for his discussions, N. Stockert for the protein disulphide isomerase antibody and the personnel at the Analytical Imaging Facility for their technical assistance. This work was supported by National Institutes of Health grants from the National Institute of Diabetes and Digestive and Kidney Diseases and National Institute on Aging, a Glenn Award and an American Liver Foundation Postdoctoral Research Fellowship Award R.

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Singh R 1 ,. Kaushik S ,. Wang Y ,. Xiang Y ,. Novak I ,. Komatsu M ,. Tanaka K ,. Cuervo AM ,. Czaja MJ. Affiliations 1. Department of Medicine, Albert Einstein College of Medicine, Morris Park Avenue, Bronx, New York , USA.

Authors Singh R 1. A comment on this article appears in " Dropping liver fat droplets. A comment on this article appears in " Cell biology: Another way to get rid of fat.

Share this article Share with email Share with twitter Share with linkedin Share with facebook. Abstract The intracellular storage and utilization of lipids are critical to maintain cellular energy homeostasis. Free full text. Author manuscript; available in PMC Apr PMCID: PMC NIHMSID: NIHMS PMID: Czaja 1, 2.

Rajat Singh 1 Department of Medicine, Albert Einstein College of Medicine, Morris Park Avenue, Bronx, New York , USA. Find articles by Rajat Singh. Susmita Kaushik 1 Department of Medicine, Albert Einstein College of Medicine, Morris Park Avenue, Bronx, New York , USA. Find articles by Susmita Kaushik.

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Correspondence and requests for materials should be addressed to M. mocea ajazc or A. In addition, autophagy plays an important role in the cellular mobilization and degradation of neutral lipids in LDs, in a process termed lipophagy Wang, ; Zechner et al.

Recent studies have demonstrated a functional link between lipolysis and lipophagy Martinez-Lopez et al. Mammalian lipophagy depends on the core macroautophagy machinery Jaishy and Abel, and is morphologically similar to macroautophagy; thus, it is referred to as macrolipophagy Singh et al. Consequently, disruption of the core ATG genes increases LD accumulation in various organs Singh et al.

Unlike the situation in mammals, lipophagy in yeast resembles microautophagy and therefore is referred to as microlipophagy van Zutphen et al. It has been suggested that microlipophagy plays an important role in maintaining cell viability van Zutphen et al. Disruption of autophagy has been shown to affect lipid turnover in maize Zea mays ; McLoughlin et al.

In plants, as in yeast and mammals, TAG is assembled in the ER and stored in LDs in the cytosol Chapman and Ohlrogge, In Arabidopsis, phospholipid:diacylglycerol acyltransferase1 PDAT1 is a key enzyme catalyzing the last step of TAG assembly Zhang et al.

TAG breakdown is catalyzed by cytosolic lipases including SUGAR-DEPENDENT1 SDP1 , a patatin domain lipase responsible for the initiation of TAG catabolism Eastmond, Disruption of SDP1 blocks TAG hydrolysis in germinating seeds Eastmond, and in vegetative tissues such as mature leaves and roots Kelly et al.

Under extended darkness, TAG levels in leaves of sugar dependent1 sdp1 mutants increased rapidly and then declined, suggesting an activation of unknown, alternative pathways for TAG hydrolysis under starvation conditions Fan et al. Lipid metabolism in photosynthetic tissues such as leaves is geared toward the supply of building blocks for organellar membrane biogenesis and maintenance.

As a result, leaf tissues do not accumulate TAG to significant amounts, although they do possess a high capacity for its synthesis and metabolism Xu and Shanklin, In Arabidopsis, two parallel pathways, compartmentalized in either the ER or the chloroplast, contribute to membrane lipid biosynthesis Browse and Somerville, ; Ohlrogge and Browse, Disruption of either pathway causes drastic changes in lipid metabolism including an increase in fatty acid synthesis and turnover and an accumulation of TAG Fan et al.

In the trigalactosyldiacylglycerol1 tgd1 mutant, a defect in the ER pathway also results in a compensatory increase in the chloroplast pathway activity Xu et al. Similarly, overexpressing PDAT1 draws lipids from the ER pathway to TAG synthesis, causing an increase in the biosynthesis of thylakoid lipids via the chloroplast pathway Fan et al.

On the other hand, the plastidic glycerolphosphate acyltransferase1 act1 mutant is defective in the initial step in the chloroplast pathway of membrane lipid synthesis Kunst et al.

To understand the role of autophagy in lipid metabolism at the mechanistic level, we generated a series of double mutants defective in autophagy in the tgd1 -, sdp1- , or PDAT1 -overexpressing-line background.

Using these mutants along with transgenic plants coexpressing an LD-targeted, GFP-tagged OLEOSIN1 OLE1 fusion protein Fan et al. We show that lipophagy occurs in a process morphologically resembling microautophagy in yeast and requires key core players in macroautophagy.

This study demonstrates the functional importance of autophagy in TAG metabolism and storage and the mechanistic basis for lipophagy in plants. To test the role of autophagy in lipid metabolism in plants, we first compared TAG levels in mature seeds, young seedlings, and leaves of adult plants between the wild type and two atg mutants defective in ATG2 or ATG5, two core protein components of the macroautophagic machinery.

Disruption of autophagy caused small but significant decreases in TAG content in seeds Figure 1A and 4-d-old seedlings Figure 1B. Seed weight was slightly decreased in atg TAG levels were low in developing leaves but increased as leaves matured and aged.

In all tissues examined, there were no significant differences in TAG content between atg and atg , suggesting the decreased TAG levels in atg mutants are associated with defects in basal autophagy.

A to C TAG levels in dry seeds A , 4-d-old seedlings B , and leaves of 5-week-old plants C. Data are means of three replicates with sd. FW, fresh weight; WT, wild type. Mutants defective in the core components of autophagy often display pleiotropic phenotypes including early senescence and defects in nutrient remobilization.

Therefore, it is possible that the observed decrease in TAG content in seeds in atg mutants is due to a decrease in resource allocation to seeds rather than to a change in seed TAG metabolism. Similarly, a decreased TAG storage in seeds may also affect TAG content in young seedlings.

To test these possibilities, we performed radiotracer labeling experiments using two different labeled substrates, 14 C-acetate and 3 H 2 O, substrates that label nascent fatty acids with 14 C or 3 H during the initial or reduction steps of fatty acid synthesis, respectively Browse et al.

Under our growth conditions, the incorporation of the radiolabel from 14 C-acetate or tritiated water 3 H 2 O into fatty acids of developing embryos was linear for at least 1 h Supplemental Figure 1. The rate of incorporation of 14 C or 3 H into TAG calculated following 1 h of incubation was similar between the wild-type and atg embryos Supplemental Figure 2.

Likewise, there was no significant difference in the rate of radiolabeled TAG accumulation between the wild-type and atg seedlings. On the other hand, the rate of radiolabel incorporation into TAG was significantly reduced in mature and senescing leaves, with the largest effect being observed in mature leaves and the least in developing leaves Figure 2 , mirroring the differences in TAG content in leaves at different ages Figure 1.

Again, leaf TAG levels and rates of radiolabel incorporation into TAG were similar between two atg mutants.

Disruption of Autophagy Reduces TAG Synthesis in Mature and Senescing, But Not in Growing Leaves. A and B Detached leaves of 5-week-old plants were incubated with 14 C-acetate A or 3 H 2 O B for 1 h, and total radioactivity in TAG was measured by scintillation counting following separation by thin layer chromatography.

The decreased rate of radiolabel incorporation into TAG in atg leaves may be due to a decrease in fatty acid synthesis or a decline in the mobilization of fatty acids from organellar membranes to TAG via autophagy.

The rate of fatty acid synthesis can be assessed by measuring the rate of 14 C-acetate or 3 H 2 O incorporation into total fatty acids Browse et al. As shown in Supplemental Figure 3 , growing leaves incorporated 14 C from 14 C-acetate or 3 H from 3 H 2 O into total lipids at a higher rate than did mature and senescing leaves, likely reflecting a higher demand for fatty acids to support membrane expansion and organellar biogenesis during rapid growth.

Rates of radiolabel incorporation following 1 h of incubation were similar in the wild-type and atg leaves. These results suggest that the decreased TAG synthesis in atg mutants is not due to a decline in the rate of fatty acid synthesis.

We next tested whether disruption of autophagy affects membrane lipid turnover. To this end, we first incubated leaves with 14 C-acetate for 1 h pulse.

After thoroughly washing with water to remove 14 C-acetate, the leaves were incubated in unlabeled solution for an additional 3 d chase. The radiolabel in leaf total membrane lipids following 1 h of pulse was similar between the wild type and atg mutants Supplemental Figure 4.

Quantification of radioactivity in total membrane lipids showed significant decreases in rates of radiolabeled fatty acid loss, particularly in mature and senescing leaves of atg mutants compared with the wild-type leaves of the same age during 3 d of chase Figure 3.

Together, results from pulse-chase labeling experiments suggest that disruption of autophagy results in a decrease in membrane lipid turnover and hence the accumulation of leaf TAG.

Disruption of Autophagy Slows Down Membrane Lipid Turnover in Mature and Senescing, but Not in Growing Leaves. Radiolabel loss was calculated as percentage of loss of radioactivity in total membrane lipids during 3 d of chase following 1 h of 14 C-acetate pulse of detached leaves of 5-week-old plants.

WT, wild type. To provide additional evidence for the involvement of autophagy in TAG synthesis and also to test the relative contribution of the chloroplast versus the ER lipid assembly pathway to autophagy-mediated TAG synthesis, we constructed double mutants between tgd1 and atg or atg Assays for PDAT activity in microsomal membranes revealed that disruption of autophagy had no significant effect on TAG formation from 14 C-labeled phosphatidylcholine PC , whereas the activity was more than fourfold higher in transgenic plants overexpressing PDAT1 compared with the wild type Supplemental Figure 5.

Analysis of lipid extracts from mature leaves of 5-week-old plants showed that TAG content was higher in tgd1 , as expected Figure 4.

Interestingly, there was also a significant increase in TAG in act1 compared with the wild type. Disruption of autophagy caused significant decreases in TAG content in atg tgd1 and atg tgd1.

TAG levels were 1. TAG content in mature leaves of 4-week-old PDAT1-overexpressing line 4 PDAT1-OE4 in the wild-type, act1 , atg , atg , atg act1 , or atg act1 background. FW, fresh weight. Together, these results suggest that basal autophagy plays an important role in regulating both fatty acid synthesis and membrane lipid turnover and that the ER lipid biosynthesis pathway contributes more to autophagy-mediated leaf TAG synthesis than the chloroplast pathway.

Disruption of Autophagy Reduces Fatty Acid Synthesis and Membrane Lipid Turnover in Growing Leaves of the tgd1 Mutant and PDAT1 -Overexpressing Lines. A Rate of 14 C-acetate incorporation into total fatty acids in growing leaves of the 4-week-old PDAT1 -overexpressing line 4 PDAT1 - OE4 in the wild-type, act1 , atg , atg , atg act1 , or atg act1 background.

B Radiolabel loss during the 3-d chase following 1 h incubation with 14 C-acetate. Our data so far indicate that autophagy contributes to TAG synthesis and membrane lipid turnover, but it is not clear whether this mechanism is also involved in the breakdown of TAG stored in LDs. As a first step toward answering this important question, we took advantage of OLE1-GFP -overexpressing lines Fan et al.

OLE1 is one of the most abundant LD proteins in seeds Huang, When ectopically expressed in leaves, OLE1-GFP is specifically targeted to the surface of LDs Wahlroos et al. When exposed to extended darkness, a starvation condition known to induce autophagy Breeze et al.

In addition, while the OLE1-GFP signals rarely overlapped with DsRed-ATG8e—labeled structures under normal growth conditions, some of the OLE1-GFP signals colocalized with DsRed-ATG8e after 3 d of dark treatment Figure 6. The extent of colocalization was quantified using the Costes image randomization test Costes et al.

The average PCC for OLE1-GFP colocalization with DsRed-ATG8e was 0. The relatively low PCC most likely reflects the large difference in size between the DsRed-labeled structures less than nm in diameter, Figure 7A and the OLE1-GFP—labeled LD clusters 5 to 10 µm in diameter, Figure 7.

Colocalization of LDs With Autophagic Structures in Leaves Under Dark-Induced Starvation. Confocal images of mature leaves of 4-week-old transgenic plants coexpressing OLE1-GFP green and DsRed-ATG8e red in tgd1 before and after 3 d of dark treatment.

Boxed areas show colocalization of green and red signals under higher magnification. Quantification of colocalization is provided by the PCC and the Costes P-value below the images. A Confocal images of mature leaves of 4-week-old transgenic plants coexpressing OLE1-GFP and DsRed-ATG8e in tgd1 after 3 d of dark treatment.

B to D Electron micrographs of LD clusters in leaf cells of tgd1 overexpressing OLE1-GFP before B and after see [C] and [D] 3 d of dark treatment. D Enlargement of the boxed area in C. Arrows indicate LDs. Under higher magnification, DsRed-ATG8e—labeled autophagic structures were clearly found to be associated with LDs Figure 7A.

After dark treatment for 3 d, autophagic vacuoles AVs appeared in LD clusters, some of which contained LDs Figure 7C , which appeared to be partially degraded Figure 7D. Free GFP is relatively resistant to degradation within the vacuole or lysosome Yoshimoto, ; Klionsky et al. Therefore, if OLE1-GFP—coated LDs are degraded in the vacuole, we would expect to observe an increased accumulation of free GFP under dark treatment.

Autophagic activity can also be assessed by monitoring the protein level of ATG8-Phosphatidylethanolamine PE; Suzuki and Ohsumi, ; Yoshimoto, , which migrates faster on SDS-PAGE in the presence of urea than does the unmodified form Chung et al.

Immunoblot analysis using antibody against ATG8 showed that ATG8-PE conjugates were absent in leaves prior to dark treatment but accumulated after 3 d of darkness Figure 8A , indicating an overall increase in autophagic activity during dark-induced starvation conditions, as expected.

Time-course analysis showed that free GFP levels were low under normal growth conditions but increased steadily during 5 d of darkness, similar to dark-induced accumulation of ATG8-PE Figure 8B. ATG5 has been shown to be essential for ATG8 lipidation Chung et al.

Consistent with this, no ATG8-PE conjugates were detected in atg leaves following 3 d of dark treatment Figure 8A. Together, these results provide evidence that lipophagy is induced during dark-induced starvation.

OLE1-GFP—Coated Leaf LDs Are Degraded in Vacuoles Under Dark-Induced Starvation. A Accumulation of free GFP and ATG8-PE in mature leaves of the 4-week-old wild-type and tgd1 plants, but not in mature leaves of atg tgd1 double mutant overexpressing OLE1 - GFP following 3 d of darkness.

B Time course of free GFP and ATG8-PE accumulation in mature leaves of tgd1 overexpressing OLE1 - GFP under dark treatment. Equal amounts of proteins were subjected to SDS-PAGE followed by immunoblot analysis with antibodies against GFP, ATG8, or the loading control actin.

The dashed lines and asterisks locate free ATG8 proteins and ATG8-PE conjugates, respectively. When these plants were exposed to dark treatment for 2 d, individual LDs or LD clusters were observed inside Figures 9A to 9D or within invagination of Figures 9E and 9F ΔTIP-DsRed—labeled tonoplasts.

Analysis of max-intensity projection images of z-stacks acquired by confocal microscopy revealed that the LDs were clearly enclosed by the tonoplast Figures 9G and 9H. A to F Confocal images of cotyledon cells of the 7-d-old wild-type transgenic plants coexpressing the tonoplast marker ΔTIP-DsRed red and OLE1-GFP green after 2 d of darkness in the presence of 0.

Overlay of red and green fluorescence showing the presence of LDs in vacuoles see [A] to [D] or within tonoplast invagination see [E] and [F]. G and H Three-dimensional images reconstructed from a series of confocal z-stack images. Therefore, to further examine the process leading to lipophagy in plants, we took advantage of sdp1 mutants, which accumulated small LDs under dark-induced starvation conditions Fan et al.

Because lipophagy and autophagy appeared to be induced after, but not within, the first 1 d of darkness Figure 8B , we focused on the subcellular morphological changes in leaf samples between 1 and 2 d of dark treatment. Consistent with changes in ATG8-PE abundance Figure 8B , very few autophagic structures were seen after 1 d of darkness Supplemental Figure 8A.

Following 2 d of dark treatment, however, we observed the occurrence of autophagosomes Supplemental Figures 8B and 8C and many small vacuoles with diameters of 0. Many of these structures contained autophagic bodies or remnants of cytoplasmic materials, suggesting that they are AVs.

LDs increased in size after 2 d of dark treatment Figures 10B to 10F and were frequently found to be in close contact with AVs Figure 10C or within invagination of AV membranes Figures 10B and 10C or inside AVs Figure 10D or the central vacuole Figure 10E.

Immunoelectron microscopy of dark-treated sdp1 plants with ATG8 antibody revealed the presence of immunogold particles on LDs Figure 10F. Interestingly, LDs appeared to undergo degradation prior to being fully internalized into AVs, along with other sequestered materials Figure 10C.

Dark treatment in the presence of concanamycin A concA also led to the appearance of LDs in the central vacuole in leaves of wild-type seedlings Supplemental Figure 9. On the other hand, we did not detect association of macroautophagic membrane structures with LDs as observed during macrolipophagy in mammals Singh et al.

Importantly, disruption of ATG2 Figures 11A and 11B or ATG5 Figures 11C and 11D in sdp1 largely blocked the formation of AVs and hence the interaction between LDs and AVs.

In atg sdp1 double mutants, most of LDs were still present in the cytosol after 2 d of dark treatment. A to F Electron micrographs of leaf cells of 4-week-old sdp plants dark treated for 1 d A and 2 d see [B] to [F]. B to D Various stages of LD internalization into AVs see [B] and [D].

Note that LD is partially degraded within invagination of the AV membrane in C. E Presence of LDs in the central vacuole. CV, central vacuole.

F Immunogold labeling of sdp seedlings dark treated for 2 d in the presence of 0. Arrowheads indicate gold particles. The inset shows higher magnification of the boxed region.

A to D Electron micrographs of leaf cells of 4-week-old atg sdp see [A] and [B] and atg sdp see [C] and [D] plants dark treated for 2 d. We next tested whether deficiency in cytosolic lipolysis affects autophagy under dark-induced starvation in plants as in mammals Sathyanarayan et al.

To do so, we crossed the wild type or sdp1 with tgd1 overexpressing DsRed-ATG8e and recovered the DsRed-ATG8e line in the wild-type or sdp1 background. As expected, the number of DsRed-ATG8e—labeled puncta increased under dark-induced starvation Supplemental Figure 10A.

Quantitative analysis showed that there was no significant difference in the number of puncta between the wild type and sdp1 after 4 d of darkness.

In addition, there was an increase in levels of faster migrating forms of ATG8-PE during dark-induced starvation conditions Supplemental Figure 10B ; and again, there were no discernible differences in levels of starvation-induced ATG8-PE between the wild type and sdp1 mutants. Together, these data suggest that disruption of SDP1 does not affect autophagic flux under dark-induced starvation conditions in Arabidopsis.

Under dark-induced starvation conditions, TAG accumulated rapidly within the initial 1 d and then started to decline in leaves of sdp1 plants, likely reflecting the induction of lipophagy after dark treatment for 1 d Fan et al. To test this possibility, we treated detached leaves of sdp1 mutants with 3-methyladenine 3-MA , a widely used inhibitor of autophagy in mammals Blommaart et al.

In untreated control leaves, TAG content increased by more than sixfold during the initial 2 d of dark treatment Figure 12A. Treatment with 3-MA did not affect TAG levels during the initial 2 d of dark incubation, suggesting an involvement of an autophagy-independent mechanism in TAG synthesis.

However, TAG content declined after day 2 of dark treatment in the untreated control but continued to increase toward the end of the experiment in 3-MA—treated leaves, such that TAG content was significantly higher at days 3 and 4 in 3-MA—treated leaves compared with the untreated control. These results suggest that autophagy contributes to TAG hydrolysis under severe starvation.

Inhibition of Autophagy Enhances TAG Accumulation in sdp under Extended Darkness. A Changes in TAG levels in detached sdp mature leaves during dark treatment in the presence or absence of 3-MA.

B Changes in TAG levels in mature leaves of 4-week-old sdp , atg sdp , and atg sdp plants during dark treatment.

To provide genetic evidence for the induction of lipophagy during dark-induced starvation and also to test whether lipophagy depends on the core autophagic machinery, we generated double mutants between sdp1 and atg or atg Under normal growth conditions, TAG levels were lower in leaves of atg sdp1 double mutants compared with sdp1 Figure 12B.

During dark treatment, TAG levels in sdp1 peaked at day 1 following dark exposure and started to decline thereafter Figure 12B , consistent with our previous report Fan et al. In contrast to sdp1 , TAG content in atg sdp1 double mutants increased steadily during the first 2 d of dark treatment and remained largely unchanged at day 3.

Statistical analysis confirmed that atg sdp1 double mutants accumulated significantly more TAG at days 2 and 3 following dark treatment compared with the sdp1 single mutant Figure 12B. TAG levels remained largely unchanged in the wild type and atg single mutants following dark incubation for 3 d Supplemental Figure The increased TAG accumulation in atg sdp1 double mutants could result from a decrease in TAG hydrolysis or an increase in the conversion of membrane lipids to TAG.

To test these possibilities, we analyzed the changes in levels of total membrane lipids during dark treatment. We detected no significant differences in leaf membrane lipid content among wild type, single, and double mutants prior to or during 3 d of darkness Supplemental Figure Since fatty acid synthesis is completely inactive in the dark Bao et al.

Total membrane lipid levels were decreased to a similar extent following 3 d of dark treatment in all genotypes analyzed Supplemental Figure 12 , apparently because of an increase in fatty acid β-oxidation Fan et al.

Together, these results suggest that the increased TAG accumulation in atg sdp1 double mutants compared with sdp1 is due to decreased lipophagic activity and that lipophagy relies on the core machinery such as ATG2 and ATG5.

We have shown that autophagy plays an important role in organellar membrane turnover, TAG synthesis, and LD accumulation under normal growth conditions. Lipophagy, the autophagic degradation of LDs, was induced following extended dark treatment as evident from increased colocalization of LDs and autophagic structures, an increase in accumulation of free GFP derived from OLE1-GFP—coated LDs, the presence of LDs in vacuoles, the association of autophagic marker protein ATG8 with LDs, and an increase in TAG levels in atg sdp1 double mutants compared with sdp1.

We show that lipophagy occurs in a process resembling microlipophagy as described in yeast and requires the core components of macroautophagy. These results provide mechanistic insight into the role of autophagy in lipid metabolism in plants and lend further support for a critical role of autophagy in quality control of cellular organelles Yang and Bassham, ; Wang et al.

Our results show that disruption of autophagy impedes membrane lipid turnover and hence TAG synthesis under normal growth conditions. These results are perhaps not surprising because, in contrast to the situation in mature and senescing leaves, organellar membranes in growing cells are newly formed and therefore may not be targeted for autophagy-mediated degradation under normal growth conditions.

In developing embryos, fatty acids in membrane lipids are known to be directed to TAG synthesis via acyl editing and headgroup exchange Bates et al. In plants, autophagy has been implicated in the degradation of peroxisomes Kim et al.

The contribution of autophagy to TAG synthesis is higher in act1 defective in the chloroplast pathway of glycerolipid biosynthesis but lower in tgd1 disrupted in the parallel ER pathway.

The importance of ER in autophagy-mediated TAG synthesis may reflect not only the role of autophagy in the degradation of this organelle Liu et al. De novo fatty acid FA synthesis in chloroplasts is mediated by a series of enzymatic reactions collectively referred to as fatty acid synthase.

The resultant FAs feed into membrane lipid synthesis via two parallel pathways localized in the chloroplast or the ER. Autophagy-mediated degradation of cellular organelles other than chloroplasts provides a source of FAs for TAG synthesis under normal and starvation conditions.

Thylakoid lipids are broken down by hydrolytic enzymes inside the chloroplast, and the released FAs are used for TAG synthesis. TAG is packaged in LDs in the cytosol. Under normal growth conditions, TAG stored in LDs is hydrolyzed by SDP1. Nutrient starvation triggers microlipophagy, which functions together with cytosolic lipolysis catalyzed by SDP1 to mediate LD breakdown into FAs for energy production through β-oxidation.

Black arrows represent processes occurring in both normal and starvation conditions. The red arrow is specific to starvation. FAS, fatty acid synthase; HEs, hydrolytic enzymes. Previous studies have shown that during autophagy-mediated chloroplast breakdown, stromal proteins Ishida et al. In line with these observations, our results showed that disruption of autophagy had no significant impact on the dark-induced synthesis of TAG Figure 12B , which is mainly derived from thylakoid lipids Kunz et al.

Similarly, treatment with 3-MA did not affect TAG content during the initial 2 d of dark treatment Figure 12A , suggesting that autophagy-independent breakdown of chloroplasts serves as a main source of fatty acids for TAG synthesis.

In addition, our microscopy analysis showed that the number of chloroplasts per cell remained unaltered during dark treatment Supplemental Figure 13 , consistent with previous reports Keech et al.

These results exclude the possibility of whole chloroplast autophagy as observed in plants under photooxidative stress Izumi et al. The autophagy-independent degradation of thylakoids is also consistent with previous reports showing an internal dismantling of thylakoid systems during senescence-induced chloroplast breakdown Evans et al.

In addition to reduced organellar membrane turnover and TAG synthesis, disruption of basal autophagy results in significant decreases in fatty acid synthesis in tgd1 or PDAT1-OE lines Figure 5A. Although the exact mechanistic basis as to how autophagy impacts fatty acid synthesis remains unclear, it is possible that blocking autophagy results in a buildup of fatty acids in the cytosol due to reduced cellular fatty acid needs for organellar membrane lipid turnover, which act as feedback signals to negatively regulate fatty acid synthesis in the chloroplast.

On the other hand, overexpression of PDAT1 or blocking the chloroplast lipid biosynthesis pathway in act1 accelerates autophagy-mediated membrane lipid turnover and hence increases the cellular demand for fatty acids.

This increased fatty acid demand may cause a decrease in fatty acids in the cytosol, thereby partially relieving feedback inhibition on plastid fatty acid synthesis.

In this context, it is worth noting that inefficient utilization of fatty acids for glycerolipid biosynthesis in the ER has been shown to cause a feedback inhibition on fatty acid synthesis by an unknown mechanism Bates et al. TAG and fatty acid synthesis are increased in tgd1 mutants Fan et al.

These results suggest that under normal growth conditions, autophagy functions in TAG synthesis, whereas the cytosolic pathway mediated by neutral lipases including SDP1 is the major mechanism for TAG catabolism Figure Under extended darkness, TAG content decreases when autophagy is induced but increases when autophagy is disabled in sdp1.

In addition, disruption of SDP1 does not impact autophagic flux under either normal growth or starvation conditions Supplemental Figure These results suggest an important and general role of lipophagy in mediating TAG hydrolysis under starvation conditions Figure TAG did not accumulate in atg mutants under extended darkness Supplemental Figure This result suggests that the SDP1-mediated cytosolic lipolytic pathway can functionally compensate for the lack of lipophagy in TAG hydrolysis under starvation.

Previous studies showed that plant autophagic organelles contain hydrolytic enzymes, including proteases and lipases, for cargo degradation at the onset of their formation Marty, , ; Buvat and Robert, and are functionally sufficient to break down the sequestered materials on their own Rose et al.

In accordance with the autophagosome-autonomous hydrolysis, our ultrastructural analysis showed that LDs and other cellular constituents were degraded in AVs Figures 7D and 10C , in addition to the central vacuole Figure 10E.

These results point to the unique aspects of plant autophagy in comparison with this catabolic process in yeast and mammals, where the autophagosome itself lacks degradative enzymes and its cargo is broken down following fusion with lytic compartments such as vacuoles and lysosomes, respectively Eskelinen, ; Suzuki and Ohsumi, ; Reggiori and Klionsky, ; Dikic, ; Galluzzi et al.

Our ultrastructural analysis showed that the autophagic degradation of LDs in Arabidopsis occurs in a process resembling microlipophagy in yeast. Disruption of autophagy genes increased TAG content in sdp1 under starvation conditions Figure These results suggest that microlipophagy in Arabidopsis depends on the core machinery of macroautophagy, similar to the situation in yeast van Zutphen et al.

At present, the exact mechanism underlying microautophagy and the role of ATG gene products in microlipophagy remain largely unknown Noda and Inagaki, ; Galluzzi et al.

Our results showed that microautophagy-like LD degradation occurs in AVs, key autophagic structures in macroautophagy Eskelinen, Therefore, it is possible that the observed dependence of starvation-induced TAG and LD accumulation on the macroautophagic machinery in Arabidopsis may simply reflect the essential role of core ATG proteins in the formation of autophagosomes and hence AVs.

In support of this possibility, disruption of the core ATG genes blocks the formation of both AVs and microlipophagy Figure Recently, vacuolar membrane lipid rafts enriched in sterols have been shown to be necessary for microlipophagy in yeast Oku and Sakai, Further studies are needed to test whether the sterol-enriched membrane rafts are involved in microlipophagy in plants, to determine how TAG is hydrolyzed in vacuoles, and to establish the regulation and physiological functions of lipophagy.

The Arabidopsis Arabidopsis thaliana plants used in this study were of the Columbia ecotype. The tgd1 mutant was previously described by Xu et al.

The PDAT1 -overexpressing lines 3 and 4 were described in Fan et al. The primers used for genotyping sdp1 were as described previously Fan et al. Genotyping of tgd1 and act1 mutants was as described previously Xu et al. For plant growth in soil, surface-sterilized seeds of Arabidopsis were germinated on 0.

For starvation treatment, whole plants, unless stated otherwise in Figure 12A , were transferred to continuous darkness at 24°C for the time indicated.

The PCR products were cloned into a binary vector pPZP Fan et al. After confirming the integrity of the construct by sequencing, plant stable transformation was performed according to Clough and Bent Lipids were extracted from leaves of 4-week-old plants grown in soil as described by Fan et al.

To quantitate low TAG levels in leaves of wild type and atg mutants, total lipid extracts were first fractionated through silica columns Discovery DSC-Si SPE tube, volume 6 mL, Supelco as described by James et al. Fatty acid methyl esters were prepared as described by Li-Beisson et al.

Separation and identification of the fatty acid methyl esters were performed on an HP gas chromatograph-mass spectrometer Hewlett-Packard fitted with a 30 m × μm DB capillary column Agilent with helium as the carrier gas as described by Fan et al.

Fatty acid methyl esters were quantified using heptadecanoic acid as an internal standard as described by Fan et al. Equal fresh weight of mature leaves of 4-week-old plants grown in soil was ground in liquid nitrogen, homogenized with 2× Laemmli sample buffer.

The extracts were incubated for 5 min in boiling water and clarified by centrifugation at 12, g for 5 min at 22°C. Immunoblot analyses were performed according to the ECL Western Blotting procedure 32,, Thermo Fisher Scientific with antibodies against GFP catalog no.

E11LF, BioLegend , ATG8a catalog no. AS, lot no. MBS, lot no. M14L06, MyBioSource. In this process, proteins, organelles, and metabolites are engulfed in a lipid vesicle and trafficked to a lysosome for degradation. Its central role in protein and organelle homeostasis has piqued interest for autophagy dysfunction as a driver of pathology for a number of diseases including cancer, muscular disorders, neurological disorders, and non-alcoholic fatty liver disease.

For much of its history, the study of autophagy has centered around proteins, however, due to advances in mass spectrometry and refined methodologies, the role of lipids in this essential cellular process has become more apparent. This review discusses the diverse endogenous lipid compounds shown to mediate autophagy.

Downstream lipid signaling pathways are also reviewed in the context of autophagy regulation. Specific focus is placed upon the Mammalian Target of Rapamycin mTOR and Peroxisome Proliferator-Activated Receptor PPAR signaling pathways as integration hubs for lipid regulation of autophagy.

Autophagy is a process by which proteins, organelles, and metabolites are broken down and turned over often as a response to starvation or as a means to protect the cell from damage.

Autophagy pathways come in three forms, macroautophagy, microautophagy, and chaperone-mediated autophagy [ 1 ]. Of these, macroautophagy is the best characterized and most well understood.

Macroautophagy hereafter referred to as autophagy was originally studied in yeast and involves the formation of lipid vesicles known as autophagosomes that engulf cargo to be degraded.

Once formed, the autophagosome is trafficked to a lysosome and a fusion event occurs resulting in the degradation of the cargo within the autophagosome Fig. Autophagy mechanism.

Autophagy is a cellular mechanism by which metabolites, organelles, proteins, and protein aggregates are enveloped by a vesicular membrane to form an autophagosome. The autophagosome is trafficked to a lysosome where fusion occurs, and lysosomal degradative enzymes break down the cargo. Autophagy is delineated into key events: initiation, nucleation, elongation and formation of a mature autophagosome, fusion of the autophagosome with a lysosome, and degradation of cargo.

The initiation of autophagy is tightly regulated by the mTOR complex 1 [ 2 ]. When the cell is in a nutrient-rich state, mTORC1 is active and autophagy is suppressed, however, during nutrient-poor conditions, mTOR is inhibited which allows for the formation of Unc like kinase ULK initiation complex composed of ULK kinases, autophagy-related protein 13 Atg13 , Autophagy related protein Atg , and RB1-inducible coiled-coil protein 1 FIP [ 3 ].

Furthermore, ULK-1 also activates a second complex composed of Beclin1-vacuolar protein sorting protein 34 VPS34 -autophagy related protein 14 Atg14L -P, which produces phosphatidylinositolphosphate PI3P Fig.

This complex is responsible for autophagic vesicles budding from the endoplasmic reticulum and forming a structure known as an omegasome. In mammals, this is the site responsible for the nucleation of autophagosomes [ 5 ].

Next, phosphatidylethanolamine PE is conjugated to microtubule-associated light protein light chain 3 LC3 by autophagy-related protein 7 Atg7 and autophagy-related protein 3 Atg3 , which are ubiquitin-like conjugating enzymes.

Then the conjugated PE-LC3 is inserted into the autophagosome membrane [ 6 ]. In addition, autophagy-related protein 12 Atg12 is conjugated to autophagy-related protein 5 Atg5 by Atg7 and autophagy-related protein 10 Atg10 also in a ubiquitin-like manner [ 7 ].

AtgAtg5 interacts with autophagy-related 16 like protein Atg16L and promotes elongation [ 8 ]. Cargo bound to p62 then binds to the p62 interacting regions of LC3 [ 9 ].

After the cargo is selected, the autophagosome matures by disassembling the autophagy-related proteins from the outer layer with the help of myotubularin 3 MTMR3 , a PI3P phosphatase [ 10 ]. Once matured, the autophagosome will fuse with early and late endosomes as well as with lysosomes this is mediated by Rubicon, UV resistance-associated gene UVRAG , Ras-related protein 7 Rab7 , snap receptor proteins SNAREs , and Lysosome-associated membrane glycoproteins LAMPs [ 11 , 12 , 13 ].

Once fusion with a lysosome is complete, the cargo is degraded. In mammals, lysosomal hydrolases break down cargo. Beneficial components, such as amino acids are then returned to the cytosol via amino acid efflux proteins such as vacuolar amino acid transporter 3 Avt3 and vacuolar amino acid transporter 4 Avt4 [ 14 ].

While autophagy has been studied extensively over the years, the role of lipids in this process is underrepresented. Historically, working with lipids has presented a challenge, leading to an emphasis on work that primarily focused upon protein contributions.

However, recent advances in both mass spectrometry capabilities and methodologies have spurred considerable progress in the study of lipids. For example, lipophagy, the targeted breakdown of lipid droplets by autophagic pathways, is currently being studied in the context of non-alcoholic fatty liver disease, aging, and cancer.

It is becoming more apparent that lipids play a prominent role in autophagy. mTOR, the master regulator of cell growth, metabolism, and autophagy is itself a part of a signaling cascade in which lipid phosphoinositides are involved.

In addition, Peroxisome Proliferator-activating factors PPARs , are nuclear receptors that respond to lipid signals and have been implicated in the control of autophagy and autophagy-related genes.

For all of these reasons, this review seeks to provide a comprehensive overview of the growing field of lipid signaling. In the subsequent sections of this article, we discuss the different lipid signaling pathways known to regulate autophagy and their implications in disease states.

Autophagy is of considerable interest as a potential target for treatment in many diseases that include cancer, muscular disorders, and neurodegenerative disease. The fundamental role for organelle, particularly mitochondria, and biomolecule turnover by autophagy provides a broad influence of this process in cellular physiology.

In addition, autophagy is the only known cellular process for removing protein aggregates making the study of this process of considerable interest in protein aggregation disorders which coincide with numerous neurodegenerative diseases. Therefore, understanding and developing tools to manipulate autophagy could yield widespread therapeutic benefits.

Due to its regulation by mammalian target of rapamycin mTOR , autophagy is intimately involved in growth, cell death, and cytoprotective processes. As a result, there is great interest in harnessing this process in the context of cancer.

In the early stages, suppression of autophagy is believed to facilitate the uncontrolled growth [ 15 ]. In later stages, cells may require increased autophagy in low-oxygen and low-nutrient conditions, such as those seen in tumors [ 16 ].

Autophagy also can protect tumors from ionizing radiation by helping to remove damaged organelles and proteins [ 17 ]. Dysfunction in the phosphatidylinositolkinase PI3K -protein kinase B Akt -mTOR pathway has been commonly seen to result in altered autophagy. This pathway, when active, suppresses autophagy and uses lipid signaling molecules such as phosphatidylinositol-3,4,5-triphosphate PIP 3 as key signal transducers [ 18 ].

Mutations in phosphatase and tensin homolog PTEN , a phosphatase that antagonizes PI3K and causes positive regulation of autophagy, result in aberrant inhibition of autophagy that has been associated with excessive growth and tumor formation [ 19 ]. Another common mutation in cancers that leads to autophagy dysfunction is Beclin A high percentage of human breast, ovarian, and prostate cancers have a heterozygous mutation in this gene.

Beclin-1 is a part of the initiation complex responsible for activating lipid kinases required for the formation of autophagosomes. In breast carcinoma cell line MCF7, it has been established that Beclin-1 expression is below detectable limits, and transfection of the Beclin-1 gene upregulates autophagy [ 15 ].

Studies have also shown that mice with a heterozygous deletion of Beclin-1 are more susceptible to developing tumors [ 20 , 21 ].

This is further evidence of the role of beclin-1 and autophagy play in cancer. Autophagy has also been implicated in muscular disorders. It is common for autophagy to play an important role in post-mitotic cells, such as muscle cells and neurons due to the potential for damage from the accumulation of dysfunctional or toxic molecules, protein, or organelles.

Vacuolar myopathy is a type of muscular disease in which the structure of lysosomes is abnormal either from a deficiency in lysosomal enzymes or a deficiency in lysosomal membrane proteins [ 22 ].

Therefore, it is not surprising that diseases in which lysosomal function is affected also result in altered autophagy. In fact, an accumulation autophagosomes is typically required to diagnose vacuolar myopathies [ 23 ].

LAMP-2 is a lysosomal membrane protein whose function is still not fully understood. Autophagy has long been thought to play an important role in neurodegenerative disorders.

A prominent hallmark of these diseases is the accumulation of protein aggregates associated with neuronal loss in the brain.

It is speculated that these aggregates may be substrates for autophagy. It is also thought that in these disease states, autophagy is disrupted. Several proteins have been identified and are linked to dysfunction in various steps of autophagy in each of these diseases.

This inhibition is responsible for the mislocalization of autophagy-related protein 9 Atg9 , a protein involved in the formation of autophagosomes [ 30 ]. Also, PTEN induced kinase 1 PINK1 and Parkin are proteins involved in the recognition of damaged mitochondria normally targeted for degradation mitophagy.

Loss of function mutations in these proteins can prevent the necessary destruction of damaged mitochondria through autophagy resulting in cell death [ 31 , 32 ].

In addition, the park9 gene encodes lysosomal type 5 P-type ATPase ATP13A2. Many research groups have reported changes in the expression of mRNA corresponding to genes in the autophagic pathway [ 34 ].

Phosphoinositides are a class of phospholipids derived from phosphatidylinositol, which is found in the inner layer of the cell membrane and are commonly used by the cell as signaling molecules [ 36 ]. They play a major role in the regulation of autophagy through phosphorylation and dephosphorylation at the 3,4 and 5-hydroxyl positions of the inositol ring.

They control the pathway that directly activates or deactivates mTOR [ 37 ]. mTOR itself is a master regulator of growth, anabolic processes, and autophagy. Generally, mTOR is activated in response to insulin, other nutrients such as amino acids or triglycerides, and growth factors.

When active, mTOR promotes growth and suppresses autophagy. In response to starvation, the cell inhibits mTOR, and autophagy is promoted [ 2 ]. The canonical signaling pathway that controls autophagy through PIP 3. The pathway begins as a response to insulin, other nutrients, or growth factors [ 38 ].

Phosphoinositide 3-kinases convert phosphatidylinositol 4,5- bisphosphate PIP 2 to PIP 3. PIP 3 activates phosphoinositide-dependent kinase-1 PDK1 which in turn phosphorylates Akt [ 39 , 40 ].

When bound to GTP, Rheb mediates the activation of mTOR complex 1 mTORC1 which, in turn, inhibits autophagy [ 42 ]. Activated mTORC1 inhibits autophagy by inhibiting the ULK1 initiation complex. Pro-autophagy signals result in ULK1 dissociation from mTOR and autophagy initiation is facilitated Fig.

PI3K-mTOR autophagy pathway utilizes PIP3 lipid signaling. PI3K converts the lipid PIP2 to PIP3. PIP3 mediates the phosphorylation of PDK1 causing the activation of AKT. MTORC1 inhibits the activation of the ULK activation complex leading to an inhibition of autophagy.

Inversely, phosphatase activity of PTEN converts PIP3 to PIP2 which suppresses the activation of PDK1 and downstream AKT. An active Rheb results in inhibition of mTORC1 and an activation of the ULK1 complex. PI3P also plays an integral role in the process of autophagy by interacting with VPS34 [ 44 ].

Originally identified and studied in yeast, VPS34 is a class III phosphatidylinositol 3 kinase. In yeast, VPS34 forms one of two complexes. In Mammals, VPS34 is thought to play a similar role in autophagy initiation.

However, it has been difficult to study in mouse models since pan knockouts of VPS34 are embryonically lethal, and there are no inhibitors specific to VPS34 necessitating the use of low specificity inhibitors, such as wortmannin or 3-MA [ 47 ].

Conditional knockout studies using cultured mice embryonic fibroblasts have shown that VPS34 is required for the formation of autophagosomes [ 48 ]. In addition, VPS34 is involved with mTOR regulation of autophagy. Studies using mice embryonic fibroblasts have shown that mTORC1 must be inactivated for the VPS34 initiation complex to be active and that mTORC1 can inhibit the phosphatidylinositol 3-kinase activity of this complex by phosphorylating ATG14 [ 48 ].

PI3P is also a component of the autophagosome. It has been observed to be enriched in the concave surface of early phagophores [ 49 ]. Because of this, PI3P is thought to facilitate the expansion and sealing of autophagosomes. In addition to its function in the autophagosomal membrane, PI3P is thought to mediate selected cargo capture via its interaction with autophagy linked FYVE protein Alfy , a nuclear scaffold protein with a FYVE domain that binds PI3P [ 50 ].

In the autophagic process, Alfy interacts with Ath5 and P62 through its WD40 domain and with PI3P through its FYVE domain. Metabolites formed from the breakdown of phospholipids are also involved in autophagy.

Phosphatidic acid is formed by the breakdown of phosphatidylcholine into choline and phosphatidic acid by Phospholipase D [ 52 ].

Phosphatidic acid plays a role in autophagy by inducing membrane curvature due to its cone shape. In addition, Phosphatidic acid is formed as a result of an absence of nutrients and serves as an inhibitor of mTORC1 thus acting as a positive regulator of autophagy [ 53 ].

Phosphatidic acid can also be converted into diacylglycerol by the Phosphatidic Acid Phosphatases which has other autophagy regulating properties [ 54 ]. Diacylglycerol modulates autophagy by activating Protein Kinase C which induces autophagy by disrupting the B-cell lymphoma protein 2 Bcl-2 -Beclin-1 complex via c-Jun N terminal kinase JNK and Nicotinamide adenine dinucleotide phosphate NADPH oxidase [ 54 ].

Sphingolipids are a class of lipids involved in several processes ranging from apoptosis to cell proliferation to differentiation, inflammation, and autophagy [ 55 ]. Ceramide activates autophagy by inhibiting Akt and resulting in an inactivation of mTOR and an upregulation of Beclin-1 function [ 56 ].

Sphingosinephosphate is formed by the hydrolysis of ceramide into sphingosine followed by its phosphorylation by Sphingosine Kinases 1 and 2 [ 57 ]. Sphingosine Kinase 1 is activated by starvation signals and drives the formation of sphingosinephosphate [ 57 ].

In addition, sphingosine kinase 1 inhibits mTORC1 independently of Akt while upregulating beclin-1 expression, ultimately promoting cell survival [ 57 ].

PPARs are a family of nuclear receptor proteins that act as transcription factors. There are 3 isoforms of PPARs in mammals, PPARα, PPARδ, and PPARγ [ 58 ].

All 3 isoforms of PPARs must bind with a Retinoid Receptor X RXR and a lipid ligand in order to act as transcription factors Fig. Generally, they have been reported to bind to oleic acid, linoleic acids, linolenic acids, prostaglandins, eicosanoids, and oxidized lipids with the help of fatty acid-binding proteins which bind lipophilic ligands in the cytoplasm and shuttle them to their target PPAR [ 60 , 61 ].

PPARs bind to their ligands through the ligand-binding domain LBD. The ligand-binding site is located in the core of the ligand-binding domain that is formed by helices 3,5,7,11, and The cavity formed by these helices is T-shaped [ 62 ].

In order for ligands to bind to PPAR-α or PPAR-γ, they must be able to form a U-shaped conformation, and to bind to PPAR-delta ligands must form an L-shaped conformation [ 63 ].

All PPARs isoforms have been shown to modulate autophagy in the context of different diseases and cellular responses. Overview of PPAR signaling and mechanism for PPARα-mediated autophagy activation in innate immune system.

Initially, lipid molecule enters the cell and is quickly bound by a fatty acid binding protein. The fatty acid binding protein transports the lipid to a PPAR which in turn activates a corresponding RXR.

The PPAR-RXR complex crosses into the nucleus and facilitate expression of required genes. In the case of tuberculosis infection, PPARα upregulates the expression of TFEB which, in turn, drives the expression of autophagy related genes, LAMP3 and RAB7 thus stimulating autophagy.

PPARα is primarily expressed in the liver, brown adipose tissue, heart, and kidney. It promotes uptake and catabolism of fatty acids by helping to express fatty acid transport and binding genes [ 58 , 64 ].

It has been thought to be involved in the innate immune response during mycobacterium infection [ 65 ]. In studies with tuberculosis infected bone-derived macrophages, PPARα was shown to stimulate autophagy and autophagosomal maturation, while suppressing inflammatory responses.

It was determined that following PPARα activation, Transcription Factor EB TFEB was activated and a series of autophagy and lysosomal genes were expressed such as LAMP3 and Rab7 [ 65 ]. Based on this work, it is thought that, in mycobacterial infections, such as tuberculosis, PPARα is activated and in turn activates TFEB.

Together they promote the expression of autophagy-related genes that stimulate autophagy Fig. PPAR δ has high expression levels in the colon, small intestine, liver, heart, lung, and brain.

It plays an important role in diseases such as diabetes, obesity, atherosclerosis, and cancer [ 58 , 64 ]. This is especially poignant because there are great efforts in exploiting autophagy as possible treatments for cancer and diabetes.

Studies in mice cells have shown a marked decrease in autophagic markers associated with the knockout of PPAR δ suggesting its involvement in autophagy [ 66 ]. Finally, PPARγ is expressed in adipose tissue, the intestines, and macrophages.

It is usually involved in fatty acid storage, glucose uptake, and adipogenesis [ 58 , 64 ]. Because of its role in controlling the availability of nutrients, there has been an interest in targeting it as a treatment for cancer.

In Colorectal cancer, studies with Caco-2, a common colorectal cancer cell line, have shown that activation of autophagy occurs following treatment with PPARγ agonist rosiglitazone [ 67 ]. In addition, inhibition of autophagy with 3-MA was observed to induce the expression of PPARγ.

PPARγ was determined to cause the induction of PTEN, an antagonist to PI3K which dephosphorylates and reduces the concentration of PIP 3 [ 67 ]. This results in the overall inhibition of the mTOR pathway and induces autophagy.

In the context of breast cancer, PPARγ has also been implicated to modulate autophagy. Activation of PPARγ by agonist troglitazone was shown to induce autophagy in MDA-MB cells as determined by the measurement of acidic vesicular organelles by staining with Acridine orange [ 68 ].

In addition, studies of constitutively active PPARγ suggest that it is sufficient for the activation of autophagy leading to the belief that autophagy acts to protect cancer cells Fig. PPARγ mediated activation of autophagy.

PPARγ promotes the expression of PTEN. High amounts of PTEN lead to lower concentrations of PI 3,4,5 P3. Less PI 3,4,5 P3 inhibits the activation of PDK1 and ultimately results in inhibition of mTORC1 which causes an activation of autophagy.

Autophagy is intricately related to the metabolism of lipids, namely triglycerides because it responds to the presence or absence of nutrients in the cell.

Furthermore, it is involved with the breakdown of stored lipids in the cell. Triglycerides are stored in organelles known as lipid droplets. They are used to generate energy, building blocks for membranes, and for lipid signaling [ 69 ]. Lipid droplets are broken down for use by the cell via lipophagy.

This process is mediated by the GTPase Rab7 in hepatocytes and results in the release of free fatty acids under starvation conditions to be used as fuel in the mitochondria and undergo β-oxidation.

Rab7 was shown to mediate the docking of autophagosome to lipid droplets facilitating their catabolism [ 70 ]. In addition, Adipose triglyceride lipase ATGL is a regulator of lipophagy. When knocked down in hepatocytes, ATGL causes decreased lipophagy. This ATGL signaling has been observed to occur through sirtuin 1.

Together, these two proteins drive lipophagy and fatty acid oxidation [ 71 ]. Additionally, the breakdown of lipid droplets by lipophagy can be regulated by transcription factors.

TFEB mediates the activation of PPAR alpha as a response to nutrient deprivation in order to activate lipophagy [ 72 ] Additionally, forkhead homeobox protein O1 FOXO1 becomes upregulated in nutrient restricted conditions and increases lipophagy of lipid droplets. The FOXO1 mediated lipophagy activation is facilitated by an increased expression of lysosomal acid lipase LIPA resulting in a release of free fatty acids through adenosine monophosphate kinase AMPK -dependent β-oxidation in adipocytes in nutrient restricted conditions [ 73 ].

Conversely, autophagy is linked to the biosynthesis of new triglycerides as well. Not only does autophagy drive the breakdown of lipid droplets, but it is also tied to the metabolic balance of liver triglycerides.

Diets low in protein result in reduced expression of autophagy receptor SQstm1 and increases the expression of LC3-II. This correlates to the induction of autophagy.

It is speculated that, in the case of low protein availability, autophagy does not catabolize lipids and instead may help triglycerides to accumulate in the liver [ 74 ]. Additionally, Perilipin-2, a protein that associates with lipid droplets, has been observed to protect lipid droplets from autophagy.

Perilipin-2 has been observed to inhibit lipogenesis and triglyceride production as well as upregulating autophagy when it is depleted in the cell [ 75 ]. Free fatty acids have also been implicated in the autophagic pathway. Although they usually act as nutrients, fatty acids can induce cell death when they accumulate in excessive levels in non-adipose cells and tissues.

This is known as lipotoxicity and has been observed in diseases such as obesity, diabetes, and non-alcoholic fatty liver disease [ 76 ]. As a result, levels of free fatty acids are thought to be regulated inside the cell through lipophagy [ 69 ].

Palmitic acid PA and its effects on diabetes has been studied in rat pancreatic beta-cell line INS-1 [ 77 ]. It was determined to trigger autophagy independently of the mTOR pathway. For instance, autophagy was shown to be promoted by stimulating JNK which leads to phosphorylation of Bcl-2 a resulting in its dissociation from Beclin-1 which in turn allowed for the initiation of autophagy and autophagosome formation [ 77 ].

In addition, protein kinase C PKC isoforms δ, ɑ, and Θ have also been implicated in PA-mediated autophagy regulation [ 78 ]. Studies suggest that in mice embryonic fibroblasts, PA, a saturated fatty acid can induce autophagy [ 79 ].

It was reported that palmitic acid was able to increase the amount of LC3, suggesting the induction of autophagy. However, there was no increase in phosphorylation of P70S6K or S6, two downstream proteins in the mTOR signaling pathway [ 79 ].

This suggests that PA induces autophagy independent of mTOR. PKCɑ was identified and shown to be involved in the autophagy inducing process. When it was knocked down with siRNA, LC3 detection fell [ 79 ]. Furthermore, studies show that while prolonged exposure to PA causes cell death, short term exposure induces autophagy, this suggests that autophagy is an important protective measure against lipotoxicity caused by PA [ 79 ].

PA has been shown to modulate autophagy via a secondary signaling pathway. Its effects have been studied in the context of hepatic steatosis; a condition caused by high amounts of fat in the liver [ 80 ].

In hepatic steatosis, high lipid levels cause lipotoxicity. Non-alcoholic steatohepatitis mice were fed a high-fat diet. These mice were shown to exhibit high autophagy mediated by PA [ 80 ].

In these studies, autophagy was determined to be regulated by the activation of mitogen-activated protein kinase MAPK , extracellular signal-regulated kinase ERK , P38, JNK.

Interplay Between Lipid Metabolism and Autophagy

Glycosphingolipid is a key component of the eukaryotic cell membrane and is necessary for cavernous-mediated endocytosis and the function of glycosphingolipid-binding toxins Sillence, Glycosphingolipid biosynthesis is restricted by enhanced autophagy, while its catabolism increases Ghidoni et al.

De novo sphingolipid biosynthesis is essential for autophagy induction Wang et al. Administering inhibitors to the first step of sphingolipid synthesis reduces autophagic activity by affecting autophagosome formation rather than the pre-structure formation of autophagosomes Yamagata et al.

Ceramide, a sphingolipid metabolite, serves as a strong autophagy activator Scarlatti et al. Inhibiting synthesis of inositol phosphorylceramide reduces autophagy Yamagata et al. Mitophagy, the degradation of mitochondria via selective autophagy, is linked to the phospholipid biosynthesis pathway for the conversion of PE to PC by the two methyltransferases, EBP cholestenol delta-isomerase EBP, also known as CHO2 and phosphatidylethanolamine N -methyltransferase PEMT Sakakibara et al.

In addition, the autophagic digestion of LDs through lipophagy in liver is an essential process to obtain energy Cai et al. Thus, the composition of membrane lipid seems to be a hallmark of autophagy induction. Cell death has multiple forms, each exhibiting different molecular mechanisms and signal transductions Tang et al.

Although autophagy generally promotes cell survival through removing damaged organelles and oxidized molecules, it can also cause cell death under certain circumstances. This type of regulated cell death requires autophagy machinery and is termed as autophagy-dependent cell death by the Nomenclature Committee on Cell Death Galluzzi et al.

Lipid peroxidation is a chain reaction of the oxidative degradation of lipids. In the reaction, an initiator radical first takes an allylic hydrogen of the unsaturated lipid and generates a corresponding radical.

The free radical then reacts with an oxygen molecule to generate a corresponding peroxy radical, which captures the allyl hydrogen of another molecule and converts it into a hydroperoxide. These lipid peroxidation products influence cell fate partly through the activation of autophagy.

For example, 4HNE can induce autophagy through the activation of c-Jun amino-terminal kinase JNK Csala et al. The activation of JNK is accompanied by BCL2 being dissociated from BECN1 or by the induction of heme oxygenase 1 HMOX1, also known as HO1 expression and MAP1LC3-II formation Velez et al.

Other signaling associated with 4HNE-induced autophagy are the MAPK, mTOR, and protein kinase C pathways Martinez-Useros and Garcia-Foncillas, In addition to inducing autophagy at lower concentrations, 4HNE can inhibit autophagic flux at higher concentrations Dodson et al.

Lipid peroxidation is implicated in various kinds of regulated cell death Kang et al. In particular, increased lipid peroxidation is an important signal for triggering ferroptosis, an iron-dependent form of cell death that was first identified in mutated RAS cancer cells Dixon et al.

The molecular mechanism of ferroptosis is complicated, depending on the context Xie et al. There are many connections between lipid metabolism and ferroptosis. Lipid biosynthesis that depends on acyl-CoA synthetase long-chain family member 4 ACSL4 Yuan et al.

NADPH oxidases NOXs and other oxidases may also facilitate membrane oxidative injury during ferroptosis Gaschler and Stockwell, ; Xie et al. In contrast, several antioxidant or membrane repair mechanisms can prevent ferroptosis.

The main anti-ferroptosis mechanisms include system xc — -mediated glutathione peroxidase 4 GPX4 activation Dixon et al. Early studies indicate that ferroptosis is different from other forms of regulated cell death, such as apoptosis, necroptosis, and autophagy Dixon et al.

However, increasing studies suggest that ferroptosis exhibits a particular relationship with autophagy during anticancer therapies, tumorigenesis, inflammatory injury, and tissue fibrosis Kang and Tang, ; Zhou et al. Several types of selective autophagy, such as ferritinophagy, clockophagy, lipophagy, and mitophagy, promote ferroptotic cell death through degradation of the iron-storing protein ferritin, the core circadian clock protein aryl hydrocarbon receptor nuclear translocator-like ARNTL, also known as BMAL1 , LDs, and mitochondria, respectively Hou et al.

CMA also promotes ferroptosis through HSPmediated GPX4 degradation Wu et al. The stimulator of interferon response cGAMP interactor 1 STING1, also known as TMEM , an ER-associated protein involved in immunity, infection, and coagulation, connects mitochondrial DNA stress to autophagy-dependent ferroptosis Li et al.

Nanoparticle ferritin-bound erastin and rapamycin NFER , a nanodrug, exhibits a robust ability to induce ferroptosis and autophagy to inhibit tumor growth Li et al.

The release of damage-associated molecular patterns DAMPs from ferroptotic cells serves as a mediator implicated in immune cell activation Wen et al. In addition to cancer biology, autophagy-mediated ferroptosis is also implicated in hepatic fibrosis and neurodegenerative disease Zhang et al.

These findings may provide a useful framework for understanding the pathological characteristics of autophagy-mediated ferroptosis in diseases. Figure 3.

The role of selective autophagy in ferroptosis. Ferritinophagy, clockophagy, lipophagy, and mitophagy promote the degradation of the iron-storing protein ferritin, the core circadian clock protein ARNTL, lipid droplets, and mitochondria, respectively.

Activating these types of selective autophagy results in iron accumulation and lipid peroxidation, which finally induces ferroptotic cell death. Autophagy is tightly regulated by ATG genes. When these genes are mutated, a series of diseases, such as cancer, infectious disease, and neurodegenerative disease, can be induced.

In addition, impaired autophagy is also closely related to the pathology of several lipid metabolic disorders discussed below. Lysosomal storage diseases LSDs are a class of genetic disorders in which proteins responsible for digestion or absorption of endocytosed material do not function or localize properly.

LSDs consist of a group of rare inherited metabolic disorder diseases, such as Niemann-Pick C1 NPC1 disease, G M1 -gangliosidosis, Gaucher disease, Danon disease, Pompe disease, mucolipidosis type IV disease, and neuronal ceroid lipofuscinoses NCLs.

Impaired autophagy activity is commonly responsible for these LSDs Seranova et al. For example, NCLs can be caused by mutations in lysosomal proteases, which leads to a deficiency in the autophagy-dependent degradation of NCL proteins Brandenstein et al.

Mutated NPC1 protein can block autophagy induction through the inhibition of SNARE-dependent membrane fusion, whereas ATG5-deficient cells exhibit increased NPC1protein accumulation Sarkar et al.

Thus, the pharmacological induction of autophagy may ameliorate the phenotypes of LSDs. Preeclampsia is a pregnancy complication characterized by high blood pressure and signs of multiple organ damage e.

Preeclampsia is associated with increased oxidative stress, which can cause autophagy-dependent cell death in extravillous trophoblasts. Mechanistically, oxidative stress reduces lysosomal activities and enhances de novo sphingolipids synthesis, which finally results in ceramide overload-dependent autophagic cell death and subsequent inflammation response Melland-Smith et al.

In addition to excessive autophagy-mediated cellular damage in extravillous trophoblasts, mild levels of autophagy may promote cell survival under hypoxic and low-nutrient conditions Nakashima et al.

It remains unknown whether a systemic autophagy response affects pregnant women. The liver is the hub of fat transport. After fat is digested and absorpted, a portion of it enters the liver, and then it is converted into body fat and stored.

The liver is also one of the main organs for the synthesis of FAs, cholesterol, and phospholipids in the body. Excess cholesterol is excreted with bile. Lipid metabolic imbalance leads to lipid accumulation in the liver, resulting from steatosis due to non-alcoholic fatty liver disease NAFLD.

The level of lipids in the liver is modulated by lipophagy, and impaired lysosomal pathways are involved in the pathogenesis of NAFLD. In contrast, the activation of autophagic pathways has been shown to ameliorate steatosis and NAFLD in animal models Ma et al.

These findings suggest that autophagy activators may have therapeutic potential in NAFLD, which includes a spectrum of hepatic disorders associated with obesity. Specific gene mutations, such for as PTEN-induced kinase 1 PINK1 , increase the risk of PD. PINK1 is an important regulator of mitochondrial quality through multiple mechanisms, including mitophagy Rub et al.

Depleted or mutated PINK1 can increase mitochondrial oxidative injury, ER stress, and mitophagy deficient, which leads to cell death, inflammation, and immune suppression in various diseases Kang et al.

Of note, reduced hydrolase activity has shown to increase cholesterol accumulation during PD development Garcia-Sanz et al. Thus, reducing lipid storage may restore the activity of autophagy, especially mitophagy, to alleviate mitochondrial damage in PD Han et al.

Metabolic syndrome includes a cluster of conditions, such as hypertension, hyperglycemia, excessive waist fat, and abnormal cholesterol levels. Autophagic activity is significantly reduced in metabolic syndrome, which increases the risk of obesity, type 2 diabetes, and atherosclerosis.

The inhibition of autophagy promotes lipid accumulation, mitochondria dysfunction, and ER stress Perrotta and Aquila, ; Zhang et al.

In contrast, the activation of autophagy may decrease metabolic syndrome-related diseases. Autophagy is a conserved adaptive response to environmental changes and plays a pivotal role in cell survival and death.

It can degrade aging organelles and proteins to produce amino acids, nucleotides, and FFAs for cell survival.

At the same time, it can also be used as an active mechanism to induce autophagy-dependent cell death. Generally, ceramides are involved in pro-survival autophagy, while PUFAs are involved in pro-death autophagy. The process of autophagy is regulated by a series of complex signaling molecules and metabolic pathways.

Lipid metabolism plays an important role in regulating multiple cell processes. In the past 10 years, there have been major breakthroughs in understanding the crosstalk between lipid metabolism e.

In particular, lipid metabolism has been found to be involved in the formation of membrane structures related to autophagy.

Moreover, autophagy promotes lipid catabolism and lipid peroxidation-induced cell death, such as ferroptosis. Targeting the autophagy pathway has received extensive attention in human diseases, including lipid metabolism-related disorders.

Although these advances in knowledge have propelled the field forward, there is still much to explore. For example, how does autophagy function in lipid metabolism pathways in different cells or tissues? To what extent does the lipid context around membranes affect autophagy induction? How does autophagy switch from pro-survival mode to a pro-death one that ruptures the membranes?

To what degree is selective autophagy specially linked to ferroptotic cell death? Which ATG modifications are responsible for lipid disorder phenotypes? YX and DT conceived of the topic for this review.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. YX was supported by the National Natural Science Foundation of China , , , and The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We thank Dave Primm Department of Surgery, University of Texas Southwestern Medical Center for his critical reading of the manuscript. Adeva-Andany, M. Mitochondrial beta-oxidation of saturated fatty acids in humans. Mitochondrion 46, 73— doi: PubMed Abstract CrossRef Full Text Google Scholar.

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Cytoplasmic components in hepatic cell lysosomes. CrossRef Full Text Google Scholar. Bai, Y. Lipid storage and lipophagy regulates ferroptosis. Basit, F. Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells. Cell Death Dis.

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Autophagy 10, 1—2. Bersuker, K. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature , — Brandenstein, L. Lysosomal dysfunction and impaired autophagy in a novel mouse model deficient for the lysosomal membrane protein Cln7.

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We thank D. Silver for his discussions, N. Stockert for the protein disulphide isomerase antibody and the personnel at the Analytical Imaging Facility for their technical assistance. This work was supported by National Institutes of Health grants from the National Institute of Diabetes and Digestive and Kidney Diseases and National Institute on Aging, a Glenn Award and an American Liver Foundation Postdoctoral Research Fellowship Award R.

Author Contributions R. performed biochemical analyses and immunoblots. performed the imaging studies and subcellular fractionations.

generated the shRNAs and performed immunoblotting. performed biochemical analyses. and I. all contributed to the in vivo studies. and K. provided the knockout mice. and M. conceived and planned the study, analysed data and wrote the paper.

Department of Developmental and Molecular Biology,,. Department of Pediatrics, Albert Einstein College of Medicine, Morris Park Avenue, Bronx, New York , USA,.

Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo , Japan ,. You can also search for this author in PubMed Google Scholar. Correspondence to Ana Maria Cuervo or Mark J. Cells were imaged at 30 sec intervals.

AVI kb. Arrow points to colocalization event with lysosomes leading to a reduced size of the lipid droplet.

Frontiers | Interplay Between Lipid Metabolism and Autophagy For Autophgay, Autophagy and lipid metabolism can be caused Autophagy and lipid metabolism Vegan diet recipes in Auto;hagy proteases, which leads mdtabolism a Autopahgy in the autophagy-dependent degradation of Metabolismm proteins Brandenstein et al. Department of Developmental and Molecular Biology. To test these possibilities, we performed radiotracer labeling experiments using two different labeled substrates, 14 C-acetate and 3 H 2 O, substrates that label nascent fatty acids with 14 C or 3 H during the initial or reduction steps of fatty acid synthesis, respectively Browse et al. RAB18 loss interferes with lipid droplet catabolism and provokes autophagy network adaptations. Han, X. PDF Split View Views.
Autophagy and lipid metabolism

Autophagy and lipid metabolism -

Thus, the pharmacological induction of autophagy may ameliorate the phenotypes of LSDs. Preeclampsia is a pregnancy complication characterized by high blood pressure and signs of multiple organ damage e.

Preeclampsia is associated with increased oxidative stress, which can cause autophagy-dependent cell death in extravillous trophoblasts. Mechanistically, oxidative stress reduces lysosomal activities and enhances de novo sphingolipids synthesis, which finally results in ceramide overload-dependent autophagic cell death and subsequent inflammation response Melland-Smith et al.

In addition to excessive autophagy-mediated cellular damage in extravillous trophoblasts, mild levels of autophagy may promote cell survival under hypoxic and low-nutrient conditions Nakashima et al.

It remains unknown whether a systemic autophagy response affects pregnant women. The liver is the hub of fat transport. After fat is digested and absorpted, a portion of it enters the liver, and then it is converted into body fat and stored. The liver is also one of the main organs for the synthesis of FAs, cholesterol, and phospholipids in the body.

Excess cholesterol is excreted with bile. Lipid metabolic imbalance leads to lipid accumulation in the liver, resulting from steatosis due to non-alcoholic fatty liver disease NAFLD. The level of lipids in the liver is modulated by lipophagy, and impaired lysosomal pathways are involved in the pathogenesis of NAFLD.

In contrast, the activation of autophagic pathways has been shown to ameliorate steatosis and NAFLD in animal models Ma et al. These findings suggest that autophagy activators may have therapeutic potential in NAFLD, which includes a spectrum of hepatic disorders associated with obesity.

Specific gene mutations, such for as PTEN-induced kinase 1 PINK1 , increase the risk of PD. PINK1 is an important regulator of mitochondrial quality through multiple mechanisms, including mitophagy Rub et al.

Depleted or mutated PINK1 can increase mitochondrial oxidative injury, ER stress, and mitophagy deficient, which leads to cell death, inflammation, and immune suppression in various diseases Kang et al. Of note, reduced hydrolase activity has shown to increase cholesterol accumulation during PD development Garcia-Sanz et al.

Thus, reducing lipid storage may restore the activity of autophagy, especially mitophagy, to alleviate mitochondrial damage in PD Han et al. Metabolic syndrome includes a cluster of conditions, such as hypertension, hyperglycemia, excessive waist fat, and abnormal cholesterol levels.

Autophagic activity is significantly reduced in metabolic syndrome, which increases the risk of obesity, type 2 diabetes, and atherosclerosis.

The inhibition of autophagy promotes lipid accumulation, mitochondria dysfunction, and ER stress Perrotta and Aquila, ; Zhang et al. In contrast, the activation of autophagy may decrease metabolic syndrome-related diseases.

Autophagy is a conserved adaptive response to environmental changes and plays a pivotal role in cell survival and death. It can degrade aging organelles and proteins to produce amino acids, nucleotides, and FFAs for cell survival.

At the same time, it can also be used as an active mechanism to induce autophagy-dependent cell death. Generally, ceramides are involved in pro-survival autophagy, while PUFAs are involved in pro-death autophagy.

The process of autophagy is regulated by a series of complex signaling molecules and metabolic pathways. Lipid metabolism plays an important role in regulating multiple cell processes. In the past 10 years, there have been major breakthroughs in understanding the crosstalk between lipid metabolism e.

In particular, lipid metabolism has been found to be involved in the formation of membrane structures related to autophagy. Moreover, autophagy promotes lipid catabolism and lipid peroxidation-induced cell death, such as ferroptosis. Targeting the autophagy pathway has received extensive attention in human diseases, including lipid metabolism-related disorders.

Although these advances in knowledge have propelled the field forward, there is still much to explore. For example, how does autophagy function in lipid metabolism pathways in different cells or tissues?

To what extent does the lipid context around membranes affect autophagy induction? How does autophagy switch from pro-survival mode to a pro-death one that ruptures the membranes? To what degree is selective autophagy specially linked to ferroptotic cell death?

Which ATG modifications are responsible for lipid disorder phenotypes? YX and DT conceived of the topic for this review. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. YX was supported by the National Natural Science Foundation of China , , , and The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We thank Dave Primm Department of Surgery, University of Texas Southwestern Medical Center for his critical reading of the manuscript. Adeva-Andany, M. Mitochondrial beta-oxidation of saturated fatty acids in humans. Mitochondrion 46, 73— doi: PubMed Abstract CrossRef Full Text Google Scholar.

Alphonse, P. Revisiting human cholesterol synthesis and absorption: the reciprocity paradigm and its key regulators. Lipids 51, — Ashford, T. Cytoplasmic components in hepatic cell lysosomes. CrossRef Full Text Google Scholar. Bai, Y. Lipid storage and lipophagy regulates ferroptosis.

Basit, F. Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells. Cell Death Dis. Bejarano, E. Connexins modulate autophagosome biogenesis.

Cell Biol. Bekbulat, F. RAB18 loss interferes with lipid droplet catabolism and provokes autophagy network adaptations. Bernard, A. Defining the membrane precursor supporting the nucleation of the phagophore. Autophagy 10, 1—2. Bersuker, K. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis.

Nature , — Brandenstein, L. Lysosomal dysfunction and impaired autophagy in a novel mouse model deficient for the lysosomal membrane protein Cln7. Cabodevilla, A. Cell survival during complete nutrient deprivation depends on lipid droplet-fueled beta-oxidation of fatty acids.

Cai, Z. Activation of cell-surface proteases promotes necroptosis, inflammation and cell migration. Cell Res. Caron, A. The roles of mTOR complexes in lipid metabolism.

Cerqueira, N. Cholesterol biosynthesis: a mechanistic overview. Biochemistry 55, — Chai, C. Metabolic circuit involving free fatty acids, microRNA , and triglyceride synthesis in liver and muscle tissues. Gastroenterology , — Chirala, S. Structure and function of animal fatty acid synthase.

Lipids 39, — Csala, M. On the role of 4-hydroxynonenal in health and disease. Acta , — Dai, C. Transcription factors in ferroptotic cell death. Cancer Gene Ther. Dai, E. Autophagy-dependent ferroptosis drives tumor-associated macrophage polarization via release and uptake of oncogenic KRAS protein.

Autophagy 4, 1— ESCRT-III-dependent membrane repair blocks ferroptosis. AIFM2 blocks ferroptosis independent of ubiquinol metabolism. DeBose-Boyd, R. Significance and regulation of lipid metabolism. Diao, J. ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes.

Dikic, I. Mechanism and medical implications of mammalian autophagy. Dixon, S. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell , — Dodson, M. NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis. Redox Biol. Regulation of autophagy, mitochondrial dynamics, and cellular bioenergetics by 4-hydroxynonenal in primary neurons.

Autophagy 13, — Doll, S. FSP1 is a glutathione-independent ferroptosis suppressor. Dugail, I. Biochimie 96, — Fahy, E.

A comprehensive classification system for lipids. Lipid Res. PubMed Abstract Google Scholar. Update of the LIPID MAPS comprehensive classification system for lipids. Fan, W. Galluzzi, L. Molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death Cell Death Differ.

Garcia-Sanz, P. Gaschler, M. Lipid peroxidation in cell death. Gatticchi, L. The Tm7sf2 gene deficiency protects mice against endotoxin-induced acute kidney injury. PLoS One e Ghidoni, R. The metabolism of sphingo glyco lipids is correlated with the differentiation-dependent autophagic pathway in HT cells.

Girardi, J. De novo synthesis of phospholipids is coupled with autophagosome formation. Hypotheses 77, — Haberzettl, P. Oxidized lipids activate autophagy in a JNK-dependent manner by stimulating the endoplasmic reticulum stress response.

Halama, A. Accelerated lipid catabolism and autophagy are cancer survival mechanisms under inhibited glutaminolysis. Cancer Lett. Han, X.

Hatakeyama, R. Spatially distinct pools of TORC1 balance protein homeostasis. Cell 73, — Herman, N. Enzymes for fatty acid-based hydrocarbon biosynthesis.

Holczer, M. A double negative feedback loop between mTORC1 and AMPK kinases guarantees precise autophagy induction upon cellular stress. Hou, W. III, et al. Autophagy promotes ferroptosis by degradation of ferritin.

Autophagy 12, — Hung, Y. DGAT1 deficiency disrupts lysosome function in enterocytes during dietary fat absorption.

Acta Mol. Lipids , — Iershov, A. The class 3 PI3K coordinates autophagy and mitochondrial lipid catabolism by controlling nuclear receptor PPARalpha. Irungbam, K.

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Cell Sci. On the other hand, overexpression of PDAT1 or blocking the chloroplast lipid biosynthesis pathway in act1 accelerates autophagy-mediated membrane lipid turnover and hence increases the cellular demand for fatty acids.

This increased fatty acid demand may cause a decrease in fatty acids in the cytosol, thereby partially relieving feedback inhibition on plastid fatty acid synthesis.

In this context, it is worth noting that inefficient utilization of fatty acids for glycerolipid biosynthesis in the ER has been shown to cause a feedback inhibition on fatty acid synthesis by an unknown mechanism Bates et al. TAG and fatty acid synthesis are increased in tgd1 mutants Fan et al.

These results suggest that under normal growth conditions, autophagy functions in TAG synthesis, whereas the cytosolic pathway mediated by neutral lipases including SDP1 is the major mechanism for TAG catabolism Figure Under extended darkness, TAG content decreases when autophagy is induced but increases when autophagy is disabled in sdp1.

In addition, disruption of SDP1 does not impact autophagic flux under either normal growth or starvation conditions Supplemental Figure These results suggest an important and general role of lipophagy in mediating TAG hydrolysis under starvation conditions Figure TAG did not accumulate in atg mutants under extended darkness Supplemental Figure This result suggests that the SDP1-mediated cytosolic lipolytic pathway can functionally compensate for the lack of lipophagy in TAG hydrolysis under starvation.

Previous studies showed that plant autophagic organelles contain hydrolytic enzymes, including proteases and lipases, for cargo degradation at the onset of their formation Marty, , ; Buvat and Robert, and are functionally sufficient to break down the sequestered materials on their own Rose et al.

In accordance with the autophagosome-autonomous hydrolysis, our ultrastructural analysis showed that LDs and other cellular constituents were degraded in AVs Figures 7D and 10C , in addition to the central vacuole Figure 10E.

These results point to the unique aspects of plant autophagy in comparison with this catabolic process in yeast and mammals, where the autophagosome itself lacks degradative enzymes and its cargo is broken down following fusion with lytic compartments such as vacuoles and lysosomes, respectively Eskelinen, ; Suzuki and Ohsumi, ; Reggiori and Klionsky, ; Dikic, ; Galluzzi et al.

Our ultrastructural analysis showed that the autophagic degradation of LDs in Arabidopsis occurs in a process resembling microlipophagy in yeast.

Disruption of autophagy genes increased TAG content in sdp1 under starvation conditions Figure These results suggest that microlipophagy in Arabidopsis depends on the core machinery of macroautophagy, similar to the situation in yeast van Zutphen et al. At present, the exact mechanism underlying microautophagy and the role of ATG gene products in microlipophagy remain largely unknown Noda and Inagaki, ; Galluzzi et al.

Our results showed that microautophagy-like LD degradation occurs in AVs, key autophagic structures in macroautophagy Eskelinen, Therefore, it is possible that the observed dependence of starvation-induced TAG and LD accumulation on the macroautophagic machinery in Arabidopsis may simply reflect the essential role of core ATG proteins in the formation of autophagosomes and hence AVs.

In support of this possibility, disruption of the core ATG genes blocks the formation of both AVs and microlipophagy Figure Recently, vacuolar membrane lipid rafts enriched in sterols have been shown to be necessary for microlipophagy in yeast Oku and Sakai, Further studies are needed to test whether the sterol-enriched membrane rafts are involved in microlipophagy in plants, to determine how TAG is hydrolyzed in vacuoles, and to establish the regulation and physiological functions of lipophagy.

The Arabidopsis Arabidopsis thaliana plants used in this study were of the Columbia ecotype. The tgd1 mutant was previously described by Xu et al. The PDAT1 -overexpressing lines 3 and 4 were described in Fan et al. The primers used for genotyping sdp1 were as described previously Fan et al.

Genotyping of tgd1 and act1 mutants was as described previously Xu et al. For plant growth in soil, surface-sterilized seeds of Arabidopsis were germinated on 0.

For starvation treatment, whole plants, unless stated otherwise in Figure 12A , were transferred to continuous darkness at 24°C for the time indicated.

The PCR products were cloned into a binary vector pPZP Fan et al. After confirming the integrity of the construct by sequencing, plant stable transformation was performed according to Clough and Bent Lipids were extracted from leaves of 4-week-old plants grown in soil as described by Fan et al.

To quantitate low TAG levels in leaves of wild type and atg mutants, total lipid extracts were first fractionated through silica columns Discovery DSC-Si SPE tube, volume 6 mL, Supelco as described by James et al.

Fatty acid methyl esters were prepared as described by Li-Beisson et al. Separation and identification of the fatty acid methyl esters were performed on an HP gas chromatograph-mass spectrometer Hewlett-Packard fitted with a 30 m × μm DB capillary column Agilent with helium as the carrier gas as described by Fan et al.

Fatty acid methyl esters were quantified using heptadecanoic acid as an internal standard as described by Fan et al. Equal fresh weight of mature leaves of 4-week-old plants grown in soil was ground in liquid nitrogen, homogenized with 2× Laemmli sample buffer. The extracts were incubated for 5 min in boiling water and clarified by centrifugation at 12, g for 5 min at 22°C.

Immunoblot analyses were performed according to the ECL Western Blotting procedure 32,, Thermo Fisher Scientific with antibodies against GFP catalog no.

E11LF, BioLegend , ATG8a catalog no. AS, lot no. MBS, lot no. M14L06, MyBioSource. Targeted proteins were visualized using an ImageQuant LAS biomolecular imager GE Healthcare Life Sciences.

In vivo labeling experiments with 14 C-acetate or 3 H 2 O were done as described previously by Fan et al. Developing seeds of 50 siliques were directly harvested into labeling medium containing 20 mM MES, pH 5.

The assay was started by the addition of 0. After incubation for 1 h, tissues were washed two times with water and immediately used for lipid extraction. For pulse-chase labeling experiments, leaves were labeled for 1 h with 14 C-acetate. Total lipids were extracted and separated as described previously by Fan et al.

Radiolabel loss was calculated by correcting for the dilution of radioactivity caused by tissue growth during the chase period. Microsomal membranes were isolated from 3-week-old seedlings as described previously Xu et al.

Radioactive PC for PDAT activity assays was prepared after incubating 2-week-old seedlings overnight in 20 mM MES-KOH, pH 6. Lipids were extracted and separated by TLC as described by Fan et al. Radiolabeled PC was eluted from silica gel using chloroform:methanol:formic acid The reaction mixture contained 0.

The reaction solution was thoroughly mixed and incubated at room temperature for 30 min. Lipid extraction and TLC separation were done as described previously by Fan et al.

Radioactivity in TAG was determined by scintillation counting. Detached leaves of 4-week-old plants grown in soil were floated on water with or without the addition of 5 mM 3-MA dissolved in water, Sigma-Aldrich and 0. Samples were taken every 24 h over 4 d for lipid analysis as described previously Fan et al.

For the colocalization study, leaf samples were mounted in water on slides and were directly examined using a Leica TCS SP5 laser scanning confocal microscope with sequential scanning.

GFP was excited with a wavelength of nm and detected at to nm. DsRed was excited at nm and detected at to nm. For tonoplast imaging, transgenic plants coexpressing OLE1-GFP and ΔTIP-DsRed were germinated on 0.

Six-day-old seedlings were dark treated for 1 d and then transferred to half-strength MS medium with or without 0. The hypocotyls or cotyledons were observed under confocal microscopy.

For transmission electron microscopy, leaf tissues were fixed with 2. For chloroplast counting, leaf tissues were fixed and embedded. The number of chloroplasts was counted from at least 60 mesophyll cell cross sections for each time point of dark treatment. Colocalization analysis of OLE1-GFP and ATG8e-DsRed signals was done with the Coloc 2 plugin for ImageJ.

Background subtraction from image pairs was performed using rolling ball subtraction with a pixel ball size. Statistical significance of the PCC of the image pairs was analyzed using the Costes image randomization test as described previously Costes et al.

Regions of interest were selected for colocalization analysis with Costes randomizations using a point spread function of 3. Five-day-old seedlings grown on 0. The seedlings were then transferred to half-strength MS medium containing 0.

The fixed hypocotyls were washed twice with 0. After dehydration, the tissues were embedded in LR White resin CA, Electron Microscopy Sciences, London Resin Company in gelatin capsules.

Resin polymerization was performed at 50 to 55°C. Ultrathin sections 70 to 90 nm of LR White—embedded hypocotyls were collected with formvar-coated mesh nickel grids.

The grids were first washed with 1× PBS containing 0. After blocking, the grids were incubated with the primary antibody:rabbit polyclonal anti-ATG8a catalog no.

After rinsing with blocking solution five times, 1 min each, the grids were then incubated in the secondary antibody of goat anti-rabbit immunoglobulin G conjugated with nm gold particles catalog no.

G, lot no. SLBW, Sigma-Aldrich; dilution in blocking solution for 1 h at room temperature. Following washing with 1× PBS and 0. Supplemental Figure 1. Time course of the incorporation of radiolabel from 14 C-acetate or 3 H 2 O into total fatty acids in wild-type developing embryos.

Supplemental Figure 2. Rate of the incorporation of radiolabel from 14 C-acetate or 3 H 2 O into TAG in developing embryos and seedlings. Supplemental Figure 3. Rate of the incorporation of radiolabel from 14 C-acetate or 3 H 2 O into total fatty acids in leaves.

Supplemental Figure 4. Rate of the incorporation of radiolabel from 14 C-acetate into total membrane lipids in leaves. Supplemental Figure 5. PDAT activity in microsomal membranes isolated from seedlings.

Supplemental Figure 6. Disruption of autophagy reduces TAG content in mature leaves of 4-week-old PDAT1 -overexpressing transgenic plants. Supplemental Figure 7.

Increased accumulation of DsRed-ATG8e—labeled structures in leaves of tgd1 plants under dark treatment. Supplemental Figure 8.

Accumulation of autophagosomes and autophagic vacuoles in mature leaves of 4-week-old sdp plants under dark treatment. Supplemental Figure 9. The appearance of LDs in the central vacuole in wild-type seedlings after dark treatment in the presence of concA.

Supplemental Figure Autophagic activity in 4-week-old sdp plants under dark-induced starvation. TAG levels in mature leaves of 4-week-old wild type, atg and atg plants under dark-induced starvation.

Membrane lipid levels in mature leaves of 4-week-old sdp , atg sdp , and atg sdp plants under dark-induced starvation. Chloroplast number in mature leaves of sdp plants under dark-induced starvation. Supplemental Data Set. Results of statistical analyses.

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper: SDP1 Gramene: At5g SDP1 Araport: At5g ATG10 Gramene: AT3G ATG10 Araport: AT3G ATG3 Gramene: AT5G ATG3 Araport: AT5G LAS Gramene: AT1G LAS Araport: AT1G ACT1 Gramene: AT2G ACT1 Araport: AT2G TGD1 Gramene: AT1G TGD1 Araport: AT1G ATG5 Gramene: AT5G ATG5 Araport: AT5G PDAT1 Gramene: at5g PDAT1 Araport: at5g ATG2 Gramene: AT3G ATG2 Araport: AT3G ATG8 Gramene: AT4G ATG8 Araport: AT4G This work was supported by the U.

Department of Energy , Office of Science, Office of Basic Energy Sciences DE-SC , specifically through the Physical Biosciences program of the Chemical Sciences, Geosciences and Biosciences Division. Use of the transmission electron microscope and the confocal microscope at the Center of Functional Nanomaterials was supported by the Office of Basic Energy Sciences, U.

Department of Energy DE-SC and J. designed the experiments. performed the research. and C. participate in data analysis. wrote the article with contributions from J. and L. Anding , A. Cleaning house: Selective autophagy of organelles.

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AAutophagy in Health metabolusm Disease volume 19 Collagen and Muscle Recovery, Article number: Metabolixm this article. Metrics details. The process of autophagy is integral Recovery solutions cellular metaboolism. In this process, proteins, Recovery solutions, and metabolites Autphagy engulfed in a lipid Mental resilience building and Ketabolism to a lysosome for degradation. Its central role in protein and organelle homeostasis has piqued interest for autophagy dysfunction as a driver of pathology for a number of diseases including cancer, muscular disorders, neurological disorders, and non-alcoholic fatty liver disease. For much of its history, the study of autophagy has centered around proteins, however, due to advances in mass spectrometry and refined methodologies, the role of lipids in this essential cellular process has become more apparent. The Autopgagy is the metaboilsm organ of lipid Mental resilience building and plays a vital role Lipud cellular Atophagy, such as lipid digestion, absorption, transport, and decomposition [ Recovery solutions ]. Chronically disturbed hepatic metabolism may lead to obesity and metabolic syndrome, causing non-alcoholic fatty liver disease NAFLD [ 2 ]. Approximately 1 in 30 patients diagnosed with NAFLD develops cirrhosis or a liver-associated complication [ 3 ]. Autophagy plays a crucial role during hepatic lipid metabolism. Diet, environment, and drugs strongly affect hepatic lipid metabolism through autophagy [ 4 ]. Therefore, a further understanding of the specific molecular mechanisms of autophagy and various cell types involved has been deeply explored.

Autophagy and lipid metabolism -

The process of lipophagy involves subcellular structures, including LD, autophagosomes, and lysosomes, dynamic changes in the membrane structure and vesicle transport, and synergism with lipid intake and hydrolysis [ 9 ]. Hormones, natural compounds, and metal ions can control lipophagy.

There are several relatively specific molecules involved in lipophagy in cells. The deacetylation of proteins and histones upregulates or downregulates gene transcription and protein function [ 56 ].

A previous study showed that the regulation of the deacetylation of SIRT1 can induce autophagy and decrease lipid accumulation in the liver [ 57 ]. Berberine stimulates SIRT1 deacetylation and induces autophagy in an autophagy protein 5-dependent manner.

It also promotes gene expression and circulation of fibroblast growth factor 21 FGF21 and ketone bodies within mouse liver in a SIRT1-dependent way. Autophagy and FGF21 activation in the liver regulate lipid storage as well as the utilization and whole-body energy metabolism [ 58 ].

Both resveratrol and caloric restriction upregulate the SIRT1-autophagy pathway to reduce hepatic steatosis in rats fed with a high-fat diet. Autophagy is later induced by the SIRT1-FOXO3 signal transduction pathway [ 59 ]. A recent study observed that the natural polyphenol resveratrol enhances the levels of cAMP, SIRT1, phosphorylated protein kinase A pPRKA , phosphorylated AMP-activated protein kinase pAMPK , and SIRT1 activity in HepG2 cells.

Moreover, it can induce autophagy through the cAMP-PRKA-AMPK-SIRT1 signal pathway, whereas autophagy inhibition markedly abolishes resveratrol-mediated hepatic steatosis improvement [ 60 ]. Caloric restriction and resveratrol in animal studies can affect the sirtuin system. A randomized trial on healthy people revealed that the reduction of calories and supplementation with resveratrol significantly increases SIRT1 plasma levels [ 60 ].

Resveratrol increases the expression of SIRT1 mRNA in the liver of mice fed with a high-fat diet and controls the number and function of human adipocytes in a SIRT1-dependent manner [ 59 ].

However, SIRT1 also down-regulates the sterol regulatory element-binding protein SREBP homologs during fasting, inhibiting lipid synthesis and storage. SREBPs are steroid regulatory element binding proteins and transcription factors.

A previous study revealed that SIRT1 can directly deacetylate SREBP [ 61 ]. Therefore, the modulation of SIRT1 activity may affect SREBP ubiquitination, protein stability, and target gene expression.

SREBPs undergo cleavage-induced activation due to the low sterol levels in the cell, thus promoting the transcription of enzymes critical to sterol biosynthesis [ 62 ]. TGs in the liver are sequestered into the LDs, which are the main lipid storage organelles.

LDs are home to proteins, the most abundant of which are the perilipins PLINs [ 7 ]. When mice are hungry, the degradation of PLIN2 and PLIN3 in hepatocytes is enhanced using autophagy. In contrast, the increase of adipose triglyceride lipase ATGL and LC3 is observed on the surface of LD [ 64 ].

In addition, PLIN1 can colocalized with the selective autophagy receptor p62, mediating the recognition of LD through the autophagy initiation membrane [ 65 ].

Neutral lipids in mouse liver and cultured hepatocytes are mobilized through the sequestration of perilipin 2-coated LDs in autophagosomes. This involves that LDs are delivered to the lysosomes, where triacylglycerols are hydrolyzed by lysosomal lipases [ 66 ].

The research identified that unphosphorylated perilipin 1 prevents Rab7 from docking to LDs and inhibits lipophagy. Moreover, Rab7 is recruited to lysosomes through conformational changes in perilipin 1, which are induced by phosphorylation with the association of LD with lysosomes [ 67 ].

ATGL is closely associated with lipophagy. Although ATGL is an essential molecule in direct lipid decomposition, it mediates lipid decomposition synergistically through lipophagy [ 68 ]. When lipophagy is inhibited, the increase in lipid decomposition due to ATGL overexpression is also inhibited.

It is speculated that ATGL may decrease the size of LDs through lipolysis and develop the conditions for lipophagy [ 69 ]. In addition, ATGL is a selective autophagy receptor and mediates the specific recognition of LD by LC3 on the autophagy membrane through the LC3-interacting region LIR [ 70 ].

Autophagy stimulates lipophagy by the co-localization of ATGL and LD. Similarly, autophagy activation increases the co-localization of ATGL and LD [ 71 ].

Moreover, the correlation between ATGL and LD in autophagy-deficient cells significantly decreased, indicating that the localization ability of the lipase on LD is deficient [ 72 ].

The decrease in lipolytic activity of ATGL in autophagy-deficient cells cannot be attributed to the change in its association with LD.

Moreover, its binding to the autophagy activation gene marker 58 CGI increases. These phenomena indicate that the relationship between ATGL and liver LD affects lipophagy activation [ 72 ].

Peroxisome proliferator-activated receptor- α PPAR α is a ligand-induced nuclear receptor protein and a PPAR subtype. PPAR α controls liver autophagy-regulating gene transcription of autophagy-related proteins, thereby regulating fatty acid β -oxidation and autophagy [ 73 ].

Researchers speculate that PPAR α can link autophagy signal and gene transcription [ 74 ]. PPAR α is the main isomer inside the liver. PPARs are from the nuclear receptor family of ligand-activated transcription factors.

PPAR ligands include endogenous lipids like FFA and lipid metabolites [ 75 ]. After ligand binding, PPAR binds to the PPAR response element inside the promoter of the target gene and heterodimerizes with another nuclear receptor, the retinoid X receptor RXR [ 76 ].

Autophagy-lysosome-mediated lipophagy of TG in the liver is controlled by PPAR α [ 77 ]. A study revealed that zinc is an effective promoter of fat phagocytosis.

The administration of zinc significantly reduces lipid accumulation in hepatocytes. It enhanced the release of free fatty acids, which is associated with elevated fatty acid oxidation and inhibition of fat formation, followed by autophagy activation.

High-fat diet significantly induces hepatic steatosis in rats. In contrast, pioglitazone reverses hepatic steatosis due to a high-fat diet.

Pioglitazone combined with a high-fat diet dramatically decreases the content of serum insulin and liver TG [ 78 ]. Pioglitazone significantly increases the expression of lipolysis-related proteins ATGL, HSL , β -oxidation-related proteins CPT-1A , and autophagy-related proteins ATG7, LC3, LAL in hepatocytes [ 79 ].

Pioglitazone attenuates hepatic steatosis by promoting intracellular lipolysis, β -oxidation, and autophagy, depending on PPAR α and PPAR γ [ 5 ]. Some researchers also identified a nuclear receptor, farnesoid X receptor FXR , which has an opposite effect than that exerted by PPAR. PPAR α and FXR are activated in fasting and fed livers.

Both PPAR α and FXR regulate liver autophagy in mice. PPAR α induces autophagic lipid degradation or lipophagy, while FXR strongly suppresses the induction of autophagy in the fasting state. Although PPAR α and FXR compete to bind to the common sites in the autophagy gene promoter, they possess an opposite regulatory effect [ 74 ].

The Rab GTPase protein family is a crucial regulator of intracellular vesicular trafficking. The Rab family members regulate a series of molecular events by cycles between active GTP-states and inactive GDP-states [ 80 ].

The Rab protein family is vital in LD metabolism. Approximately 30 Rab family proteins have been identified on the LD surface [ 81 ]. Among them, Rab7 protein is widely involved in the regulation of the autophagosome maturation and intracellular transport. Hepatocyte starvation activates Rab7 on the surface of LDs and promotes lysosomal transport to LDs [ 82 ].

Rab7 expression in the liver of rats subjected to alcohol liquid diet is also significantly reduced.

These results indicate that Rab7 regulates lipid metabolism through lipophagy [ 83 ]. Rab10 is a member of the Rab family of small GTPase located on the LD surface.

Activated Rab10 in starved hepatocytes is co-located on the LD surface with the autophagy proteins Lc3 and Atg In addition, Rab25 and Rab32 can be involved in the molecular mechanism of lipophagy [ 84 ]. However, the involvement of other members of the RAB family in regulating lipophagy requires further exploration.

It has been observed that specific neurons within the central nervous system CNS can remotely control lipophagy in the liver [ 85 ]. Lysosomal-associated membrane protein 3 LAMP3 is overexpressed, activating Akt and upregulating the expression of the lipogenase FASN and SCD-1 in HepG2 cells.

Oleic acid in unsaturated fatty acids can enhance the formation of triglyceride-rich LD while inducing autophagy. Palmitic acid in saturated fatty acids is rarely converted into triglyceride-rich LD, thereby inhibiting autophagy and inducing apoptosis [ 87 ].

However, palmitic acid can induce autophagy through the protein kinase C-mediated signal transduction pathway. Moreover, palmitic acid can phosphorylate the autophagy-promoting factor Hsp27 and enhance its induced autophagy [ 88 ].

TFEB is a basic-helix-loop-helix-leucine zipper bHLH-Zip transcription factor in the MiT family. Many lysosomal genes are regulated by the single transcription factor TFEB, such as lipid metabolism-related genes PGC-1 α , autophagy-related genes-Lamp1, cathepsin B, Atg9B, and LC3.

TFEB plays a crucial role in the regulation of lipid homeostasis through the link of autophagy to energy metabolism at the transcriptional level [ 89 ]. TFEB expression is induced by starvation and inhibited by alcohol, while its activation is associated with the inactivation of mTORC and other TFEB dephosphorylations [ 90 ].

The transcription factor E3 also belongs to the MiT family, its function is similar to TFEB, and it is usually located in the cytoplasm.

After activation, it translocates inside the nucleus and decreases the steatosis of hepatocytes by enhancing lipophagy [ 48 ]. Many other factors affect autophagy, including calcium channel blockers, phosphatidylinositolphosphate 4-kinase, and thyroid hormones [ 91 , 92 ].

In addition, diets rich in polyunsaturated fatty acids PUFA , excessive exercise, and a certain degree of radiation directly or indirectly regulate lipophagy [ 93 ]. Autophagy and lipid metabolism can influence each other and are crucial in maintaining the homeostasis of lipid metabolism [ 4 ].

Lipophagy can produce free fatty acids and remove excess lipids to maintain the balance of lipid metabolism along with the physiological function of hepatocytes [ 10 ].

In addition, lipid metabolism feedback induces autophagy to start or stop various abnormal lipid metabolisms, liver injury repair, and other pathological changes, thereby regulating lipid metabolism [ 94 ].

Lipophagy is a pathway of lipid catabolism in the liver. The inhibition of autophagy increases the content of intracellular TG and the number and size of LD [ 95 ].

In a situation of continuous starvation, liver autophagosomes prioritize the phagocytosis of lipids to provide energy, and lipophagy is activated when lipids accumulate excessively, inhibiting the excessive accumulation of fat inside cells [ 96 ].

The selectivity of autophagosomes in hepatocytes gradually increased with the extension of time during in vitro and in vivo experiments to form lipid-based selective autophagy, regardless of starvation or oleic acid incubation [ 97 ].

Hepatocyte in vitro experiments revealed that lipopolysaccharide-induced autophagy can enhance LD degradation and inhibit lipid accumulation in mouse liver [ 12 ]. In addition to the classical degradation pathway of LD by cellular solute lipase, LD is also isolated in autophagosomes.

Autophagosomes selectively uptake LD and regulate the LD isolation membrane curvature through autophagy-associated proteins. The autophagic degradation of LD is also involved in the formation of LD themselves [ 98 ].

Lysosomal enzymes decompose the LD components after the fusion of autophagosomes and lysosomes [ 99 ]. In addition, experiments have revealed that cold-induced central autophagy activates liposomes and cellular solute lipase; thus, the inhibition of autophagy can prevent lipid utilization [ ].

In contrast to mammalian cells, microlipophagy has been documented in Saccharomyces cerevisiae , in which the lipid droplet is trafficked to the vacuole by microtubules and not by Atg proteins [ ]. Despite this, the latter is still necessary to catalyze the interactions between the lipid droplet and the vacuole, as evidenced by the formation of aggregates of larger droplets in their absence.

Vac8 and Trs85 are special adaptor proteins used in microlipophagy to selectively dissolve lipids. Atg11, a component of other specialized autophagic machinery pexophagy and Cvt pathway , also increases the efficiency of macrolipophagy [ ]. A study observed that autophagy mediates hepatic lipid degradation and is involved in hepatic lipid anabolism to maintain lipid homeostasis.

The main components of very low-density lipoprotein VLDL are TGs synthesized by hepatocytes using sugars and fatty acids from lipid mobilization or residual chylous particles , and apolipoproteins APOB, APOAI and APOE synthesized by hepatocytes plus a small amount of phospholipids, cholesterol and their esters [ ].

In contrast, the inhibition of autophagy causes a decrease in VLDL production [ ]. In addition to lipid catabolism, lipophagy is also vital in lipogenesis, lipid synthesis, and lipid droplet biogenesis. The autophagy machinery participates in lipid droplet formation in hepatocytes and cardiomyocytes.

A research found that the LC3 conjugation system is involved in lipid droplet formation [ ]. Genetical ablation of Atg7 highly suppresses the cytosolic accumulation of LDs in hepatocytes and cardiac myocytes under both 12 and 24 h of starvation [ ].

The researchers speculated that lipophagy enhances lipid droplet formation [ ]. Lipophagy prevents fat accumulation by degrading intracellular LD, which are closely associated with various pathways of lipophagy.

However, the excessive activation of autophagy can induce apoptosis and aggravate the disease in the later stage of the fatty liver [ ]. Therefore, the proper regulation of lipophagy is more conducive to maintaining normal lipid metabolism. There are many ways to regulate hepatocyte lipid metabolism.

One keyway is to increase lipid deposition in the liver by controlling lipophagy. However, the related mechanism is not precise and requires further research [ ]. SCD1 is a crucial enzyme for controlling lipid metabolism.

Sodium palmitate-treated hepatocytes had an increased SCD1 expression, AMPK inactivation, and lipophagy deficiency [ ].

We found that disruption of basal autophagy impedes organellar membrane lipid turnover and hence fatty acid mobilization from membrane lipids to TAG. We show that lipophagy is induced under starvation as indicated by colocalization of LDs with the autophagic marker and the presence of LDs in vacuoles.

We additionally show that lipophagy occurs in a process morphologically resembling microlipophagy and requires the core components of the macroautophagic machinery.

Together, this study provides mechanistic insight into lipophagy and reveals a dual role for autophagy in regulating lipid synthesis and turnover in plants.

Eukaryotic cells use a highly evolutionarily conserved mechanism named autophagy to deliver cytoplasmic constituents into lysosomes or vacuoles for degradation. This catabolic process enables the removal of obsolete or damaged macromolecules, defective organelles, and invading microorganisms and at the same time the recycling of cellular components into needed nutrients and is therefore essential for homeostasis, development, and survival Jaishy and Abel, ; Anding and Baehrecke, ; Dikic, Autophagy is functional at basal levels in virtually all cell types under favorable growth conditions but can also be massively induced by a wide array of developmental and environmental stimuli including nutrient starvation, senescence, pathogens, metabolic stress, and many other abiotic and biotic cues Levine and Kroemer, ; Jaishy and Abel, ; Anding and Baehrecke, ; Wang et al.

The functional importance of both inducible and basal autophagy is well illustrated in plants using autophagy-defective mutants of Arabidopsis Arabidopsis thaliana and other plants Liu et al.

These mutants display early senescence, shortened lifespan, reduced seed yield, defective reproductive growth, and altered phytohormone signaling under normal growth conditions and are also hypersensitive to nutrient deprivation, oxidative stress, pathogen infection, drought, and high salinity, although many mechanistic details underlying these phenotypes and the stress sensitivity remain largely unknown.

Two major types of autophagy-related pathways, namely, macroautophagy and microautophagy, have been described in yeast, plants, and mammals Yoshimoto, ; Noda and Inagaki, ; Yang and Bassham, ; Antonioli et al. Macroautophagy is the most extensively studied form of autophagy in diverse organisms and is characterized by the formation of a double-membrane structure named an autophagosome.

Initial steps in macroautophagy involve the assembly and expansion of an isolated membrane structure called the phagophore. This membrane structure eventually closes to enwrap a portion of cytoplasmic content as cargo.

Subsequently, the outer membrane of the autophagosome is fused with the vacuolar membrane to release the inner membrane along with cargo as an autophagic body into the vacuole for degradation and recycling. Macroautophagy is mediated by a group of proteins encoded by AUTOPHAGY-RELATED ATG genes that are highly conserved from yeast to mammals and plants.

Among them, ATG3, ATG4, ATG5, ATG7, ATG8, ATG10, ATG12, and ATG16 are components of two ubiquitin-like conjugation systems essential for the formation of autophagosomes, their fusion with the tonoplast, and vacuolar degradation Yoshimoto, ; Üstün et al.

These two systems facilitate the formation of ATG8-phosphatidylethanolamine PE conjugates, whose abundance is often used as an indicator of autophagic activity Suzuki and Ohsumi, ; Yoshimoto, In particular, because ATG8 proteins are membrane associated, residing in phagophores, autophagosomes, and autophagic bodies, green fluorescent protein GFP —tagged ATG or autophagic cargo proteins are often used to monitor the delivery of autophagosomes to or the breakdown of autophagic cargos in the vacuole or lysosome Yoshimoto, ; Klionsky et al.

In contrast to macroautophagy, our understanding of microautophagy in terms of the underlying mechanism, the molecular machinery involved, and its functional role is still fragmentary, even in simple model systems such as yeast. At the ultrastructural level, microautophagy involves direct engulfment of cytoplasmic components via tonoplast invagination and subsequent release of the cargo into the vacuolar lumen for degradation Noda and Inagaki, ; Dikic, ; Galluzzi et al.

In yeast, various cellular organelles including peroxisomes, mitochondria, the endoplasmic reticulum ER , and the nucleus were identified as targets of microautophagy Suzuki and Ohsumi, ; Reggiori and Klionsky, ; Noda and Inagaki, ; Galluzzi et al.

Several forms of yeast microautophagy have been shown to require at least some components of the core machinery of macroautophagy Suzuki and Ohsumi, ; Reggiori and Klionsky, ; van Zutphen et al. In plants, microautophagy has been shown to participate in the degradation of cytoplasmic anthocyanin aggregates Chanoca et al.

Recent studies indicate that autophagy is functionally connected to lipid metabolism and storage in diverse model systems Jaishy and Abel, ; Shatz et al.

Lipids in membranous organelles are used as alternative substrates for energy production via β-oxidation of fatty acids in mitochondria in mammals Shatz et al. Emerging evidence suggests that rather than directly being used for β-oxidation, fatty acids released from cellular membranes are first stored in the form of triacylglycerol TAG in lipid droplets LDs , and TAG in LDs is then hydrolyzed by a process named lipolysis to supply the cell with fatty acids for the generation of energy Cabodevilla et al.

In mammalian cells, autophagic digestion of membranous organelles is the major source of fatty acids for TAG synthesis and LD biogenesis under starvation conditions Rambold et al. In addition, autophagy plays an important role in the cellular mobilization and degradation of neutral lipids in LDs, in a process termed lipophagy Wang, ; Zechner et al.

Recent studies have demonstrated a functional link between lipolysis and lipophagy Martinez-Lopez et al. Mammalian lipophagy depends on the core macroautophagy machinery Jaishy and Abel, and is morphologically similar to macroautophagy; thus, it is referred to as macrolipophagy Singh et al.

Consequently, disruption of the core ATG genes increases LD accumulation in various organs Singh et al. Unlike the situation in mammals, lipophagy in yeast resembles microautophagy and therefore is referred to as microlipophagy van Zutphen et al.

It has been suggested that microlipophagy plays an important role in maintaining cell viability van Zutphen et al. Disruption of autophagy has been shown to affect lipid turnover in maize Zea mays ; McLoughlin et al. In plants, as in yeast and mammals, TAG is assembled in the ER and stored in LDs in the cytosol Chapman and Ohlrogge, In Arabidopsis, phospholipid:diacylglycerol acyltransferase1 PDAT1 is a key enzyme catalyzing the last step of TAG assembly Zhang et al.

TAG breakdown is catalyzed by cytosolic lipases including SUGAR-DEPENDENT1 SDP1 , a patatin domain lipase responsible for the initiation of TAG catabolism Eastmond, Disruption of SDP1 blocks TAG hydrolysis in germinating seeds Eastmond, and in vegetative tissues such as mature leaves and roots Kelly et al.

Under extended darkness, TAG levels in leaves of sugar dependent1 sdp1 mutants increased rapidly and then declined, suggesting an activation of unknown, alternative pathways for TAG hydrolysis under starvation conditions Fan et al.

Lipid metabolism in photosynthetic tissues such as leaves is geared toward the supply of building blocks for organellar membrane biogenesis and maintenance.

As a result, leaf tissues do not accumulate TAG to significant amounts, although they do possess a high capacity for its synthesis and metabolism Xu and Shanklin, In Arabidopsis, two parallel pathways, compartmentalized in either the ER or the chloroplast, contribute to membrane lipid biosynthesis Browse and Somerville, ; Ohlrogge and Browse, Disruption of either pathway causes drastic changes in lipid metabolism including an increase in fatty acid synthesis and turnover and an accumulation of TAG Fan et al.

In the trigalactosyldiacylglycerol1 tgd1 mutant, a defect in the ER pathway also results in a compensatory increase in the chloroplast pathway activity Xu et al.

Similarly, overexpressing PDAT1 draws lipids from the ER pathway to TAG synthesis, causing an increase in the biosynthesis of thylakoid lipids via the chloroplast pathway Fan et al.

On the other hand, the plastidic glycerolphosphate acyltransferase1 act1 mutant is defective in the initial step in the chloroplast pathway of membrane lipid synthesis Kunst et al. To understand the role of autophagy in lipid metabolism at the mechanistic level, we generated a series of double mutants defective in autophagy in the tgd1 -, sdp1- , or PDAT1 -overexpressing-line background.

Using these mutants along with transgenic plants coexpressing an LD-targeted, GFP-tagged OLEOSIN1 OLE1 fusion protein Fan et al. We show that lipophagy occurs in a process morphologically resembling microautophagy in yeast and requires key core players in macroautophagy. This study demonstrates the functional importance of autophagy in TAG metabolism and storage and the mechanistic basis for lipophagy in plants.

To test the role of autophagy in lipid metabolism in plants, we first compared TAG levels in mature seeds, young seedlings, and leaves of adult plants between the wild type and two atg mutants defective in ATG2 or ATG5, two core protein components of the macroautophagic machinery.

Disruption of autophagy caused small but significant decreases in TAG content in seeds Figure 1A and 4-d-old seedlings Figure 1B. Seed weight was slightly decreased in atg TAG levels were low in developing leaves but increased as leaves matured and aged.

In all tissues examined, there were no significant differences in TAG content between atg and atg , suggesting the decreased TAG levels in atg mutants are associated with defects in basal autophagy. A to C TAG levels in dry seeds A , 4-d-old seedlings B , and leaves of 5-week-old plants C.

Data are means of three replicates with sd. FW, fresh weight; WT, wild type. Mutants defective in the core components of autophagy often display pleiotropic phenotypes including early senescence and defects in nutrient remobilization.

Therefore, it is possible that the observed decrease in TAG content in seeds in atg mutants is due to a decrease in resource allocation to seeds rather than to a change in seed TAG metabolism.

Similarly, a decreased TAG storage in seeds may also affect TAG content in young seedlings. To test these possibilities, we performed radiotracer labeling experiments using two different labeled substrates, 14 C-acetate and 3 H 2 O, substrates that label nascent fatty acids with 14 C or 3 H during the initial or reduction steps of fatty acid synthesis, respectively Browse et al.

Under our growth conditions, the incorporation of the radiolabel from 14 C-acetate or tritiated water 3 H 2 O into fatty acids of developing embryos was linear for at least 1 h Supplemental Figure 1. The rate of incorporation of 14 C or 3 H into TAG calculated following 1 h of incubation was similar between the wild-type and atg embryos Supplemental Figure 2.

Likewise, there was no significant difference in the rate of radiolabeled TAG accumulation between the wild-type and atg seedlings. On the other hand, the rate of radiolabel incorporation into TAG was significantly reduced in mature and senescing leaves, with the largest effect being observed in mature leaves and the least in developing leaves Figure 2 , mirroring the differences in TAG content in leaves at different ages Figure 1.

Again, leaf TAG levels and rates of radiolabel incorporation into TAG were similar between two atg mutants. Disruption of Autophagy Reduces TAG Synthesis in Mature and Senescing, But Not in Growing Leaves. A and B Detached leaves of 5-week-old plants were incubated with 14 C-acetate A or 3 H 2 O B for 1 h, and total radioactivity in TAG was measured by scintillation counting following separation by thin layer chromatography.

The decreased rate of radiolabel incorporation into TAG in atg leaves may be due to a decrease in fatty acid synthesis or a decline in the mobilization of fatty acids from organellar membranes to TAG via autophagy.

The rate of fatty acid synthesis can be assessed by measuring the rate of 14 C-acetate or 3 H 2 O incorporation into total fatty acids Browse et al.

As shown in Supplemental Figure 3 , growing leaves incorporated 14 C from 14 C-acetate or 3 H from 3 H 2 O into total lipids at a higher rate than did mature and senescing leaves, likely reflecting a higher demand for fatty acids to support membrane expansion and organellar biogenesis during rapid growth.

Rates of radiolabel incorporation following 1 h of incubation were similar in the wild-type and atg leaves. These results suggest that the decreased TAG synthesis in atg mutants is not due to a decline in the rate of fatty acid synthesis.

We next tested whether disruption of autophagy affects membrane lipid turnover. To this end, we first incubated leaves with 14 C-acetate for 1 h pulse.

After thoroughly washing with water to remove 14 C-acetate, the leaves were incubated in unlabeled solution for an additional 3 d chase. The radiolabel in leaf total membrane lipids following 1 h of pulse was similar between the wild type and atg mutants Supplemental Figure 4.

Quantification of radioactivity in total membrane lipids showed significant decreases in rates of radiolabeled fatty acid loss, particularly in mature and senescing leaves of atg mutants compared with the wild-type leaves of the same age during 3 d of chase Figure 3.

Together, results from pulse-chase labeling experiments suggest that disruption of autophagy results in a decrease in membrane lipid turnover and hence the accumulation of leaf TAG. Disruption of Autophagy Slows Down Membrane Lipid Turnover in Mature and Senescing, but Not in Growing Leaves.

Radiolabel loss was calculated as percentage of loss of radioactivity in total membrane lipids during 3 d of chase following 1 h of 14 C-acetate pulse of detached leaves of 5-week-old plants. WT, wild type. To provide additional evidence for the involvement of autophagy in TAG synthesis and also to test the relative contribution of the chloroplast versus the ER lipid assembly pathway to autophagy-mediated TAG synthesis, we constructed double mutants between tgd1 and atg or atg Assays for PDAT activity in microsomal membranes revealed that disruption of autophagy had no significant effect on TAG formation from 14 C-labeled phosphatidylcholine PC , whereas the activity was more than fourfold higher in transgenic plants overexpressing PDAT1 compared with the wild type Supplemental Figure 5.

Analysis of lipid extracts from mature leaves of 5-week-old plants showed that TAG content was higher in tgd1 , as expected Figure 4. Interestingly, there was also a significant increase in TAG in act1 compared with the wild type. Disruption of autophagy caused significant decreases in TAG content in atg tgd1 and atg tgd1.

TAG levels were 1. TAG content in mature leaves of 4-week-old PDAT1-overexpressing line 4 PDAT1-OE4 in the wild-type, act1 , atg , atg , atg act1 , or atg act1 background.

FW, fresh weight. Together, these results suggest that basal autophagy plays an important role in regulating both fatty acid synthesis and membrane lipid turnover and that the ER lipid biosynthesis pathway contributes more to autophagy-mediated leaf TAG synthesis than the chloroplast pathway. Disruption of Autophagy Reduces Fatty Acid Synthesis and Membrane Lipid Turnover in Growing Leaves of the tgd1 Mutant and PDAT1 -Overexpressing Lines.

A Rate of 14 C-acetate incorporation into total fatty acids in growing leaves of the 4-week-old PDAT1 -overexpressing line 4 PDAT1 - OE4 in the wild-type, act1 , atg , atg , atg act1 , or atg act1 background. B Radiolabel loss during the 3-d chase following 1 h incubation with 14 C-acetate. Our data so far indicate that autophagy contributes to TAG synthesis and membrane lipid turnover, but it is not clear whether this mechanism is also involved in the breakdown of TAG stored in LDs.

As a first step toward answering this important question, we took advantage of OLE1-GFP -overexpressing lines Fan et al. OLE1 is one of the most abundant LD proteins in seeds Huang, When ectopically expressed in leaves, OLE1-GFP is specifically targeted to the surface of LDs Wahlroos et al.

When exposed to extended darkness, a starvation condition known to induce autophagy Breeze et al. In addition, while the OLE1-GFP signals rarely overlapped with DsRed-ATG8e—labeled structures under normal growth conditions, some of the OLE1-GFP signals colocalized with DsRed-ATG8e after 3 d of dark treatment Figure 6.

The extent of colocalization was quantified using the Costes image randomization test Costes et al. The average PCC for OLE1-GFP colocalization with DsRed-ATG8e was 0. The relatively low PCC most likely reflects the large difference in size between the DsRed-labeled structures less than nm in diameter, Figure 7A and the OLE1-GFP—labeled LD clusters 5 to 10 µm in diameter, Figure 7.

Colocalization of LDs With Autophagic Structures in Leaves Under Dark-Induced Starvation. Confocal images of mature leaves of 4-week-old transgenic plants coexpressing OLE1-GFP green and DsRed-ATG8e red in tgd1 before and after 3 d of dark treatment.

Boxed areas show colocalization of green and red signals under higher magnification. Quantification of colocalization is provided by the PCC and the Costes P-value below the images. A Confocal images of mature leaves of 4-week-old transgenic plants coexpressing OLE1-GFP and DsRed-ATG8e in tgd1 after 3 d of dark treatment.

B to D Electron micrographs of LD clusters in leaf cells of tgd1 overexpressing OLE1-GFP before B and after see [C] and [D] 3 d of dark treatment.

D Enlargement of the boxed area in C. Arrows indicate LDs. Under higher magnification, DsRed-ATG8e—labeled autophagic structures were clearly found to be associated with LDs Figure 7A.

After dark treatment for 3 d, autophagic vacuoles AVs appeared in LD clusters, some of which contained LDs Figure 7C , which appeared to be partially degraded Figure 7D. Free GFP is relatively resistant to degradation within the vacuole or lysosome Yoshimoto, ; Klionsky et al.

Therefore, if OLE1-GFP—coated LDs are degraded in the vacuole, we would expect to observe an increased accumulation of free GFP under dark treatment. Autophagic activity can also be assessed by monitoring the protein level of ATG8-Phosphatidylethanolamine PE; Suzuki and Ohsumi, ; Yoshimoto, , which migrates faster on SDS-PAGE in the presence of urea than does the unmodified form Chung et al.

Immunoblot analysis using antibody against ATG8 showed that ATG8-PE conjugates were absent in leaves prior to dark treatment but accumulated after 3 d of darkness Figure 8A , indicating an overall increase in autophagic activity during dark-induced starvation conditions, as expected.

Time-course analysis showed that free GFP levels were low under normal growth conditions but increased steadily during 5 d of darkness, similar to dark-induced accumulation of ATG8-PE Figure 8B. ATG5 has been shown to be essential for ATG8 lipidation Chung et al.

Consistent with this, no ATG8-PE conjugates were detected in atg leaves following 3 d of dark treatment Figure 8A. Together, these results provide evidence that lipophagy is induced during dark-induced starvation. OLE1-GFP—Coated Leaf LDs Are Degraded in Vacuoles Under Dark-Induced Starvation.

A Accumulation of free GFP and ATG8-PE in mature leaves of the 4-week-old wild-type and tgd1 plants, but not in mature leaves of atg tgd1 double mutant overexpressing OLE1 - GFP following 3 d of darkness. B Time course of free GFP and ATG8-PE accumulation in mature leaves of tgd1 overexpressing OLE1 - GFP under dark treatment.

Equal amounts of proteins were subjected to SDS-PAGE followed by immunoblot analysis with antibodies against GFP, ATG8, or the loading control actin. The dashed lines and asterisks locate free ATG8 proteins and ATG8-PE conjugates, respectively. When these plants were exposed to dark treatment for 2 d, individual LDs or LD clusters were observed inside Figures 9A to 9D or within invagination of Figures 9E and 9F ΔTIP-DsRed—labeled tonoplasts.

Analysis of max-intensity projection images of z-stacks acquired by confocal microscopy revealed that the LDs were clearly enclosed by the tonoplast Figures 9G and 9H. A to F Confocal images of cotyledon cells of the 7-d-old wild-type transgenic plants coexpressing the tonoplast marker ΔTIP-DsRed red and OLE1-GFP green after 2 d of darkness in the presence of 0.

Overlay of red and green fluorescence showing the presence of LDs in vacuoles see [A] to [D] or within tonoplast invagination see [E] and [F]. G and H Three-dimensional images reconstructed from a series of confocal z-stack images.

Therefore, to further examine the process leading to lipophagy in plants, we took advantage of sdp1 mutants, which accumulated small LDs under dark-induced starvation conditions Fan et al. Because lipophagy and autophagy appeared to be induced after, but not within, the first 1 d of darkness Figure 8B , we focused on the subcellular morphological changes in leaf samples between 1 and 2 d of dark treatment.

Consistent with changes in ATG8-PE abundance Figure 8B , very few autophagic structures were seen after 1 d of darkness Supplemental Figure 8A. Following 2 d of dark treatment, however, we observed the occurrence of autophagosomes Supplemental Figures 8B and 8C and many small vacuoles with diameters of 0.

Many of these structures contained autophagic bodies or remnants of cytoplasmic materials, suggesting that they are AVs. LDs increased in size after 2 d of dark treatment Figures 10B to 10F and were frequently found to be in close contact with AVs Figure 10C or within invagination of AV membranes Figures 10B and 10C or inside AVs Figure 10D or the central vacuole Figure 10E.

Immunoelectron microscopy of dark-treated sdp1 plants with ATG8 antibody revealed the presence of immunogold particles on LDs Figure 10F. Interestingly, LDs appeared to undergo degradation prior to being fully internalized into AVs, along with other sequestered materials Figure 10C.

Dark treatment in the presence of concanamycin A concA also led to the appearance of LDs in the central vacuole in leaves of wild-type seedlings Supplemental Figure 9.

On the other hand, we did not detect association of macroautophagic membrane structures with LDs as observed during macrolipophagy in mammals Singh et al. Importantly, disruption of ATG2 Figures 11A and 11B or ATG5 Figures 11C and 11D in sdp1 largely blocked the formation of AVs and hence the interaction between LDs and AVs.

In atg sdp1 double mutants, most of LDs were still present in the cytosol after 2 d of dark treatment. A to F Electron micrographs of leaf cells of 4-week-old sdp plants dark treated for 1 d A and 2 d see [B] to [F].

B to D Various stages of LD internalization into AVs see [B] and [D]. Note that LD is partially degraded within invagination of the AV membrane in C. E Presence of LDs in the central vacuole.

CV, central vacuole. F Immunogold labeling of sdp seedlings dark treated for 2 d in the presence of 0. Arrowheads indicate gold particles. The inset shows higher magnification of the boxed region. A to D Electron micrographs of leaf cells of 4-week-old atg sdp see [A] and [B] and atg sdp see [C] and [D] plants dark treated for 2 d.

We next tested whether deficiency in cytosolic lipolysis affects autophagy under dark-induced starvation in plants as in mammals Sathyanarayan et al. To do so, we crossed the wild type or sdp1 with tgd1 overexpressing DsRed-ATG8e and recovered the DsRed-ATG8e line in the wild-type or sdp1 background.

As expected, the number of DsRed-ATG8e—labeled puncta increased under dark-induced starvation Supplemental Figure 10A. Quantitative analysis showed that there was no significant difference in the number of puncta between the wild type and sdp1 after 4 d of darkness.

In addition, there was an increase in levels of faster migrating forms of ATG8-PE during dark-induced starvation conditions Supplemental Figure 10B ; and again, there were no discernible differences in levels of starvation-induced ATG8-PE between the wild type and sdp1 mutants.

Together, these data suggest that disruption of SDP1 does not affect autophagic flux under dark-induced starvation conditions in Arabidopsis. Under dark-induced starvation conditions, TAG accumulated rapidly within the initial 1 d and then started to decline in leaves of sdp1 plants, likely reflecting the induction of lipophagy after dark treatment for 1 d Fan et al.

To test this possibility, we treated detached leaves of sdp1 mutants with 3-methyladenine 3-MA , a widely used inhibitor of autophagy in mammals Blommaart et al. In untreated control leaves, TAG content increased by more than sixfold during the initial 2 d of dark treatment Figure 12A.

Treatment with 3-MA did not affect TAG levels during the initial 2 d of dark incubation, suggesting an involvement of an autophagy-independent mechanism in TAG synthesis. However, TAG content declined after day 2 of dark treatment in the untreated control but continued to increase toward the end of the experiment in 3-MA—treated leaves, such that TAG content was significantly higher at days 3 and 4 in 3-MA—treated leaves compared with the untreated control.

These results suggest that autophagy contributes to TAG hydrolysis under severe starvation. Inhibition of Autophagy Enhances TAG Accumulation in sdp under Extended Darkness. A Changes in TAG levels in detached sdp mature leaves during dark treatment in the presence or absence of 3-MA.

B Changes in TAG levels in mature leaves of 4-week-old sdp , atg sdp , and atg sdp plants during dark treatment. To provide genetic evidence for the induction of lipophagy during dark-induced starvation and also to test whether lipophagy depends on the core autophagic machinery, we generated double mutants between sdp1 and atg or atg Under normal growth conditions, TAG levels were lower in leaves of atg sdp1 double mutants compared with sdp1 Figure 12B.

During dark treatment, TAG levels in sdp1 peaked at day 1 following dark exposure and started to decline thereafter Figure 12B , consistent with our previous report Fan et al. In contrast to sdp1 , TAG content in atg sdp1 double mutants increased steadily during the first 2 d of dark treatment and remained largely unchanged at day 3.

Statistical analysis confirmed that atg sdp1 double mutants accumulated significantly more TAG at days 2 and 3 following dark treatment compared with the sdp1 single mutant Figure 12B.

TAG levels remained largely unchanged in the wild type and atg single mutants following dark incubation for 3 d Supplemental Figure The increased TAG accumulation in atg sdp1 double mutants could result from a decrease in TAG hydrolysis or an increase in the conversion of membrane lipids to TAG.

To test these possibilities, we analyzed the changes in levels of total membrane lipids during dark treatment. We detected no significant differences in leaf membrane lipid content among wild type, single, and double mutants prior to or during 3 d of darkness Supplemental Figure Since fatty acid synthesis is completely inactive in the dark Bao et al.

Total membrane lipid levels were decreased to a similar extent following 3 d of dark treatment in all genotypes analyzed Supplemental Figure 12 , apparently because of an increase in fatty acid β-oxidation Fan et al. Together, these results suggest that the increased TAG accumulation in atg sdp1 double mutants compared with sdp1 is due to decreased lipophagic activity and that lipophagy relies on the core machinery such as ATG2 and ATG5.

We have shown that autophagy plays an important role in organellar membrane turnover, TAG synthesis, and LD accumulation under normal growth conditions. Lipophagy, the autophagic degradation of LDs, was induced following extended dark treatment as evident from increased colocalization of LDs and autophagic structures, an increase in accumulation of free GFP derived from OLE1-GFP—coated LDs, the presence of LDs in vacuoles, the association of autophagic marker protein ATG8 with LDs, and an increase in TAG levels in atg sdp1 double mutants compared with sdp1.

We show that lipophagy occurs in a process resembling microlipophagy as described in yeast and requires the core components of macroautophagy. These results provide mechanistic insight into the role of autophagy in lipid metabolism in plants and lend further support for a critical role of autophagy in quality control of cellular organelles Yang and Bassham, ; Wang et al.

Our results show that disruption of autophagy impedes membrane lipid turnover and hence TAG synthesis under normal growth conditions. These results are perhaps not surprising because, in contrast to the situation in mature and senescing leaves, organellar membranes in growing cells are newly formed and therefore may not be targeted for autophagy-mediated degradation under normal growth conditions.

In developing embryos, fatty acids in membrane lipids are known to be directed to TAG synthesis via acyl editing and headgroup exchange Bates et al. In plants, autophagy has been implicated in the degradation of peroxisomes Kim et al. The contribution of autophagy to TAG synthesis is higher in act1 defective in the chloroplast pathway of glycerolipid biosynthesis but lower in tgd1 disrupted in the parallel ER pathway.

The importance of ER in autophagy-mediated TAG synthesis may reflect not only the role of autophagy in the degradation of this organelle Liu et al. De novo fatty acid FA synthesis in chloroplasts is mediated by a series of enzymatic reactions collectively referred to as fatty acid synthase.

The resultant FAs feed into membrane lipid synthesis via two parallel pathways localized in the chloroplast or the ER. Autophagy-mediated degradation of cellular organelles other than chloroplasts provides a source of FAs for TAG synthesis under normal and starvation conditions.

Thylakoid lipids are broken down by hydrolytic enzymes inside the chloroplast, and the released FAs are used for TAG synthesis. TAG is packaged in LDs in the cytosol. Under normal growth conditions, TAG stored in LDs is hydrolyzed by SDP1. Nutrient starvation triggers microlipophagy, which functions together with cytosolic lipolysis catalyzed by SDP1 to mediate LD breakdown into FAs for energy production through β-oxidation.

Black arrows represent processes occurring in both normal and starvation conditions. The red arrow is specific to starvation. FAS, fatty acid synthase; HEs, hydrolytic enzymes. Previous studies have shown that during autophagy-mediated chloroplast breakdown, stromal proteins Ishida et al.

In line with these observations, our results showed that disruption of autophagy had no significant impact on the dark-induced synthesis of TAG Figure 12B , which is mainly derived from thylakoid lipids Kunz et al.

Similarly, treatment with 3-MA did not affect TAG content during the initial 2 d of dark treatment Figure 12A , suggesting that autophagy-independent breakdown of chloroplasts serves as a main source of fatty acids for TAG synthesis. In addition, our microscopy analysis showed that the number of chloroplasts per cell remained unaltered during dark treatment Supplemental Figure 13 , consistent with previous reports Keech et al.

These results exclude the possibility of whole chloroplast autophagy as observed in plants under photooxidative stress Izumi et al. The autophagy-independent degradation of thylakoids is also consistent with previous reports showing an internal dismantling of thylakoid systems during senescence-induced chloroplast breakdown Evans et al.

In addition to reduced organellar membrane turnover and TAG synthesis, disruption of basal autophagy results in significant decreases in fatty acid synthesis in tgd1 or PDAT1-OE lines Figure 5A. Although the exact mechanistic basis as to how autophagy impacts fatty acid synthesis remains unclear, it is possible that blocking autophagy results in a buildup of fatty acids in the cytosol due to reduced cellular fatty acid needs for organellar membrane lipid turnover, which act as feedback signals to negatively regulate fatty acid synthesis in the chloroplast.

On the other hand, overexpression of PDAT1 or blocking the chloroplast lipid biosynthesis pathway in act1 accelerates autophagy-mediated membrane lipid turnover and hence increases the cellular demand for fatty acids.

This increased fatty acid demand may cause a decrease in fatty acids in the cytosol, thereby partially relieving feedback inhibition on plastid fatty acid synthesis. In this context, it is worth noting that inefficient utilization of fatty acids for glycerolipid biosynthesis in the ER has been shown to cause a feedback inhibition on fatty acid synthesis by an unknown mechanism Bates et al.

TAG and fatty acid synthesis are increased in tgd1 mutants Fan et al. These results suggest that under normal growth conditions, autophagy functions in TAG synthesis, whereas the cytosolic pathway mediated by neutral lipases including SDP1 is the major mechanism for TAG catabolism Figure Under extended darkness, TAG content decreases when autophagy is induced but increases when autophagy is disabled in sdp1.

In addition, disruption of SDP1 does not impact autophagic flux under either normal growth or starvation conditions Supplemental Figure These results suggest an important and general role of lipophagy in mediating TAG hydrolysis under starvation conditions Figure TAG did not accumulate in atg mutants under extended darkness Supplemental Figure This result suggests that the SDP1-mediated cytosolic lipolytic pathway can functionally compensate for the lack of lipophagy in TAG hydrolysis under starvation.

Previous studies showed that plant autophagic organelles contain hydrolytic enzymes, including proteases and lipases, for cargo degradation at the onset of their formation Marty, , ; Buvat and Robert, and are functionally sufficient to break down the sequestered materials on their own Rose et al.

In accordance with the autophagosome-autonomous hydrolysis, our ultrastructural analysis showed that LDs and other cellular constituents were degraded in AVs Figures 7D and 10C , in addition to the central vacuole Figure 10E. These results point to the unique aspects of plant autophagy in comparison with this catabolic process in yeast and mammals, where the autophagosome itself lacks degradative enzymes and its cargo is broken down following fusion with lytic compartments such as vacuoles and lysosomes, respectively Eskelinen, ; Suzuki and Ohsumi, ; Reggiori and Klionsky, ; Dikic, ; Galluzzi et al.

Our ultrastructural analysis showed that the autophagic degradation of LDs in Arabidopsis occurs in a process resembling microlipophagy in yeast. Disruption of autophagy genes increased TAG content in sdp1 under starvation conditions Figure These results suggest that microlipophagy in Arabidopsis depends on the core machinery of macroautophagy, similar to the situation in yeast van Zutphen et al.

At present, the exact mechanism underlying microautophagy and the role of ATG gene products in microlipophagy remain largely unknown Noda and Inagaki, ; Galluzzi et al. Our results showed that microautophagy-like LD degradation occurs in AVs, key autophagic structures in macroautophagy Eskelinen, Therefore, it is possible that the observed dependence of starvation-induced TAG and LD accumulation on the macroautophagic machinery in Arabidopsis may simply reflect the essential role of core ATG proteins in the formation of autophagosomes and hence AVs.

In support of this possibility, disruption of the core ATG genes blocks the formation of both AVs and microlipophagy Figure Recently, vacuolar membrane lipid rafts enriched in sterols have been shown to be necessary for microlipophagy in yeast Oku and Sakai, Further studies are needed to test whether the sterol-enriched membrane rafts are involved in microlipophagy in plants, to determine how TAG is hydrolyzed in vacuoles, and to establish the regulation and physiological functions of lipophagy.

The Arabidopsis Arabidopsis thaliana plants used in this study were of the Columbia ecotype. The tgd1 mutant was previously described by Xu et al. The PDAT1 -overexpressing lines 3 and 4 were described in Fan et al.

The primers used for genotyping sdp1 were as described previously Fan et al. Genotyping of tgd1 and act1 mutants was as described previously Xu et al. For plant growth in soil, surface-sterilized seeds of Arabidopsis were germinated on 0.

For starvation treatment, whole plants, unless stated otherwise in Figure 12A , were transferred to continuous darkness at 24°C for the time indicated. The PCR products were cloned into a binary vector pPZP Fan et al.

After confirming the integrity of the construct by sequencing, plant stable transformation was performed according to Clough and Bent Lipids were extracted from leaves of 4-week-old plants grown in soil as described by Fan et al. To quantitate low TAG levels in leaves of wild type and atg mutants, total lipid extracts were first fractionated through silica columns Discovery DSC-Si SPE tube, volume 6 mL, Supelco as described by James et al.

Fatty acid methyl esters were prepared as described by Li-Beisson et al. Separation and identification of the fatty acid methyl esters were performed on an HP gas chromatograph-mass spectrometer Hewlett-Packard fitted with a 30 m × μm DB capillary column Agilent with helium as the carrier gas as described by Fan et al.

Fatty acid methyl esters were quantified using heptadecanoic acid as an internal standard as described by Fan et al.

Equal fresh weight of mature leaves of 4-week-old plants grown in soil was ground in liquid nitrogen, homogenized with 2× Laemmli sample buffer. The extracts were incubated for 5 min in boiling water and clarified by centrifugation at 12, g for 5 min at 22°C. Immunoblot analyses were performed according to the ECL Western Blotting procedure 32,, Thermo Fisher Scientific with antibodies against GFP catalog no.

E11LF, BioLegend , ATG8a catalog no. AS, lot no. MBS, lot no. M14L06, MyBioSource. Targeted proteins were visualized using an ImageQuant LAS biomolecular imager GE Healthcare Life Sciences. In vivo labeling experiments with 14 C-acetate or 3 H 2 O were done as described previously by Fan et al.

Developing seeds of 50 siliques were directly harvested into labeling medium containing 20 mM MES, pH 5. The assay was started by the addition of 0.

After incubation for 1 h, tissues were washed two times with water and immediately used for lipid extraction. For pulse-chase labeling experiments, leaves were labeled for 1 h with 14 C-acetate. Total lipids were extracted and separated as described previously by Fan et al.

Radiolabel loss was calculated by correcting for the dilution of radioactivity caused by tissue growth during the chase period. Microsomal membranes were isolated from 3-week-old seedlings as described previously Xu et al.

Radioactive PC for PDAT activity assays was prepared after incubating 2-week-old seedlings overnight in 20 mM MES-KOH, pH 6. Lipids were extracted and separated by TLC as described by Fan et al. Radiolabeled PC was eluted from silica gel using chloroform:methanol:formic acid The reaction mixture contained 0.

The reaction solution was thoroughly mixed and incubated at room temperature for 30 min. Lipid extraction and TLC separation were done as described previously by Fan et al. Radioactivity in TAG was determined by scintillation counting. Detached leaves of 4-week-old plants grown in soil were floated on water with or without the addition of 5 mM 3-MA dissolved in water, Sigma-Aldrich and 0.

Samples were taken every 24 h over 4 d for lipid analysis as described previously Fan et al. For the colocalization study, leaf samples were mounted in water on slides and were directly examined using a Leica TCS SP5 laser scanning confocal microscope with sequential scanning.

GFP was excited with a wavelength of nm and detected at to nm. DsRed was excited at nm and detected at to nm. For tonoplast imaging, transgenic plants coexpressing OLE1-GFP and ΔTIP-DsRed were germinated on 0.

Six-day-old seedlings were dark treated for 1 d and then transferred to half-strength MS medium with or without 0. The hypocotyls or cotyledons were observed under confocal microscopy.

For transmission electron microscopy, leaf tissues were fixed with 2. For chloroplast counting, leaf tissues were fixed and embedded.

The number of chloroplasts was counted from at least 60 mesophyll cell cross sections for each time point of dark treatment. Colocalization analysis of OLE1-GFP and ATG8e-DsRed signals was done with the Coloc 2 plugin for ImageJ. Background subtraction from image pairs was performed using rolling ball subtraction with a pixel ball size.

Statistical significance of the PCC of the image pairs was analyzed using the Costes image randomization test as described previously Costes et al. Regions of interest were selected for colocalization analysis with Costes randomizations using a point spread function of 3.

Five-day-old seedlings grown on 0. The seedlings were then transferred to half-strength MS medium containing 0. The fixed hypocotyls were washed twice with 0. After dehydration, the tissues were embedded in LR White resin CA, Electron Microscopy Sciences, London Resin Company in gelatin capsules.

Resin polymerization was performed at 50 to 55°C. Ultrathin sections 70 to 90 nm of LR White—embedded hypocotyls were collected with formvar-coated mesh nickel grids. The grids were first washed with 1× PBS containing 0. After blocking, the grids were incubated with the primary antibody:rabbit polyclonal anti-ATG8a catalog no.

After rinsing with blocking solution five times, 1 min each, the grids were then incubated in the secondary antibody of goat anti-rabbit immunoglobulin G conjugated with nm gold particles catalog no. G, lot no. SLBW, Sigma-Aldrich; dilution in blocking solution for 1 h at room temperature.

Following washing with 1× PBS and 0. Supplemental Figure 1. Time course of the incorporation of radiolabel from 14 C-acetate or 3 H 2 O into total fatty acids in wild-type developing embryos. Supplemental Figure 2. Rate of the incorporation of radiolabel from 14 C-acetate or 3 H 2 O into TAG in developing embryos and seedlings.

Supplemental Figure 3. Rate of the incorporation of radiolabel from 14 C-acetate or 3 H 2 O into total fatty acids in leaves. Supplemental Figure 4.

Rate of the incorporation of radiolabel from 14 C-acetate into total membrane lipids in leaves. Supplemental Figure 5. PDAT activity in microsomal membranes isolated from seedlings.

Supplemental Figure 6. Disruption of autophagy reduces TAG content in mature leaves of 4-week-old PDAT1 -overexpressing transgenic plants. Supplemental Figure 7. Increased accumulation of DsRed-ATG8e—labeled structures in leaves of tgd1 plants under dark treatment. Supplemental Figure 8. Accumulation of autophagosomes and autophagic vacuoles in mature leaves of 4-week-old sdp plants under dark treatment.

Supplemental Figure 9. The appearance of LDs in the central vacuole in wild-type seedlings after dark treatment in the presence of concA. Supplemental Figure Autophagic activity in 4-week-old sdp plants under dark-induced starvation. TAG levels in mature leaves of 4-week-old wild type, atg and atg plants under dark-induced starvation.

Membrane lipid levels in mature leaves of 4-week-old sdp , atg sdp , and atg sdp plants under dark-induced starvation. Chloroplast number in mature leaves of sdp plants under dark-induced starvation. Supplemental Data Set.

Results of statistical analyses. The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper: SDP1 Gramene: At5g SDP1 Araport: At5g ATG10 Gramene: AT3G ATG10 Araport: AT3G ATG3 Gramene: AT5G ATG3 Araport: AT5G LAS Gramene: AT1G LAS Araport: AT1G ACT1 Gramene: AT2G ACT1 Araport: AT2G TGD1 Gramene: AT1G TGD1 Araport: AT1G ATG5 Gramene: AT5G ATG5 Araport: AT5G PDAT1 Gramene: at5g PDAT1 Araport: at5g ATG2 Gramene: AT3G ATG2 Araport: AT3G

Autophagy is Autophagy and lipid metabolism self-eating process of using metabolisk to degrade macromolecular substances e. Lipid Greek yogurt toppings is the Recovery solutions and Autophayy of metabolizm e. There lipis a complex interplay between lipid metabolism e. In particular, lipid metabolism is involved in the formation of autophagic membrane structures e. Moreover, autophagy, especially selective autophagy e. A better understanding of the mechanisms of autophagy and possible links to lipid metabolism will undoubtedly promote potential treatments for a variety of diseases.

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1 thoughts on “Autophagy and lipid metabolism

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