Macroautophagy

Macroautophagy, the better characterized autophagic pathway, starts with the formation of the limiting membrane of the autophagosome, a double membrane vesicle that delivers the cytosolic material targeted for degradation (cargo) to lysosomes. This limiting membrane, or nascent phagophore, forms through the assembly of proteins and lipids from different cellular organelles such as the endoplasmic reticulum (ER), Golgi, mitochondria, endocytic system or plasma membrane (Fig. 1). Cargo sequestration by autophagy in response to starvation occurs, for the most part, in bulk. However, a better characterization of autophagy activated in response to other stimuli has led to the discovery of selective forms of macroautophagy based on the cargo recruited for degradation¹⁸. In these instances, formation of the pre-autophagosome structure occurs in close proximity to the cargo thanks to autophagy receptors, such as p62/SQSTM1, NBR1, NDP52 and Optineurin¹⁸. These receptors bind simultaneously cargo and key components of the autophagy machinery known as autophagy–related proteins or ATGs¹⁸. The elongation of this membrane around the cargo occurs through the coordinated action of a cascade of ATGs that conjugate among themselves and with lipids¹⁹. Upon sealing of the autophagosome membrane, this cargo-containing vesicle moves along microtubules to fuse with the lysosome and deliver the sequestered cargo in this hydrolytic organelle¹⁶ (Fig. 1). After hydrolysis, the resultant molecules, amino acids, lipids and carbohydrate moieties reach the cytosol through transporters and permeases for recycling¹⁶.

How cells sense the need for a boost in autophagy activity is yet to be well defined and may likely occur through multiple mechanisms depending on the type of stimulus. Recent studies have shed some light on one of such mechanisms by showing that nutrient deprivation can be sensed by signaling cascades present on the primary cilia, such as the Sonic Hedgehog pathway²⁰. Signaling by this pathway activates autophagy by recruiting ATGs to the base of the cilia using ciliary trafficking proteins. The unique position of this single ciliary structure that protrudes from the cellular surface and the highly specialized nature of the membrane that surrounds the cilia, supports the idea that primary cilia may also contribute to modulate autophagy by transducing other signals such as those initiated by growth factors, hormone ligands or even physical stimuli that lead to ciliary bending.

This association of ATGs with the plasma membrane may also assist the activation of autophagy in response to changes in the neighboring cells. Thus, for example, under rich nutritional conditions, connexins, the main structural component of intercellular gap junctions, serve as endogenous repressors of autophagy by directly binding to ATG complexes involved in autophagosome formation. During nutrient scarcity, these connexins become platforms for recruitment of additional ATGs (i.e. ATG14 and ATG9), driving the internalization of connexin-ATGs from the plasma membrane to the recycling endocytic compartment for autophagosome formation²¹.

Once the autophagy-activating signal is transduced, ATGs control each of the steps of the autophagy pathway, from autophagosome formation to cargo degradation and recycling. Consequently, in addition to their reorganization from inactive to active complexes, changes in ATG expression levels in response to different transcriptional programs modulate the magnitude and duration of autophagy activation. One of the master transcriptional regulators of the autophagic program is the transcription factor EB, or TFEB²², that enhances autophagy flux at multiple levels. First, TFEB transcriptionally manages expression of ATGs genes during sustained autophagy to prevent ATG protein depletion. Second, TFEB increases lysosomal biogenesis to avoid overwhelming of this degradative organelle by the arriving autophagosomes. Lastly, TFEB, also controls genes required in other autophagy steps such as molecular motors for trafficking, SNARES for membrane fusion and specific hydrolases, to accommodate the lysosomal enzymatic load to the type of arriving cargo^23,24. However, TFEB and the recently described more potent members of this family such as TFE3²⁵ are not the only transcriptional regulators of autophagy. The transcriptional autophagy network is also controlled by the sensing nuclear receptor farnesoid X receptor (FXR) in the fed state, guaranteeing physiological proteostasis through basal recycling of proteins²⁶. During fasting, transcriptional regulation of the autophagy program is monitored by cAMP response element-binding protein (CREB) and peroxisome proliferator-activated receptor-alpha (PPARα)^{26, 27}.

Chaperone-mediated autophagy (CMA)

CMA degrades a specific subset of proteins that cross the lysosomal membrane through the CMA receptor, the lysosome-associated membrane protein type 2A (LAMP-2A)¹⁷. All CMA substrate proteins contain in their amino acid sequence a pentapeptide motif (KFERQ)²⁸ that is selectively recognized by the cytosolic heat shock cognate protein of 70kDa (hsc70) (Fig. 1). The substrate/chaperone complex docks at the lysosomal membrane through binding to monomeric LAMP-2A proteins that then organize into a multimeric complex required for translocation²⁹. After unfolding and internalization, the substrate protein is rapidly degraded in the lysosomal lumen¹⁷ (Fig. 1). LAMP-2A levels, limiting for CMA activity, are controlled both through transcriptional activation, but more frequently, through direct changes in the stability of LAMP-2A at the lysosomal membrane³⁰. CMA was originally described in liver as part of the hepatocyte response to nutritional changes³¹. Later studies have revealed that CMA also functions as a defense mechanism against cellular insults in an effort to remove damaged proteins and thereby ensuring appropriate hepatic proteostasis ^{reviewed in 32}.

The signaling mechanisms that regulate CMA activity are, for the most part, still unknown. Signaling through the nuclear retinoic acid receptor alpha negatively regulates CMA activity³³, while recent studies in T-cells have identified the NFAT-calcineurin axis behind CMA induction during T-cell activation³⁴. Metabolites such as ketone bodies activate CMA³⁵ but whether it is because of their pro-oxidizing effects on proteins or by acting as second messengers in signaling pathways requires further investigation. Free fatty acids (FFA) also activate CMA but, their effect is bi-phasic and, as their levels increase, they inhibit CMA through destabilization of LAMP-2A at the lysosomal membrane³⁶. Changes in the lipid composition of the lysosomal membrane with age are also behind the lower LAMP-2A levels and reduced CMA activity in aging³⁶. Whether LAMP-2A instability is secondary to age-related changes in lipid metabolism, or a primary defect behind some of the metabolic changes of aging remains unclear. In any case, this reciprocal interplay between lipid metabolism and CMA may constitute a vicious cycle that contributes to perpetuate the metabolic syndrome associated with aging. Interestingly, in support of the potential anti-aging effect of breaking this vicious circle by enhancing CMA activity, restoration of LAMP-2A levels in livers from old mice improves cellular homeostasis, enhances liver response to stress and preserves normal hepatic function until late in life³⁷.

Energy homeostasis through degradation and recycling by autophagy

Starvation is the best-characterized trigger for both macroautophagy and CMA activation. In the first 4–6 hours of nutrient shortage inactivation of mechanistic target of rapamycin (mTOR), one of the best-characterized endogenous inhibitors of autophagy, leads to macroautophagy activation⁴³. Amino acids resulting from lysosomal degradation of cytosolic and organelle proteins sustain protein synthesis or directly feed the Krebs cycle in order to produce ATP and/or glucose³ (Fig. 2). Interestingly, some of the released amino acids inhibit autophagy creating an auto-inhibitory feedback.

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Figure 2

Three main functions of hepatic autophagy in the control of the energetic balance

A: Autophagy recycles essential components through degradation of cellular proteins and energy stores. The type of cargo selected by autophagy, at least in liver, changes depending on the duration of nutrient scarcity. While in bulk autophagy of cytosolic proteins and organelles is predominant early in starvation and constitutes an important source of amino acids, if nutrients shortage persists, there is a switch toward glycogen and lipid droplets as preferential cargos. Glycophagy and lipohagy contribute thus glucose and free fatty acids that can be utilized to sustain a positive energetic balance in absence of nutrients. B: Autophagy also manages the cellular energetic balance through the fine-tuned regulation of mitocondrial number and quality control. Mitophagy can eliminate non-functional mitochondria but also controls mitochondrial mass through a coordinated balance with mitochondrial biogenesis. C: Autophagy contributes to accommodation to starvation and other nutritional challenges through selective removal via chaperone-mediated autophagy (CMA) of enzymes that control metabolic pathways such as glycolysis or lipogenesis. In addition, CMA also regulates hepatic lipolytic capacity through degradation of perilipins in the surface of lipid droplets. Removal of these proteins is necessary for cytosolic lipases and autophagy factors to gain access to the lipids in the core of the lipid droplets.

The events connecting mTOR inactivation and autophagy induction have been elucidated. mTOR represses autophagy through direct phosphorylation and sequestration of ULK1, a kinase essential for autophagosome formation. Upon amino acid depletion, inactivation of mTOR is followed by cytosolic release of ULK1 and its relocation to sites of autophagosome biogenesis⁴⁴. Once amino acids become available again, mTOR reactivation suppresses autophagy. Interestingly, the mechanism for autophagy inhibition seems to be amino acid-specific. For example, changes in leucine levels sensed by the leucyl-tRNA synthetase promote the relocation of the cytosolic mTOR complex to the lysosomal and endosomal membranes through binding to Rag-GTPase and Rheb⁴⁵. mTOR activation in this location results in inhibitory phosphorylation of TFEB, thus preventing its translocation into the nucleus, and ending the transcriptional autophagic program⁴⁶. The modulatory effect of other amino acids is less well characterized and apparently more complex. For example, both intracellular uptake of glutamine through SLC1A5 as well as its efflux activate mTOR; this is due to coupling of the exit of this amino acid through the bidirectional transporter SLC7A5/SLC3A2 to intracellular influx of leucine and essential amino acids. Blockage of these transporters results in glutamine depletion and autophagy activation⁴⁷.

The physiological relevance of autophagy activation as a source of amino acids is exemplified by the death of autophagy-deficient neonate mice during the period between the interruption at birth of the transplacental-nutrient supply and lactation. Administration of amino acids is sufficient to overcome the negative energetic balance of these autophagy-deficient neonates and guarantee their survival⁴.

In many cell types, if starvation persists beyond 8h, the contribution of macroautophagy to protein breakdown gradually decreases and it is replaced by activation of CMA, which peaks at 24h and can persist up to 3 days into starvation³¹. Studies in CMA-incompetent models have confirmed that absence of CMA results in a negative energetic balance during starvation¹³. However, as described in later sections, CMA does not only supplies amino acids under this conditions, but it is also mediates changes in the proteome that facilitate the metabolic switch required for cellular adaptation to persistent nutrient scarcity.

Beyond protein breakdown

Although protein breakdown has been by far the most studied autophagic catalytic activity, degradation of other cellular energy stores by autophagy has recently gained attention. Lysosomes contain a broad repertoire of hydrolases capable of degrading not only proteins but also carbohydrates, lipids and nucleic acids.

Hepatic glycogen stores become an important source of glucose, when no longer available through dietary intake. Glycogen breakdown during starvation is attained in large extent by the lysosomal enzyme acid alpha-glucosidase⁴⁸ after glycogen delivery to this organelle by selective forms of macro- and microglycophagy (Fig. 2). How the cell senses the decline in glucose levels and switches its machinery to selective glycophagy remains poorly understood. Nonetheless, improved methods to dynamically track the autophagic process in intact cells and the ability to chemically and genetically modulate autophagy has facilitated the identification of some glycophagy effectors. Starch Binding Domain-containing Protein 1/Genethonin 1 (STBD1) binds to glycogen and drives it to autophagosomes through direct interaction with one of the ATGs (γ-aminobutyric acid receptor-associated protein-like 1 or GABARAPL1)⁴⁹. Both cAMP and mTOR signaling pathways regulate neonatal hepatic glycophagy⁵⁰ and a link between growth hormone and hepatic glucose modulation has also been proposed⁵¹. Studies in skeletal muscle have highlighted the contribution of microautophagy to mobilization of glycogen stores though microglycophagy⁵, but the involvement of this pathway in hepatic glycogen turnover is still controversial.

Lysosomal breakdown of hepatic lipids has also gained considerable attention in recent years. In addition to the well-characterized arrival of extracellular lipids to lysosomes through endocytosis, intracellular triglycerides and cholesterol stored in the form of lipid droplets can also reach this compartment through a selective form of macroautophagy known as lipophagy⁶ (Fig. 2). The FFA resulting from lipid droplet breakdown are used as cellular fuel in case of prolonged nutrient scarcity. In fact, after 8h of starvation there is a gradual shift in the autophagosome cargo from cytosolic proteins and glycogen to lipid droplets⁶. Identification of lipophagy has promoted a change from a model in which cytosolic lipases were solely responsible for triglyceride catabolism towards a cooperative action of cytosolic and lysosomal lipases in this process. Still, the interconnection between both pathways and the conditions that favor one over the other are under debate.

Although the molecular determinants of autophagic selectivity for lipid droplets are not fully understood, recent studies have shown that RAB7 drives the lipid droplets to the perinuclear region where lipophagy is initiated through kiss-and-run processes⁵². However, access of the autophagic machinery, and by the same token, of the neutral cytosolic lipases, to the lipid core of lipid droplets requires the CMA-mediated removal of perilipins, the structural proteins that cover the surface of lipid droplets. This step is a prerequisite before lipid breakdown can occur⁵³, thus placing CMA upstream of the two main types of hepatic lipolysis, the one mediated by neutral cytosolic lipases and the other dependent on lipophagy. Degradation of lipid droplets through microautophagy has, to date, only been described in yeast⁴¹.

Ongoing investigations have shed light on some of the signaling mechanisms that modulate lipophagy. TFEB exerts an exquisite fine-tuning control on this process by adapting the lysosomal enzymatic load to the arrival of the lipid cargo, and by sustaining the activation of PGC1α and PPARα-dependent programs^23,24. Interestingly, lipophagy is activated in the two extremes of nutritional status since, besides starvation, arrival of high levels of dietary lipids to the liver also activates lipophagy to prevent hepatic lipotoxicity⁶. The well-established FFA sensor PPARα is behind the potent activation of autophagy under these conditions²⁷. Similarly, CREB activation increased lipophagy probably through a mechanism involving AMPK phosphorylation of ULK1^{26, 54}. Contrarily, the end-products of lipid metabolism, bile acids, have an inhibitory effect on lipophagy both at the transcriptional level, through FXR sensing in the basal nutritional state²⁷, and by reducing autophagosome-lysosome fusion in hepatocytes through changes in Rab7 association with autophagosomes⁵⁵. The specific signals that trigger selective removal of perilipins by CMA to initiate lipolysis remain unknown. However, phosphorylation of perilipins in localized regions of the lipid droplets is a pre-requisite for their delivery to lysosomes through this pathway, supporting the possible participation of lipid-sensor kinases in this process⁵³.

Although lipophagy and CMA are activated in response to acute increases in intracellular lipids, pronounced or chronically sustained lipid challenges inhibit both autophagic pathways via changes in the membrane lipid composition of autophagosomes and lysosomes^{36, 56}. In fact, this inhibitory effect along with this newly described role of autophagy in the control of intracellular lipid content may contribute to perpetuate the dysfunctional lipid metabolism characteristic of old organisms. Thus, the decrease in autophagic activity with age may lead to accumulation of intracellular lipids that in turn further inhibits lipophagy, creating a vicious cycle.

Autophagic adjustment of mitochondrial metabolic capacity

Mitochondria, the main source of intracellular ATP, contribute to cellular metabolism through different processes including FFA oxidation, citric acid cycle, biosynthesis of heme and phospholipids and calcium storage. Therefore, adequate maintenance of mitochondria fitness and number is essential for cellular homeostasis. High membrane potential/proton motive force across the inner mitochondrial membrane can promote an excessive production of reactive oxygen species, which in turn results in mitochondrial dysfunction through oxidative damage of membrane proteins and mitochondrial DNA. These events often lead to mitochondrial permeability transition (MPT)¹¹ which causes full and irreversible mitochondrial depolarization⁵⁷. Selective removal of depolarized and damaged mitochondria through autophagy (mitophagy) under these conditions prevents futile consumption of ATP by the mitochondrial ATP synthase working in reverse and also limits generation of free radicals. Mitophagy contributes in this way to restore the energetic balance and to reduce cellular damage averting in this way activation of cell death pathways (Fig. 2).

The recent detailed dissection of mitophagy has revealed likely co-existent mechanisms for the recognition and elimination of mitochondria via autophagy ^{reviewed in 11, 12, 58}. The best described is the PINK1/PARKIN pathway, activated in response to membrane depolarization. Membrane damage leads to PINK1 accumulation on the mitochondria surface which facilitates recruitment of the ubiquitin ligase PARKIN. Ubiquitination of a variety of mitochondrial outer membrane proteins by PARKIN serves as tag for ATG recognition and mitochondria engulfment⁵⁹. Additional receptors also facilitate mitophagy in response to different environmental and cellular cues, such as hypoxia or nutritional changes. These proteins include BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), BNIP3 like (NIX) or FUN14 domain containing 1 (FUNDC1)⁵⁸. The regulation of mitochondria turnover to accommodate energetic demand occurs at least in part through ULK1 phosphorylation by AMPK⁶⁰ and by the tight balance between mitochondrial biogenesis and mitophagy orchestrated by the TFEB-PGC1 alpha axis⁶¹. Future comparative studies of the different types of mitophagy may help to further understand whether specific forms of mitophagy are more dedicated to basal quality control while others are preferentially engaged to modify the mass of still functional mitochondria.

Fatty liver/Non-alcoholic steatohepatitis

Obesity and type two diabetes are often associated with non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). Increased lipid flux into the liver, augmented de novo lipid synthesis or decreased lipid catabolism all contribute to the hepatic accumulation of lipids and NAFLD. Chronic persistence of this lipid overload leads to liver injury with inflammation, cell death and fibrosis characteristic of NASH. As mentioned in the previous section, reduced macroautophagy and CMA in liver both result in marked hepatosteatosis although through different mechanisms.

In the case of macroautophagy, part of the alterations in lipid metabolism are at the level of lipid mobilization since hepatic ATG7 deletion, decreases TGs breakdown resulting in lipid droplets accumulation⁶. Furthermore, macroautophagy blockage also leads to deficiencies in proteostasis (increased polyubiquitinated protein content⁶⁷) and in organelle ⁶⁸,⁶⁹. ER stress and defective ER functioning in the context of the chronic metabolic dysfunction that associates with NASH contributes to exacerbate the defects in glucose metabolism⁶⁸. Failure of mitochondria quality control, due to their reduced turnover through mitophagy, can promote oxidative stress through ROS production and activation of downstream inflammatory pathways such as the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)⁶⁹. The combination of lipotoxicity, oxidative stress and chronic activation of the inflammatory response upon macroautophagy failure often leads to hepatocyte cell death, thus recapitulating the hallmarks of NASH (inflammation, oxidative stress, cell death and fibrosis).

Reduced hepatic CMA promotes hepatosteatosis due to an increase in lipogenic enzymes and failure in the timely removal of perilipins^13,53. Added to lipotoxicity, oxidative damage can also contribute to hepatic injury upon CMA blockage. Thus, CMA is upregulated in liver in response to oxidative stress to selectively remove damaged proteins⁷⁰. Defects in proteostasis upon persistent blockage of hepatic CMA promote accumulation of oxidized protein aggregates and thus contribute to perpetuate chronic oxidative stress in this organ⁷¹.

Most of the changes that characterize the metabolic syndrome and that include obesity, hyperglycemia, dyslipidemia and high blood pressure, have also shown to exert a negative effect on autophagy. In the presence of insulin resistance and hyperinsulinemia, the regulatory effect of forkhead box O1 (FoxO1) on the expression of several Atg genes is lost resulting in autophagy malfunction⁷². The increase in intracellular lipids also reduces both macroautophagy and CMA due to changes in intracellular membrane composition^36,56. High dietary lipids alter the lysosomal stability of the CMA receptor and lead to reduced activity of this pathway³⁶. Likewise, lipid changes in the autophagosome limiting membrane, reduce the ability of these vesicles to fuse with lysosomes and leads to a decrease in macroautophagic flux⁵⁶. This reduced clearance of autophagosomes could explain the accumulation in LC3-II and p62 observed in patients diagnosed with non-alcoholic steatosis (NAS) and NASH and that positively correlate with the severity of the disease⁷³. Studies in obese mice and NALD patients have also demonstrated that fatty liver decreases lysosome activity through inhibition of cathepsin expression which reduces lysosomal degradation of cargo delivered by all types of autophagy and endocytosis⁷⁴.

Given the above-described essential role of the different types of autophagy in liver metabolism, their dysregulation during dietary challenges or abnormal metabolic conditions can further aggravate the metabolic malfunction, thus creating a deleterious vicious cycle. This continuous negative feedback makes it often difficult to determine if the autophagic failure is primary or secondary.

We thank Drs. Susmita Kaushik for critical reading of the manuscript. We apologize to those whose work could not be cited owing to space limitations.

FUNDING SOURCES: Work in our laboratory is supported by National Institutes of Health grants AG021904, AG031782 and DK098408 and the generous support of R&R Belfer (to A.M.C.). JMM is supported by a postdoctoral fellowship from the American Diabetes Association.