Targeting the Lysosome: Mechanisms and Treatments for Non- Alcoholic Fatty Liver Disease

Abstract

The multiple functions of the lysosome, including degradation, nutrient sensing, signaling, and gene regulation, enable the lysosome to regulate lipid metabolism at different levels. In this review, I summarize the recent studies on lysosomal regulation of lipid metabolism and the alterations of the lysosome functions in the livers affected by non-alcoholic fatty liver disease (NAFLD). NAFLD is a highly prevalent lipid metabolic disorder. The progression of NAFLD leads to non-alcoholic steatohepatitis (NASH) and other severe liver diseases, and thus the preventions and treatments of NAFLD progression are critically needed. Targeting the lysosome is a promising strategy. I also discuss the current manipulations of the lysosome functions in the pre-clinical studies of NAFLD and propose my perspectives on potential future directions.

Introduction

The lysosome is a cellular organelle ubiquitously existing in eukaryotic cells. Since it was discovered in 1955, the lysosome has been known as the organelle responsible for the degradation of a various of biomacromolecules. Until recent decades, molecular cell biology studies uncovered new functions of the lysosome in autophagy, nutrient sensing and transport, signaling, and gene regulation. With these functions, the lysosome plays a central role in the regulation of cell growth and metabolism. Recently, its role in regulating lipid metabolism has also arisen[1, 2].

Dysregulated lipid metabolism is the major cause of many metabolic disorders, such as non-alcoholic fatty liver disease (NAFLD), which is also known as metabolic dysfunction-associated liver disease (MAFLD) in a recently proposed change of terminology[3]. We use NAFLD in this review, for it is still most commonly used in the field. NAFLD is characterized by accumulated cellular lipids in the liver (steatosis) and consequent hepatic lipotoxicity. Lipotoxicity is a series of toxic effects that damage membranes, induce cell stress, promote inflammation, and cause cell death[4]. Lipotoxicity is a leading cause of NAFLD progression, from benign steatosis to more severe non-alcoholic steatohepatitis (NASH), increasing the risk of developing worse liver diseases, such as cirrhosis and hepatocellular carcinoma (HCC)[4].

Cellular free fatty acids are the major cause of lipotoxicity. Adipose tissues store fatty acids as esterified lipids. When needed, adipocytes release free fatty acids into the circulation to fuel other tissues and organs, including the liver. A combination of passive diffusion and protein-facilitated uptake mediates the ingestion of plasma free fatty acids. The cytosolic fatty acid binding proteins then receive and deliver the free fatty acids to their destinations: the endoplasmic reticulum (ER) for esterification, mitochondria and peroxisomes for oxidation, and lipid droplets for storage (Fig. 1). Obesity-related elevation of free fatty acid levels in the circulation is tightly associated to NAFLD pathology[5]. Excess cytosolic free fatty acids cause ER stress with increased unfolded protein response, mitochondrion/peroxisome dysfunction with accumulated reactive oxygen species (ROS), chronic inflammation, and cell death. Although inhibiting direct free fatty acid uptake seems like a promising strategy for NAFLD treatment, studies on plasma fatty acid profiles in both healthy people and NASH patients reveal that the plasma free fatty acids comprise ~5% of the total plasma fatty acids, and ~ 95% of fatty acids exist in esterified lipids[6, 7], such as in chylomicron remnants and lipoproteins. These lipidic particles enter the liver cells through the endocytic pathway and release free fatty acids in the lysosome. When the plasma lipidic particles are elevated in NAFLD progression[8], the lysosome could contribute a considerable amount of cellular free fatty acids.

Fig. 1. The lysosome functions in lipid metabolism and non- alcoholic fatty liver disease (NAFLD).

Lysosomal acid lipase (LAL) degrades the ingested low-density lipoproteins (LDL) and the autophagic lipids and membranes. The degradation products are oxidated in the mitochondria and peroxisomes or esterified in the endoplasmic reticulum and stored in the lipid droplet. The degradation products and cytosolic nutrients regulate mTOR activation, which requires a group of proteins localized on the lysosome. Active mTOR promotes lipogenesis and inhibits autophagy and TFEB. TFEB is a transcriptional factor, which regulates the expression of genes that are related to autophagy and lysosome biogenesis. Thus, the mTOR-TFEB-autophagy axis controls both lipid anabolism and catabolism. In the livers of NAFLD patients, excess cellular lipids stimulate mTOR activation and consequently increase lipid anabolism and decrease catabolism, resulting in lipid accumulation in the lipid droplets. Additionally, excess cellular lipids cause lipotoxicity, damaging organelles and inducing inflammation. The impaired lysosomes have reduced LAL activity and the ability of fusion with autophagosomes. These conditions result in the accumulation of lipids in the lysosomes and destruction of autophagic turnover of the lipid droplets and clearance of the damaged organelle, which worsens the hepatic steatosis and lipotoxicity.

In addition to free fatty acids, the lysosome produces free cholesterol through the degradation of chylomicron remnants and lipoproteins. The lysosome uses the cholesterol transport proteins to export cholesterol and deliver it to different cellular compartments. Accumulation of free cholesterol is associated to NAFLD via interfering with cholesterol homeostasis, inducing cell stress, and activating the liver macrophage Kupffer cells and the mesenchymal cells Stellate cells, which promotes inflammation and fibrogenesis[4, 9].

Lysosome-released free fatty acids and cholesterol not only contribute to the cellular lipid pool and induce lipotoxicity but also serve as signaling molecules to influence lipid metabolism through the lysosome functions of signaling and gene regulation. Thus, the lysosome integrates lipid receiving, processing, distributing, signaling, and downstream feedback regulations to regulate lipid metabolism at different aspects and levels. In this article, I am summarizing how lysosome functions influence lipid metabolism based on recent studies, as well as discussing the role of the lysosome as a target for NAFLD treatment.

Lysosomal acid lipase (LAL)

Dietary lipids are packed into chylomicron remnants and lipoprotein particles and are then ingested by liver cells through receptor-mediated endocytosis. At the end of endocytic pathway, the lysosome receives these lipidic particles and digests them using the lipid hydrolase Lysosomal Acid Lipase (LAL) (Fig. 1). LAL is encoded by the gene LIPA and is highly expressed in multiple tissues including the liver[10]. So far, it is the only identified lysosomal lipase, and its activity largely depends on pH. Similar to other lysosomal hydrolases, LAL is synthesized as a precursor polypeptide in the endoplasmic reticulum (ER), and delivered to the lysosome via the Golgi apparatus. In the lysosomal lumen, the matured LAL hydrolyses triglycerides, cholesteryl esters[11], and retinyl esters[12] and releases the degradation products. These include the hydrophobic lipid molecules free fatty acids, cholesterol, and retinol. Genetic deficiency of LAL causes cholesteryl ester storage disease (CESD) and Wolman disease[13]. In Wolman disease, LAL mutation results in a complete loss of enzyme activity or no enzyme presence, thus accumulating both triglycerides and cholesteryl esters in the lysosome.

Reduction of serum LAL activity has often been observed with the development of NAFLD[14–17]. A recent study shows that in NAFLD patients, hepatic LAL activity corelates to serum LAL activity, but not the LAL expression level[17]. This suggests that the reduction of LAL activity cannot be completely attributed to LAL expression alteration. The impaired LAL activity appears to be a consequence of lysosome defects, which are probably caused by lipotoxicity. Cytosolic free fatty acids damage cellular membranes and membrane-associated proteins, including those of the lysosomes. Lysosome membrane permeabilization[18] and disassembly of the V-ATPase protein complex[19], that acts as the lysosomal proton pump, impair luminal acidification[19], which results in deficient LAL maturation and activity. Reduced LAL activity slows down lipid catabolism and results in lipid accumulation. The accumulated lipids further impair lysosome functions, which harms LAL activity even more and thus forms a vicious cycle. Deletion of LAL in mice increases both cholesteryl esters and triglycerides in plasma and tissue, and increases the weights of the mice’s livers[20–22]. Liver-specific deletion of LAL causes hepatic inflammations[23]. Analogously, patients with CESD patients exhibit hepatomegaly (enlarged livers) caused by hepatic steatosis, which is a commonly associated disorder[13].

The reduction of LAL activity seen in NAFLD alludes to a potential strategy of increasing LAL activity to improve steatosis. Transit expression of human LAL[20] or liver-specific expression of LAL[24] in LAL-null mice reduces steatosis-caused hepatomegaly and liver inflammation. However, overexpression of LAL in wildtype mice fed with NAFLD-inducing diets does not relieve lipid accumulation-caused hepatomegaly and worsens hepatic inflammation[25], indicating the detrimental effects caused by hyperactivation of LAL. Enhanced LAL activity probably increases the degradation products, but the lysosome cannot store those products, which need to be exported by specific transportation machineries. Excessive amounts of free fatty acids and cholesterol can overwhelm the cells and cause lipotoxicity. Therefore, along with increasing LAL activity, other strategies are probably required to induce reactions that make the degradation products less harmful, like neutralization (lipid droplet storage), oxidation, and exporting the degradation products outside the cell. So far, only cholesterol export from the lysosome has been well studied. The mechanisms behind the transport of lysosome-released free fatty acids and retinol remains unclear.

Autophagy

In addition to the ingested lipids, the lysosome degrades cellular lipids through autophagy. Autophagy is a conserved cellular pathway for breaking down cytosolic materials via lysosomal degradation. Autophagy protects cells from starvation, stress, damage, and infection by recycling nutrients and removing harmful substances, such as damaged organelles, aggregated proteins, and pathogens[26]. Based on the specific mechanisms of how the autophagy occurs, there are three subtypes that have been described in the literature, i.e., macroautophagy, microautophagy, and chaperon-mediated autophagy. In macroautophagy, specific membranes named phagophors are formed from the ER and expand using the lipid resources from various cellular compartments. After phagophores grow long enough to wrap around autophagic substrates, the two ends of a phagophore close to form a vesicle termed autophagosome. Autophagosomes deliver the substrates to the lysosome by directly fusing with the lysosomes, forming autolysosomes. Autolysosomes degrade the substrates and release the final products[27]. So far, macroautophgy is the best understood autophagy type. There are a group of genes, namely autophagy-related genes (ATG), which control and regulate each step of macroautophagy. In many cases, the term of autophagy is used specifically for macroautophagy. Microautophagy is the process in which lysosomes invaginate their membranes to engulf cytosolic substrates directly into the lysosome lumen for degradation[28]. Compared to macroautohphagy, much less is known about the mechanism of microautophagy. Chaperone-mediated autophagy (CMA) selectively degrades the proteins with a KFEQR-like motif. A chaperone complex, containing the heat shock cognate 71 kDa protein (HSC70) and other cochaperones, recognizes and binds to the KFEQR-like motif and delivers the substrate proteins to the lysosome. Lysosomal transmembrane protein LAMP2A assists the translocation of substrate proteins crossing the membrane and enter into lysosome lumen for degradation[29].

All of above three types of autophagy mediate or participate in the degradation of selected autophagy substrates, such as lipid droplets[30]. Accumulation of lipid droplets in the liver is a major cause of hepatic steatosis. It has been known that cytosolic lipases, such as ATGL[31, 32], catalyze lipolysis to break down the lipid droplets. In 2009, Singh et. al. first reported that liver cells use the macroautophagy pathway for lipid droplet degradation[33], which is termed as macrolipophagy or lipophagy. Later studies demonstrated that macrolipophagy is an important lipid catabolism mechanism in the liver and plays a critical role in physiology and pathology[34]. Additionally, a recent work shows that microlipophagy is also active in isolated hepatocytes, in which lipid droplets are directly engulfed into the lysosome via physical contacts between the organelles[35]. Furthermore, lipid droplet surface proteins can be degraded by chaperon-mediated autophagy to allow cytosolic lipases access to the lipid contents for lipolysis[36]. Some key questions for autophagy-mediated lipid droplet turnover are, what is the importance of each type of autophagy in lipid droplet turnover in the liver, and what are the determinant and regulation factors? To answer these questions, studies on the molecular mechanisms of each type autophagy are urgently required. The crosstalk between lysosomal lipid degradation and cytosolic lipolysis is also important to be addressed.

In addition to lipophagy, defects of other selective autophagy, such as mitophagy (autophagic degradation of mitochondria) and pexophagy (autophagic degradation of peroxisomes), play important roles in lipid metabolism[37, 38]. When cells are overloaded with free fatty acids, incomplete oxidation products and ROS are produced in the oxidation processes and thus cause oxidative stress. This causes damage to organelles and triggers inflammation and apoptosis. Mitophagy and pexophagy remove the damaged organelles, eliminates cell oxidation stress, and restores normal lipid oxidation and redox condition (Fig. 1).

In NAFLD, the impaired lysosomal acidification[39] and the reduction of LAL activity[14–16] attenuate the lysosomal digestion ability and thus unavoidably impairs autophagic degradation and clearance. These defects result in accumulations of autophagic substrates in the lysosome and thus worsen lysosomal damage. Additionally, the lipids accumulated in the lysosome impair lysosomal fusion ability [40, 41], which reduces the delivery of autophagic substances into the lysosome. Indeed, the livers of NAFLD patients have accumulated abnormal lysosomes filled with lipids[42], cytoplasmic autophagosomes with lipid droplets inside[42], and enlarged mitochondria[43, 44], positively correlated to the NAFLD progression in the patients[42, 44]. Along with the fusion and degradation steps of autophagy, the autophagy-related genes[44, 45] and the factors[46, 47] that regulate autophagy are also altered under NAFLD conditions, suggesting that the upstream signaling of autophagy is changed. The alteration of the autophagy-related genes is in line with the decreased activity of autophagy-regulating factor TFEB (see below).

It logically follows that with impaired autophagy in NAFLD, induction of autophagy became a potential approach for NAFLD treatment. Many pre-clinical and clinical studies show that promoting autophagy improves hepatic steatosis, which has been reviewed recently[45, 48]. Restoring the lysosome pH could be another approach to increase lysosomal degradation ability and autophagy flux[49]. Similar to increasing LAL activity, the possibility of lysosome-related lipotoxicity caused by excess lysosome degradation products needs to be evaluated. A recent study reported that liver cells with overexpression of ATGL export autophagy-derived fatty acids to the exterior of the cells via lysosome exocytosis, suggesting a possible fate of lysosomal fatty acids in the liver[50]. Further studies are required to investigate the role of ATGL in fatty acid exportation and other possible transport routes of lysosomal free fatty acids, as well as the significance of each route in NAFLD.

Transcription Factor EB (TFEB)

TFEB belongs to the MiT-TFE family of helix-loop-helix leucine zipper transcription factors. As a transcription factor, TFEB is mainly localized in the cytoplasm and translocated into the nucleus to regulate expression of the genes that function in multiple steps of autophagy and lysosome functions[51], including LAL[52]. Remarkably, overexpression of TFEB promotes lysosome biogenesis[51]. The newly synthesized lysosomes can replace and clean up the old dysfunctional lysosomes, which is crucial in disease context. TFEB controls gene expression in response to a variety of conditions, such ER stress[53], oxidative stress[54], mitophagy[55], inflammation[56], and exercise[57].

Active TFEB protects cells from stress- and inflammation-caused damage. Several pathways have been shown as necessary for TFEB activation, including the phosphatase calcineurin, which is activated by lysosomal Ca²⁺ release[58]. Severe lysosome damage, thus, hampers TFEB activation and the opportunity for lysosome refreshment. Indeed, reduced TFEB activity and levels have been observed in NAFLD mouse models[59, 60] and patients [61, 62]. Consistent with this observation, increasing TFEB activity by using pharmaceuticals, such as liraglutide[59], metformin[60], and ezetimibe[62], and by overexpression[61, 63] or epigenetically upregulation[64] of TFEB improves hepatic steatosis in pre-clinical and clinical studies.

Mechanistic target of rapamycin (mTOR)

Cell metabolism is regulated by the environmental and cellular nutrient status. The atypical Ser/Thr kinase mTOR responds to various nutrients, such as amino acids[65], growth factors[66], cholesterol[67], fatty acids[68], and the fatty acid oxidation product acetyl-CoA[69]. By phosphorylating the substrate proteins, mTOR regulates their activity in cell growth, survival, and metabolism[70].

mTOR exists in two distinct complexes, mTORC1 and mTORC2. mTORC1 shuttles between the lysosome surface and cytosol, and mTORC2 is believed to exist in cytosol, though it is also found to be associated with the lysosome[71]. A group of proteins and protein complexes are localized on the lysosome surface to mediate mTOR activation, hence, the lysosome condition and lysosome-released degradation products directly regulate mTOR activity. Active mTOR promotes lipogenesis through directly phosphorylating the triglyceride synthesis enzyme lipin, and promoting the activation of sterol regulatory element-binding protein-1 (SREBP-1)[72][73]. In addition, active mTOR inhibits autophagy initiation and blocks TFEB-regulated gene expression by phosphorylating ULK1[74] and TFEB itself[75], respectively.

Hyperactivation of mTOR in NAFLD is a critical pathological factor of NAFLD progression[76, 77]. Since mTOR promotes lipid accumulation by positive regulation of lipogenesis and negative regulation of lipid turnover, reducing mTOR activity is important for NAFLD treatment. Indeed, inhibition of mTOR through its regulatory genes[77], microRNA[78], and compounds[79] has been shown to alleviate hepatic steatosis in animal models of NAFLD/NASH/HCC.

Other lysosomal signaling

In addition to mTOR signaling, another exciting example for lysosomal signaling comes from a study in Caenorhabditis elegance shows that the lipid oleoylethanolamide (OEA) generated by lysosomal lipolysis binds to the lipid-binding protein LBP8 and translocates into the nucleus to activate the nuclear hormone receptors NHR-49 and NHR-80[80]. The mammalian ortholog of NHR-49 is peroxisome proliferator-activated receptor alpha (PPAR-α, which a well-studied transcription factor that controls a series of genes regulating lipid metabolism. Active PPAR-α promotes fatty acid catabolism through mitochondrial/peroxisome β-oxidation. The mammalian ortholog of NHR-80 is hepatocyte nuclear factor 4 (HNF4), which controls liver development, negatively regulates liver regeneration, and suppresses tumorigenesis. In line with this study, OEA is known to regulate satiety and body weight through activating PPAR-α[81], and OEA supplementation has beneficial effects to NAFLD mice[82] and patients[83, 84]. Notedly, PPAR-α, HNF4, and LBP8 (likely human fatty acid binding protein 4, FABP4[85]) are all highly expressed in the liver, suggesting that this lysosome-initiated signaling is important to the liver. This is additional evidence for the importance to maintain lysosome normal function for liver lipid metabolism. However, this pathway still requires investigations in the NAFLD models and patients.

Cholesterol is another lysosomal signaling molecule. In addition to activating mTOR[67], as mentioned above, cholesterol is well-known for its signaling to SREBP-2. SREBP-2 is a transcription factor that regulates cholesterol synthesis and uptake and thus plays a critical role in cholesterol homeostasis. SREBP-2 is localized in the ER and activated when the ER cholesterol level is low. The functions of the lysosome in hydrolyzing cholesteryl esters and distribution of free cholesterol makes SREBP-2 activity largely depend on the lysosome. Increased SREBP-2 activity and cholesterol synthesis is observed in NAFLD patients[86]. It seems to be a paradox that increased lipid input in NAFLD patients does not suppress SREBP-2 activation. It is possible that reduced LAL activity[14–17] decreases free cholesterol levels in the lysosome. Active mTORC1 is suggested to promote SREBP-2 activation by reducing lysosome cholesterol through suppression of autophagy[87]. In addition, whether there is a noncanonical pathway for SREBP-2 activation should be investigated. Also importantly, the ability of lysosomes to deliver cholesterol should be examined in NAFLD.

Lysosome dynamics

The lysosome is a highly dynamic organelle with the abilities to move within cytoplasm (motility), to stay in different area of a cell (positioning), to change morphology between vesicular and tubular, and to physically interact with other organelles. Lysosome motility is required for the other dynamics and thus is a fundamental feature. Lysosome motility is mediated by a couple of machineries that link to the motor proteins[51, 88] and is regulated by a variety of inputs, such as pH[89], calcium[90], nutrients including cholesterol[91–95], and infections[96, 97]. In response to the inputs, the lysosomes change their motility and other dynamic features to coordinate lysosome functioning[51, 88].

Studies on the role of lysosome dynamics in NAFLD are limited. However, in the context of lipid metabolism, the significance of lysosome dynamics has emerged (Fig. 2). Lysosome positioning determines their luminal pH[98] and consequently influence LAL activity. Lysosome motility and positioning regulates mTOR activity and autophagy in response to nutrients[71, 91, 95, 99, 100]. Lysosome tubulation is a critical step in autophagic lysosome reformation (ALR), which restores the lysosome pool when autophagy is terminated[101]. In addition, lysosome dynamics are required for lipid transport. Lysosome exocytosis-dependent fatty acid exportation requires the lysosome moving to the plasma membrane. Lysosome-ER and lysosome-mitochondrion contacts mediate the transport of free fatty acids and cholesterol for lipid synthesis and oxidation[102, 103]. Overexpression of the sterol-binding protein STARD3 at the lysosome-ER contact sites results in an increased free cholesterol level and apoptosis in the mouse liver[104]. Lysosome-peroxisome contact transports cholesterol to peroxisomes[105]. Lysosome-lipid droplet contact is the key step for microlipophagy in the liver[35]. Lysosome dynamics enables lysosome functioning in a spatial-stapo manner and integrates lipid uptake, signaling, transport, storage, and turnover into a highly organized cellular network, suggesting a new perspective in the studies of NAFLD.

Fig. 2. Lysosome dynamics in lipid metabolism.

Lysosome dynamics include the features of lysosomal motility, positioning, morphology changes, and physical contact with other organelles. The lysosome moves along the microtubule tracks in two directions, outwards and inwards, mediated by the motor protein complexes kinesins and dynein. Outward movement distributes the lysosomes to the periphery area of the cell, where the lysosomes have higher activities of mTOR. In contract, inward movement clusters the lysosomes at the juxtanuclear area, where the lysosomes have lower luminal pH and thus higher activity of the hydrolases. Also, the lysosomes fuse with the autophagosomes to form autolysosomes at the juxtanuclear area. In the end of autophagy, autolysosomes form tubular proto-lysosomes for lysosomal reformation. Through regulating mTOR activation and autophagy, lysosome dynamics participate in the lysosomal regulation of lipid metabolism (Fig. 1). Additionally, lysosome motility is required to encounter other organelles for the membrane contacts, which form the lipid transport channel between membranes for inter-organelle lipid exchange. The membrane contacts between the lysosomes and lipid droplets as well as the remodeling of lysosome membrane at the contact sites are important steps in microlipophagy-mediated lipid droplet turnover in the liver.

Conclusion

As a degradation organelle, the lysosomes receive, digest, and distribute lipids to provide cells with building blocks and energy. As a signaling hub, the lysosome senses lipid signals and regulates gene expression to control lipid biogenesis and turnover. Plus, the dynamic features of the lysosome enable its control and regulation of lipid metabolism, as well as make it efficient. An overabundance of cellular nutrients in NAFLD promotes mTOR activation. Hyperactivation of mTOR increases lipogenesis, inhibits autophagy, and blocks lysosome biogenesis. Increased lipogenesis, deficient autophagy, and reduced lysosome pool promote hepatic steatosis and lipotoxicity. Lipotoxicity-caused lysosome damage impairs lipid turnover and autophagic clearance of damaged organelles, worsening steatosis and cell stress (Fig. 1). Inhibiting mTOR and restoring normal lysosomal function and autophagy is therefore the current strategy of targeting the lysosome for NAFLD treatment. Notably, the lysosomal digestion activity probably needs to be maintained at a certain level to avoid the accumulation of lipid digestion products, which could aggravate lipotoxicity and cause lipid build-up in lipid droplets. Finding approaches for the safe deposition of the lysosome degradation products could be an encouraging direction for future study, which requires the exploration of lysosomal lipid transport and the mechanisms of lysosome dynamics (Figs. 1,2).