
==== Front
Exp Mol Med
Exp Mol Med
Experimental & Molecular Medicine
1226-3613
2092-6413
Nature Publishing Group UK London

38297165
1161
10.1038/s12276-024-01161-x
Review Article
Regulation of autophagy by perilysosomal calcium: a new player in β-cell lipotoxicity
Nguyen Ha Thu 12
Wiederkehr Andreas 3
Wollheim Claes B. Claes.Wollheim@unige.ch

45
http://orcid.org/0000-0003-0322-9807
Park Kyu-Sang qsang@yonsei.ac.kr

12
1 https://ror.org/01wjejq96 grid.15444.30 0000 0004 0470 5454 Department of Physiology, Yonsei University Wonju College of Medicine, Wonju, Korea
2 https://ror.org/01wjejq96 grid.15444.30 0000 0004 0470 5454 Mitohormesis Research Center, Yonsei University Wonju College of Medicine, Wonju, Korea
3 https://ror.org/02s376052 grid.5333.6 0000 0001 2183 9049 Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
4 https://ror.org/01swzsf04 grid.8591.5 0000 0001 2175 2154 Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland
5 https://ror.org/012a77v79 grid.4514.4 0000 0001 0930 2361 Department of Clinical Sciences, Lund University, Malmö, Sweden
1 2 2024
1 2 2024
2 2024
56 2 273288
3 5 2023
16 10 2023
9 11 2023
© The Author(s) 2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Autophagy is an essential quality control mechanism for maintaining organellar functions in eukaryotic cells. Defective autophagy in pancreatic beta cells has been shown to be involved in the progression of diabetes through impaired insulin secretion under glucolipotoxic stress. The underlying mechanism reveals the pathologic role of the hyperactivation of mechanistic target of rapamycin (mTOR), which inhibits lysosomal biogenesis and autophagic processes. Moreover, accumulating evidence suggests that oxidative stress induces Ca2+ depletion in the endoplasmic reticulum (ER) and cytosolic Ca2+ overload, which may contribute to mTOR activation in perilysosomal microdomains, leading to autophagic defects and β-cell failure due to lipotoxicity. This review delineates the antagonistic regulation of autophagic flux by mTOR and AMP-dependent protein kinase (AMPK) at the lysosomal membrane, and both of these molecules could be activated by perilysosomal calcium signaling. However, aberrant and persistent Ca2+ elevation upon lipotoxic stress increases mTOR activity and suppresses autophagy. Therefore, normalization of autophagy is an attractive therapeutic strategy for patients with β-cell failure and diabetes.

Autophagy defects: the hidden culprit in diabetes progression

Excessive intake of fatty acids can result in a condition known as lipotoxicity (harmful effect of fats on cells), which can harm pancreatic beta cells (cells that produce insulin) and contribute to type 2 diabetes. Researchers have found that this harm is due to a disturbance in the cells' autophagy process (a system used by cells to eliminate unnecessary or dysfunctional parts). Specifically, the research revealed that the autophagy process is hindered by the activation of a protein complex called mTORC1 and the suppression of a protein named AMPK. These proteins control the autophagy process and their disturbance leads to the buildup of harmful substances in the cell, causing cell death. The researchers propose that future treatment strategies could focus on restoring the balance of these proteins to safeguard pancreatic beta cells from lipotoxicity. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

Subject terms

Type 2 diabetes
Energy metabolism
Myung Sun Kim Memorial Foundation (2016), Yonsei University, Koreaissue-copyright-statement© Korean Society for Biochemical and Molecular Biology 2024
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pmcIntroduction

Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia due to a deficiency in insulin secretion or an increase in insulin resistance. The main subtype is type 2 diabetes, which is characterized by insulin resistance and impaired insulin secretion. Insulin resistance is triggered by overnutrition and physical inactivity, leading to pancreatic β-cell neogenesis and hypersecretion of insulin to compensate for the elevated insulin demand. However, prolonged exposure to high glucose and saturated fatty acids eventually induces a cytotoxic effect on β-cells, causing defective insulin secretion, a major determinant in disease progression1,2.

Pancreatic β-cells play a major role as sensors and rectifiers of glucose homeostasis. Insulin, the main hormone that lowers blood glucose, is secreted from β-cells upon nutrient ingestion. To precisely decode signals reflecting the extracellular metabolic environment, β-cells have a metabolic sensing system. Nutrients are metabolized in the cytosol, and their products funnel into mitochondria to generate ATP and metabolites, which induce insulin exocytosis. Furthermore, upon glucose stimulation, a β-cell produces up to a million molecules of a single-chain precursor, proinsulin, per minute, which is a more than 20-fold increase in protein under the same transcriptional level3. Proinsulin enters the endoplasmic reticulum (ER) lumen to undergo protein folding. In the ER lumen, proinsulin molecules acquire three disulfide bonds through prooxidant enzymes such as ER oxidoreductase 1α (ERO1α) and protein disulfide isomerase (PDI), which contribute to reactive oxygen species (ROS) generation under stressful conditions4.

Maintenance of functional mitochondria and the ER in β-cells could be threatened by the stress burden related to excess nutrients5. This stress causes compensatory increases in insulin synthesis and β-cell proliferation, but prolonged hyperinsulinemia can deteriorate the efficiency of insulin receptor signaling. Insulin resistance can cause β-cell failure due to long-term increased insulin demand1,2. Additionally, ROS production induced by lipotoxic conditions can contribute to mitochondrial dysfunction and ER stress, as β-cells have a weak antioxidative capacity to counteract redox insults6. Therefore, cellular stresses induced by lipotoxicity impose a double burden on β-cells: an accelerated insulin synthesizing load and cellular oxidative stress, which lead to mitochondrial dysfunction and ER stress.

In addition to stresses on mitochondria and the ER, lysosomal stress can also play a role in β-cell lipotoxicity. Defective autophagic degradation due to lysosomal stress can impair cell survival under lipotoxic stress. The intralysosomal Ca2+ concentration is known to be in the range of hundreds of micromolar, corresponding to that of the ER, even though lysosomes do not have any active Ca2+ ATPase. This large Ca2+ gradient across the lysosomal membrane is known to be dependent on the proton gradient developed by the V-ATPase H+ pump7. However, Xu et al. suggested that there may exist a direct Ca2+ transfer from the ER to lysosomes via passive Ca2+ transporters or channels8. Disturbance in lysosomal Ca2+ homeostasis impairs autophagic degradation and deteriorates cell survival under lipotoxic stress. We have previously reported oxidative stress-mediated ER Ca2+ depletion and cytotoxicity by saturated fatty acids9, consistent with the results published by others10. In this review, we will focus on autophagic defects related to lysosomal Ca2+ dysregulation and pathologic signaling in β-cell lipotoxicity, which could be effective therapeutic targets for type 2 diabetes and other metabolic diseases.

ER calcium depletion in beta-cell lipotoxicity

A large body of evidence describes the involvement of ER stress and mitochondrial dysfunction in β-cell lipotoxicity related to oxidative stress. Elevated levels of glucose and saturated fatty acids elicit pathologic oxidative stress through different mechanisms, such as activation of the protein kinase C (PKC)-NADPH oxidase (NOX) axis in the cytosol or increased superoxide generation from mitochondria. ERO1α and PDI also participate in ROS generation from the ER, which amplifies oxidative stress via a feed-forward mechanism. This redox disequilibrium induces aberrant ER Ca2+ release by activating inositol trisphosphate (IP3) receptors and ryanodine receptors11–13. Oxidative stress also decreases sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) activity14, resulting in ER Ca2+ depletion and associated ER stress.

Another critical role of the ER, in addition to protein folding, is the sensing of cellular stresses and maintaining homeostasis15. The accumulation of unfolded or misfolded proteins due to ER dysfunction activates the unfolded protein response (UPR), which attempts to attenuate pathologic progression and recover ER function within a limited range. This process is initiated by three ER membrane proteins: inositol-requiring enzyme 1α (IRE1α), protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6). During ER stress, these three distinct signal transduction arms dissociate from 78-kDa glucose-regulated protein (GRP78), also known as binding immunoglobulin (BiP), and activate downstream signaling cascades. Functional consequences of the UPR include (1) the reduction of global protein synthesis by attenuating translation, (2) the promotion of selective translation of chaperones to increase ER protein folding capacity, and (3) signaling for ER-associated protein degradation (ERAD) to eliminate misfolded proteins by the ubiquitin‒proteasome system (UPS) (Fig. 1). However, if the ER is unable to reestablish homeostasis, cell death is initiated through proapoptotic signaling.Fig. 1 ER Ca2+ depletion and stress response in β-cell lipotoxicity.

Oxidative stress due to saturated fatty acids accelerates ER Ca2+ release and suppresses Ca2+ uptake into the ER lumen. Decreased Ca2+-activated ER chaperone function resulting from Ca2+ depletion induces the ER stress response, consisting of general suppression of protein translation with selective translation of chaperones and accelerated degradation of misfolded proteins in the ER, leading to recovery from ER stress. IP3R inositol tris-phosphate receptor, RyR ryanodine receptor.

Due to active Ca2+ transport via SERCA, the luminal Ca2+ concentration of the ER remains high (100~800 μM)16. This phenomenon is important for the function of chaperones in the ER; thus, depleted ER Ca2+ levels cause accumulation of unfolded or misfolded proteins. In addition to pathologies of the ER itself, ER Ca2+ release disrupts cytosolic as well as organellar Ca2+ homeostasis, including that of mitochondria and lysosomes. Mitochondrial Ca2+ overload may be related to superoxide generation and mitochondrial dysfunction, consequently triggering the apoptotic process. The connection between oxidative stress and organellar Ca2+ homeostasis by lipotoxicity has been supported by the formation of mitochondria-associated ER membranes (MAMs) in metabolic stress. Notably, a noncanonical function of IRE1 is the regulation of the expression of the MAM protein IP3 receptor17. This spatial proximity facilitates aberrant Ca2+ transfer between the ER and mitochondria, establishing a vicious loop of organelle dysfunction. Increased MAMs have been described in palmitate-treated insulin-secreting cells, and the mechanism involves upregulation of the MAM protein GRP7518. Disturbances in lysosomal Ca2+ regulation related to ER Ca2+ release can impair lysosomal protein degradation.

Intriguingly, unresolved ER stress induces ERAD with additional activation of autophagy, which plays a physiologic protective role against pathological burdens. During the UPR, IRE1α dissociates from BiP/GRP78 and activates c-Jun N-terminal kinases (JNKs), leading to the release of Beclin1 and enhanced basal autophagy19. Stimulation of PERK induces ATF4 and CHOP, which drive the expression of autophagy-related proteins, including Atg5 and Atg12, initiating the formation of autophagosomes20. However, chronic ER stress blocks autophagic initiation and degradation, which aggravates lipotoxicity in β-cells. Continuous and excessive demand for insulin secretion due to insulin resistance or inefficient compensatory UPR leads to autophagic defects and β cell failure.

Autophagy defects in beta-cell lipotoxicity

Role of autophagy in β-cell function

Autophagy is an evolutionarily conserved self-defense process that removes toxic materials and damaged organelles to maintain cellular homeostasis21. Over many years, this process has been demonstrated to be critical in cellular physiology and stress defense in mammalian tissues. Autophagy can be classified, with respect to cargo delivery mode and biological functions, into several major categories: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). In macroautophagy and related processes (hereafter referred to as autophagy), the major lysosomal degradation pathway is used to eliminate long-lived proteins and intracellular organelles. The autophagic process occurs through a series of steps: initiation, elongation, maturation, fusion, and degradation22. The onset of autophagy is marked by the formation of an isolated double membrane (phagophore) surrounding cytoplasmic cargos. The complete engulfment of the material to be degraded by the phagophore forms the autophagosome, which has a vesicle structure that captures the cargos. The autophagosome subsequently fuses with a lysosome to form an autolysosome where material undergoing autophagy is being degraded. Degradation products are recycled back for other cellular processes23.

In pancreatic β-cells, autophagy was first described in the context of the intracellular degradation of insulin granules24,25. The disposal of aged granules in β-cells is carried out by crinophagy, a process of degrading excess secretory granules containing insulin by delivering them to lysosomes. Crinophagy is required for maintaining insulin granule pools at an optimal level26. If any abnormalities exist in the degradation pathway, the imbalance between proinsulin biosynthesis and insulin secretion leads to β-cell dysfunction. Autophagy in β-cells received additional attention after reports about large aggregates of ubiquitinated proteins in insulin-positive β-cells from Zucker diabetic fatty rats27. The degradation of these proteins was performed by lysosomes, rather than proteasomes, following the process of autophagy.

Does autophagy have a protective or a detrimental function in β-cells in response to stress conditions? Autophagy defects in a mouse model lacking Atg7 in β-cells (Atg7f/f:RIP-Cre mice) resulted in progressive β-cell loss and impaired glucose tolerance28,29. This phenotype suggests that autophagy is fundamental for β-cell survival. In addition, autophagy is indispensable for various physiological processes in β-cells, including differentiation, development, and insulin homeostasis30,31. Autophagy is also needed to remove dysfunctional mitochondria (mitophagy) to maintain a healthy mitochondrial network through mitochondrial fission and fusion32. The ER, as the crucial organelle responsible for insulin biosynthesis, also has an intimate link to autophagy33. During the unfolded protein response (UPR), severely damaged fragments become selectively eliminated by autophagy to maintain ER homeostasis, called ER-phagy34. Impaired autophagic flux renders the quality control system inefficient and serves to eliminate damaged organelles. This process explains the accumulation of swollen mitochondria and expanded ER in Atg7 knockout β-cells28,29.

While autophagy is protective in β-cells, hyperactivation of autophagy reduces β-cell function and survival both in vitro and in vivo35. For example, strong activation of autophagy by rapamycin, as a suppressor of mechanistic target of rapamycin (mTOR), decreases insulin production and exacerbates β-cell death. The autophagy inhibitor 3-methyladenine abrogates the effects of rapamycin and restores insulin secretion, suggesting that β-cell dysfunction induced by rapamycin might be mediated through the excessive induction of autophagy. A similar phenotype has been reported in a Raptor knockout model, in which the deletion induces mTOR inhibition and enhances autophagy. This mouse exhibited compromised insulin secretion, which was rescued by autophagy inhibition36. Consistently, knockdown of Atg5/7 or short-term bafilomycin A1 treatment led to autophagy inhibition and enhanced proinsulin biosynthesis and insulin secretion31. Thus, well-regulated autophagy is crucial for maintaining β-cell homeostasis. Stresses disturbing autophagic activity, either inhibition or overactivation, contribute to β-cell dysfunction and the development of diabetes (Fig. 2). A recent study showed that proper autophagic function is also needed for the regulation of glucagon secretion in alpha cells37,38. However, α-cells are resistant to lipotoxicity, partially explained by abundant expression of antiapoptotic proteins39.Fig. 2 Autophagic defects in β-cell lipotoxicity.

Both hyperactivation and inhibition of autophagy reduce the cellular functions and survival of pancreatic islets. Activation of mTOR signaling by AMPK inhibition in β-cell lipotoxicity suppresses the activities of mitophagy, ER-phagy, and lipophagy, causing mitochondrial dysfunction, ER stress, and defective insulin secretion.

Defective autophagy as a therapeutic target in lipotoxicity

As described above, several mechanisms have been implicated in lipotoxicity, such as ER stress, mitochondrial dysfunction, and oxidative stress. Recently, defective autophagy has emerged as a focus for a new pathogenic mechanism40. Consequently, how can excess fatty acids regulate autophagic activity? Could inhibition of this mechanism be a novel therapeutic strategy to protect against lipotoxicity? Images of human islets treated with nonessential fatty acids showed an accumulation of autophagic vacuoles, increased size and number of autophagosomes, and increased β-cell death41. The accumulation of autophagic vesicles was also observed in pancreatic β-cells in a mouse model of tacrolimus-induced diabetes, a side effect of the immunosuppressant drug42. An increased number of autophagosomes can result from either stimulated autophagosome formation or slowed degradation. Stimulation of autophagosome formation could be due to an increase in autophagic flux by free fatty acids (FFAs)43–45; alternatively, FFAs could inhibit autophagic turnover, consequently leading to aggregation of autophagosomes in β-cells46,47. These conflicting interpretations of the impact of FFAs on autophagy may be explained by varying time points chosen to examine autophagic activities and/or the use of different inhibitors to follow autophagy.

Autophagy was reported to be suppressed by lipotoxic conditions due to AMP-activated protein kinase (AMPK) inhibition following mTORC1 activation in different cell types48–50. Consistent with these reports, a marked downregulation of autophagy-related proteins (Atg5 and Ag7) was observed in obese mice, contributing to autophagy suppression51. Furthermore, a high-fat diet challenge in mice resulted in compromised autophagic activity associated with impaired lysosomal acidification that contributes to lipotoxicity in the kidney52. Likewise, lipotoxicity-induced inhibition of autophagy in β-cells was normalized by lysosomal acidification, which also restored mitochondrial function53. Moreover, dyshomeostasis of intracellular Ca2+ has been proposed to inhibit autophagosome-lysosome fusion54. Thus, the application of verapamil, a Ca2+ channel blocker, restored autophagic flux in the liver and attenuated inflammation and insulin resistance in obese mice54. However, the role of voltage-gated Ca2+ entry in the intracellular Ca2+ homeostasis of hepatocytes, as nonexcitable cells, identifies a Ca2+ channel-independent action of verapamil. Of note, verapamil has been shown to inhibit thioredoxin-interacting protein (TXNIP) and the NOD-like receptor pyrin domain containing-3 (NLRP3) inflammasome in a manner independent of Ca2+ channels55. Moreover, in the clonal β-cell lines INS-1 or MIN6, verapamil stimulates autophagy55,56.

The relationship between lipotoxicity and autophagy is intricate, and the pathophysiological mechanism involves multiple factors (Fig. 2). Understanding the contribution of each factor involved in regulating autophagy is critical for discovering therapeutic treatments for lipotoxicity-related disorders. Notably, dysfunctional autophagy caused by the downregulation of key regulators of the process could be reversed by antidiabetic drugs known to modulate autophagy41,42,57,58. Metformin, the most commonly prescribed antidiabetic agent, is known to prevent lipotoxic β-cell apoptosis and restore glucose-stimulated insulin secretion59. Metformin enhanced the removal of aggregated autophagic vacuoles in β-cells and AMPK-dependent protection from lipotoxicity57. The latter study mainly used 2 mM, a suprapharmacological concentration of metformin, while a 100-fold lower dose was sufficient to restore glucose-stimulated insulin secretion in islets from type 2 diabetic organ donors. The acute restoration of insulin secretion by metformin is caused by inhibition of voltage-dependent anion channel-1 (VDAC1), which is mistargeted to the β-cell plasma membrane in diabetes60. Insulin sensitizers, such as thiazolidinediones, have been reported to stimulate autophagy associated with activation of AMPK and suppression of mTORC161.

Exendin-4, a glucagon-like peptide-1 (GLP-1) analog, also protects against β-cell lipotoxicity via a number of mechanisms, including induction of the ER chaperone and antiapoptotic BiP/GRP78 protein62, inhibition of proapoptotic stress kinases63, and restoration of lysosomal function and autophagic flux42,64. Exendin-4 prevented the excessive accumulation of autophagosomes and restored autophagic clearance in defective β-cells42. GLP-1 agonists as well as the GLP-1-degrading dipeptidyl peptidase-4 inhibitor also increase LC3 II and autophagosome formation, restoring insulin secretion in beta-cells from high-fat diet obese or diabetic mice65–67. However, there was no effect of exendin-4 on autophagy in the absence of glucolipotoxicity67.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a newly introduced class of antidiabetic drugs that reduce circulating glucose levels through the induction of glycosuria. SGLT2 inhibitors were reported to activate autophagy and preserve normal morphology and function in renal cells or tissues from diabetic mice68,69. In a recent study, an SGLT2 inhibitor or knockdown of the SGLT2 transporter restored autophagic levels via AMPK activation and mTOR inhibition in a human proximal tubular cell line cultured in high-glucose medium70. The direct molecular mechanisms explaining the beneficial effects of SGLT2 inhibitors in isolated cells are not fully understood but are consistent with the concept that SGLT2 acts as a sensor of excess nutrients. Inhibition of this molecule causes intracellular signaling linked to nutrient deprivation and AMPK-mediated autophagy activation even in cell types or tissues that do not express SGLT271. In addition to currently known antidiabetic drugs, other agents that positively modulate autophagy, such as the antiarrhythmic drug amiodarone, were shown to recover β-cell function in islet amyloid polypeptide-expressing insulinoma and human islet cells72,73. These results suggest that modulating autophagy is a promising strategy to counteract beta-cell loss and the development of diabetes.

Lysosomal calcium regulation and autophagy

Regulation of lysosomal Ca2+ signaling

Lysosomes are a group of membrane-enclosed organelles containing the bulk of digestive enzymes found in most eukaryotic cells74. As lysosomes have more than fifty acid hydrolases, the primary role of lysosomes has been regarded as a degradation system for damaged organelles and cellular macromolecules. Recently, lysosomes have been shown to have many additional functions to maintain cellular homeostasis by engaging with other compartments75. Lysosomal signal sensing and degradation have been proposed to cooperate to control fundamental physiological processes76. In response to different environmental cues, the lysosomal signaling network functions to induce adaptive responses as well as secure proper cellular demand for degradation. Moreover, the degradative process provides catabolites that act as a nutrient-sensing signal to turn on adaptive responses. Given this important association, it is not surprising that any disturbance of lysosomal degradation and signal sensing leads to the development of pathological conditions, such as lysosomal storage disorders or neurodegenerative diseases.

Lysosomes carry diverse lysosomal membrane proteins which function to transport metabolites, enzymes, and ions across the membrane. The transporting activities generate luminal acidification and are directly involved in regulating proper lysosomal functions. Particularly, by harboring the H+ ATPase pump, lysosomes have a unique strongly acidic luminal pH (4.5–5.0) that favors the activity of degradation enzymes. These organelles also contain a wide range of other ion channels that transport H+, Na+, K+, Ca2+, and Cl− driven by the electrochemical gradient across the lysosomal membrane77. In the past, it was reported that the function of lysosomes was mostly dependent on the activity of proton pumps and luminal acidification, and most studies have focused on pathological changes in lysosomal pH78. Currently, this view has changed to include other ion channels with advances in lysosomal patch-clamp technique79,80.

Ca2+ signaling is critical for lysosomal functions such as lysosomal mobility, degradation, and connection at membrane contact sites. Lysosomes are considered an intracellular Ca2+ store, along with the ER, having a high luminal Ca2+ concentration of ~ 500 μM, more than 5000-fold higher than cytosolic Ca2+7. Ca2+ efflux is conducted via at least three types of lysosomal Ca2+ channels: transient receptor potential cation channels of the mucolipin family (TRPML1-3), two-pore channels (TPC1-2), and trimeric Ca2+ two transmembrane channels (P2X4) (Fig. 3). The uptake of Ca2+ into lysosomes is not yet well elucidated. Several studies support the idea that a Ca2+/H+ exchanger exists due to a notable reduction in lysosomal Ca2+ when lysosomal pH is disturbed by a V-ATPase inhibitor or alkalizing reagents such as NH4Cl7,81–83. However, the ER is proposed to serve as the main source to refill lysosomal Ca2+ via IP3R8. However, the route and mechanism by which Ca2+ is transported into lysosomes remain unclear. A putative Ca2+ channel that resides on the contact site between the ER and lysosomes has been implicated.Fig. 3 Regulation of lysosomal Ca2+ homeostasis.

The lysosome contains different classes of Ca2+ channels, including transient receptor potential cation channels of the mucolipin family (TRPML1-3), two-pore channels (TPC1-2), and ionotropic purinergic receptors (P2X4). These channels induce Ca2+ release into perilysosomal domains driven by high Ca2+ gradients, which possibly develop by Ca2+ transfer from the ER or transporters' action related to the acidic luminal pH of the lysosome.

In the history of lysosomal Ca2+ studies, nicotinic acid adenine dinucleotide phosphate (NAADP) was the first potent reagent discovered to strongly induce Ca2+ release from lysosomes. Initially, NAADP administration provoked a cytosolic Ca2+ rise originating from a Ca2+ store that is insensitive to IP3 and cyclic ADP-ribose84. Following this, the source of Ca2+ release induced by NAADP was shown to originate from the endolysosomal system85–87. With a better understanding of the important role of lysosomal Ca2+ signaling, more factors have been found to regulate lysosomal Ca2+, including pH, nutrients, stressful conditions, or small molecules such as ATP, phospholipids and sphingosine7,88–91. Each stimulus modulates lysosomal Ca2+ via different mechanisms to trigger selective Ca2+ signaling responses that favor the needs of the cell.

In addition, lysosomal signaling was shown to be regulated via contact sites with other membrane-bound organelles, such as the ER, Golgi, mitochondria, and peroxisomes. Many important functional events occur at lysosome-organelle contact sites, including lipid transfer, lysosomal positioning and trafficking. Regarding the regulation of lysosomal Ca2+, there might be Ca2+-mediated functional coupling at the microdomain between the ER and lysosomes. Indeed, NAADP triggers Ca2+ release via TPCs, which requires intact function of IP3Rs and RyRs on the ER. This observation suggested a trigger hypothesis by NAADP in which the Ca2+ mobilized from lysosomes can initiate a global Ca2+ increase via Ca2+-induced Ca2+ release from the ER87,92,93. The triggering function of Ca2+ released from lysosomes is nicely illustrated in adrenaline-induced glucagon secretion from pancreatic α-cells, which is attenuated in TPC-2 channel KO mice94, while such channel deletion does not alter insulin secretion95. In addition, a membrane contact site between mitochondria and lysosomes was identified to facilitate the direct transfer of Ca2+ from lysosomes to mitochondria96. Lysosomal TRPML1-mediated Ca2+ efflux was shown to transfer Ca2+ into mitochondria via VDAC1 and MCU. This new discovery contributes an additional mechanism for regulating Ca2+ dynamics, which might be implicated in pathological diseases, including neurodegenerative and lysosomal storage disorders96–98.

Autophagy regulation by lysosomal Ca2+

Lysosomal Ca2+ is one of the essential factors needed for various physiological processes, including endocytic membrane trafficking, autophagy, membrane repair, formation of ER-lysosomal contact sites, and protein transport75. Most studies have revealed the important role of TRPMLs, and some have highlighted TPCs and P2X4 in autophagy and lysosomal system regulation. TRPMLs belong to a large family of transient receptor potential ion channels containing three isoforms (TRPML1, 2, and 3). TRPML1 mainly localizes in lysosomes, while TRPML2 and TRPML3 are found to reside on early endosomes, late endosomes and lysosomes99.

TRPML1 is the best-studied channel regarding lysosomal adaptation and autophagy regulation. The first report about the role of TRPML1 in pathophysiology characterized a disorder affecting the lysosomal pathway, so-called mucolipidosis type IV (MLIV)100. The TRPML1-mutated cells clearly show defective autophagic processes with impaired lysosomal pH, enlargement of lysosomes and autophagosomes along with accumulation of undigested materials. The pathogenic manifestations of TRPML1 mutation include the increased formation of new autophagosomes and a delay in autophagosome-lysosome fusion101. Imaging studies revealed substantial aggregation of cytoplasmic bodies in the cerebral cortex of TRPML1 knockout mice. As a result of suppressed autophagic degradation, the levels of LC3-II and p62 were markedly increased, suggesting that macroautophagy is defective in mucolipin-1-deficient neurons. This observation, together with the characteristics of MLIV fibroblasts, contributes new insight into the neuronal pathogenesis of this disease102.

In fasting conditions, as a molecular mechanism of autophagic regulation, lysosomal Ca2+ release via TRPML1 was enhanced, generating a high Ca2+ microdomain surrounding lysosomes103. The increased Ca2+ level in the perilysosomal microdomain activates the calcium-dependent serine/threonine phosphatase calcineurin and dephosphorylates transcription factor EB (TFEB), enabling the translocation of TFEB into the nucleus, where it activates the transcription of lysosomal biogenesis and autophagy-related genes. Another study proposed TRPML1-dependent autophagy activation by reactive oxygen species (ROS)88. In response to ROS generation, TRPML1 is directly activated and releases Ca2+, followed by calcineurin stimulation and TFEB translocation into the nucleus. Genetic or pharmacological intervention to suppress TRPML1 prevents the removal of damaged and ROS-generating mitochondria. Thus, TRPML1 is a positive regulator of autophagy required for autophagosome-lysosome fusion and transcriptional upregulation of autophagy-related genes.

TRPML2 and TRPML3 have received less attention regarding autophagy regulation. Nevertheless, TRPML3 may play an important function as a regulator of membrane trafficking and autophagy104. Overexpression of TRPML3 enhances autophagy, and knockdown or loss-of-function mutation of TRPML3 was shown to inhibit autophagy. TRPML3 was reported to specifically bind to GATE16, a mammalian Atg8 homolog, to facilitate autophagosome maturation by providing Ca2+ during the membrane fusion process105. Each TRPML channel plays a distinct role in autophagy, as noted earlier. Furthermore, the role of TRPML heteromultimerization was proven to regulate starvation-induced autophagy and cell viability, indicating that hetero-TRPMLs, not a distinct type, might be more important in the context of regulating autophagy106.

In addition to TRPMLs, another lysosomal Ca2+ flux regulator of autophagy is the TPC. Mammalian cells contain two forms of TPCs: TPC1 resides on endosomes and lysosomes, and TPC2 localizes to lysosomes83,107. Due to the specific localization of TPCs on the lysosomal membrane and their regulation of Ca2+ efflux, TPCs were predicted to be involved in autophagy regulation. However, it remains controversial whether TPCs act as enhancers or inhibitors of autophagy. Several studies have proposed that Ca2+ signaling from TPCs is needed for the activation of autophagy. NAADP, a well-known agonist of TPCs, triggers Ca2+ release and regulates the autophagic process in astrocytes by increasing lysosome formation and two autophagic markers, LC3-II and Beclin1108. The Ca2+ signal evoked by NAADP is linked to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and AMPK to promote autophagosome formation109. This Ca2+-dependent signaling also influences autophagic degradation at the lysosomal level. The link between TPCs and autophagy was postulated clearly in TPC knockout mice110. In the absence of TPC activity, autophagic flux was decreased in cardiomyocytes upon starvation, suggesting the critical role of TPCs in appropriate basal and induced autophagic flux in cardiac tissues110,111.

Conversely, TPCs have been considered a negative regulator of autophagy via effects on lysosomal pH112. Lu et al. reported that TPC2 overexpression or activation of TPC2 by NAADP inhibited autophagosome-lysosome fusion. However, knockdown of TPC2 or application of a TPC2 antagonist (Ned-19) reduced TPC2-dependent autophagosome accumulation. The molecular mechanism demonstrated that TPC2/NAADP/Ca2+ signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagic progression. In another knockout experiment, TPC2−/− mice exhibited an atrophic phenotype with enhanced autophagic flux under starvation, which was different from the study mentioned above112. This discrepancy was thought to result from different strategies used in generating the TPC2 knockout mice. As a lysosomal Ca2+ channel, P2X4R (purinergic receptor P2X4) participates in the fusion step of autophagy by regulating Ca2+ release. A complex formed by the interaction between P2X4 and calmodulin (CaM) at the endolysosomal membrane promotes fusion and vacuolation in a Ca2+-dependent fashion113.

Role of mTOR in autophagy regulation

Regulation of mTOR signaling at the lysosome

The mTOR complex, a master regulator of cell growth, has two major targets of rapamycin: mTOR1 and mTOR2, which not only have distinct roles but also regulate each other to maintain growth and proliferation114. mTORC1 functions as a main mediator of protein synthesis and cell growth, whereas mTORC2 is involved in regulating cell proliferation in response to growth factors as well as metabolism. A canonical signaling cascade to activate mTORC1 requires both active Rags and GTP-bound Ras homolog enriched in brain (Rheb) residing on the lysosomal surface. Rag-GTPases, members of the Ras family of GTP-binding proteins, regulate mTORC1 in an amino acid-dependent manner (Fig. 4). In response to increased amino acid abundance, Rag heterodimers interact with the Raptor component of mTORC1, which induces the redistribution of mTORC1 to Rab7-containing lysosomes115. The movement of mTORC1 to the lysosomal surface is driven by the interaction between the trimeric Ragulator protein complex of mTORC1 and Rag GTPases116. After translocating to the lysosomal surface, mTORC1 interacts with its kinase activator, Rheb GTPase, to enhance the binding of 4E-BP1 to mTORC1. Under conditions of limited amino acids, Rap1-GTPases concentrate lysosomes in the perinuclear area and reduce the available lysosomal surface for mTORC1 activation117. The absence of Rap1 expands the lysosome population, increasing the association between mTORC1 and activators on lysosomes, resulting in mTORC1 activation.Fig. 4 Role of mTOR signaling in autophagy regulation at lysosomes.

During activation of mTOR signaling, Rheb and Ragulator induce the localization of mTOR on the lysosomal surface. Autophagic processes mediated by Unc-51-Like Kinase 1 (ULK1) and the phosphatidylinositol 3-kinase (PI3K) complex are negatively regulated by mTORC1 signaling on the lysosome. Perilysosomal Ca2+ release via the TRPML1 channel is critically involved in mTOR activation by promoting interaction with calmodulin (CaM), which in turn causes negative feedback regulation by suppressing TRPML1 activity. FIP200 FAK family kinase-interacting protein of 200 kDa, Rheb Ras homolog enriched in brain, TFEB transcription factor EB, TSC Tuberous sclerosis complex, TRAF6 TNF receptor-associated factor 6.

In addition to canonical pathways regulating mTORC1, different extracellular and intracellular signal inputs have been reported to control mTORC1 activity. Notably, the intracellular second messenger Ca2+ has been shown to activate the p70 ribosomal S6 kinase, which is situated directly downstream of mTORC1, as shown in rat liver epithelial cell lines118–120. The activating mechanism of mTORC1 by amino acids has also been demonstrated to be Ca2+-dependent121. Amino acids increase cytosolic Ca2+ binding to CaM, in turn activating hVPS34 and mTORC1 signaling. The mTOR signal was diminished by intracellular Ca2+ chelators, a CaM antagonist, or knockdown of CaM, suggesting that intracellular Ca2+ and CaM are involved in amino acid-induced mTORC1 activation122. Another mechanism has been suggested for amino acid activation of mTORC1 through regulation of the tuberous sclerosis complex 2 (TSC2)-Rheb axis by Ca2+/CaM123. These interesting findings prompted a surge of questions as to which intracellular Ca2+ source directly plays the critical role in mediating mTORC1 activation. Several studies have reported that lysosomal Ca2+ dyshomeostasis observed in lysosomal storage diseases might be responsible for the inhibition of the mTORC1 signaling pathway. Indeed, in flies lacking TRPML1 function, mTORC1 signaling was attenuated. Reactivation of mTORC1 by a high-protein diet reduced the severity of the mutant TRPML1 phenotype, indicating the interrelationship between the TRPML and TORC1 pathways124,125. Consistent with these findings, TRPML1-mediated lysosomal Ca2+ release activates mTORC1 by promoting interaction with CaM122. Furthermore, negative feedback regulation of mTORC1 activity on TRPML1 through a CaM-dependent mechanism has been shown to be important for maintaining cellular homeostasis during starvation126.

mTOR activation in β-cell lipotoxicity

With a full nutrient supply, mTORC1 is rapidly activated to promote biosynthesis, which generates new cellular materials such as proteins, lipids, and nucleic acids. In contrast, during starvation, an adaptive mechanism is turned on to suppress mTORC1 activity to conserve the limited cellular energy and enhance the production of recycled materials via the degradation pathway. Depending on the cell type, mTORC1 plays a tissue-specific role in contributing to the maintenance of energy homeostasis127–129. In pancreatic β-cells, mTORC1 has been implicated in cell proliferation by modulating critical factors of the cell cycle, including cyclin D2, cyclin D3 and Cdk4, in response to growth factors, insulin and nutrients130. In addition, the importance of mTORC1 activity in β-cells has been documented by the integration of several growth signaling pathways, such as protein kinase B (AKT), protein kinase C, Hippo, epidermal growth factor receptor, and synapses of amphids defective protein kinase A130–134. In a genetic approach, gain or loss of mTORC1 function provided important insights into the role of mTORC1 in β-cell physiology. Knockout of Raptor, a core component of mTORC1, in mouse β-cells resulted in a diabetic phenotype, impaired glucose-stimulated insulin secretion (GSIS) and decreased β-cell viability and proliferation. The underlying mechanisms revealed that the 4E-BPs/eIF4E arm of mTORC1 regulates β-cell proliferation, while S6K controls cell size, autophagy and apoptosis. In addition, the mTORC1/4EBP2/eIF4E pathway is implicated in insulin processing via cap-dependent translation of carboxypeptidase E135. Hyperactivation of mTORC1 by overexpressing Rheb resulted in an increase in β-cell mass and insulin secretion136. Furthermore, inhibition of mTORC1 by rapamycin has been shown to trigger the onset of diabetes137. Intriguingly, another study showed biphasic effects of mTORC1 overactivation by the deletion of TSC2: β-cell mass as well as an increase in insulin secretion were increased in young (up to 30 weeks), while hyperglycemia developed along with insulin resistance due to β-cell exhaustion after 40 weeks of age138. This result indicates that prolonged and continuous upregulation of mTORC1 exerts deleterious effects and causes β-cell pathology in contrast to its physiologic roles. Upon chronic exposure to excess nutrients, mTORC1 in β-cells is consistently activated, which is accompanied by an increase in β-cell death. Notably, relative to that of the controls, mTORC1 was upregulated in islets from organ donors with type 2 diabetes, while mTORC2 was downregulated139. Consistently, genetic and pharmacological suppression of mTORC1-S6K1 signaling recovered insulin secretion in diabetic patient islets. The same results were observed in islets exposed to glucotoxic conditions and in islets from diabetic mice.

How does the sustained activation of mTORC1 cause β-cell dysfunction? Constitutively active mTORC1 has a negative impact on downstream signaling, including mTORC1-S6K1-IRS and mTORC2, as well as other intracellular processes, such as ER homeostasis and autophagy140. There are negative feedback loops between mTORC1-S6K1 and the mTORC2-AKT axis that prevent excessive insulin downstream signaling cascades. Hyperactivation of mTORC1 can downregulate RTK-IRS1/2-PI3K-AKT signaling. Another suggested mechanism is the suppression of autophagy by mTORC1. Early observations using electron microscopy showed the abnormal accumulation of autophagosomes in β-cells after exposure to high fatty acid and glucose or in islets of T2D patients41,141. Further studies reported that autophagic flux was blocked by a high-fat diet or under excessive nutrient conditions141,142. In this context, inhibition of mTORC1 was shown to improve autophagic activity and β-cell survival against overnutrition stress. One of the molecular mechanisms was demonstrated to be mTORC1-mediated Unc-51-Like Kinase 1 (ULK1) phosphorylation at Ser757, which prevented autophagy initiation143. Conversely, AMPK promotes autophagy by directly activating ULK1 through phosphorylation of Ser317 and Ser777. Along with autophagic suppression under overnutrition stress, prolonged mTORC1 upregulation induces ER stress and apoptosis in human and rodent islets and clonal β-cells141,144,145. Accordingly, hyperactivation of mTORC1 in TSC2-KO mice resulted in strong induction of UPR markers, including PERK, p-eIF2α, ATF4 and CHOP.

Autophagy regulation by mTORC1

Given the important role of autophagy, the multiple signaling modalities involved in the regulation of the process have been divided into two main categories: mTOR-dependent and mTOR-independent pathways146. The first study on autophagy regulation by mTOR reported the control mechanism of Tor (a homolog of mTOR) for autophagy induction in yeast, which blocks a factor required for autophagy initiation147. Several groups have proposed that mTORC1 is regulated by phosphorylating the autophagy regulatory complex formed by ULK1-Atg13-FIP200, which subsequently inhibits autophagy initiation148–150. In addition, AMBRA1, interacting with the E3 ligase TNF receptor-associated factor 6 (TRAF6), leads to Lys63 ubiquitylation and stabilization of ULK1, which enhances its kinase activity and autophagy induction (Fig. 4). However, under nonautophagic conditions, mTORC1 inhibits AMBRA1 by phosphorylation and subsequently inhibits autophagy151. Another mechanism has been suggested according to which mTORC1 specifically inhibits the ATG14-containing autophagic class III phosphatidylinositol 3-kinase (PI3K) complex, which is involved in autophagy induction through phosphorylation of ATG14 at multiple sites152. mTORC1 has also been implicated in autophagy inhibition by the phosphorylation of NRBF2/Atg38, which has been identified as the fifth subunit of the autophagic class III phosphatidylinositol 3-kinase complex153.

Recently, mTORC1 has been shown to not only participate in the early step of autophagy but also extensively function at later stages, including autophagy elongation and maturation (summary in Table 1). The elongation step is one of three major steps in autophagy that results in complete autophagosome formation. mTORC1 has been shown to be involved in this step via the phosphorylation of WIPI2 at Ser395, a critical protein in the growth of the isolation membrane and elongation, promoting the interaction between WIPI2 and the E3 ubiquitin ligase HUWE1 for ubiquitination and proteasomal degradation154. Additionally, mTORC1 was shown to inhibit autophagy via p300 phosphorylation, which reduces the acetylation of several autophagy-related proteins such as LC3, Atg5, and Atg7155. Regarding the late stage of autophagy, mTOR regulates factors participating in autophagosome-lysosome fusion, including UV radiation resistance-associated gene (UVRAG) and proteins associated with UVRAG as an autophagy enhancer (Pacer)156,157. mTORC1 interacts with and phosphorylates UVRAG, which prevents the interaction with the HOPS complex, a component of the late endosome/lysosome fusion machinery, enhancing autophagosome and endosome maturation156. In response to nutrients, mTORC1 phosphorylates Pacer at serine 157 to disrupt its association with Stx17 and the HOPS complex, thus preventing Pacer-mediated autophagosome maturation157. The function of mTORC1 in autophagy is not only via post-translational modification but also through the transcription factors TFEB, TFE3 and microphthalmia-associated transcription factor (MITF) to regulate lysosome biogenesis and autophagy. TFEB and TFE3 were identified as transcription factors controlling genes involved in autophagosome formation, fusion of autophagosomes with lysosomes, and lysosomal biogenesis158,159. The activity of mTORC1 negatively regulates autophagy by phosphorylating different serine residues in TFEB/TFE3 (Ser211, Ser122, Ser142 on TFEB, and Ser321 on TFE3), which promotes the binding between TFEB/TFE3 and the 14-3-3 family of proteins, resulting in their retention in the cytosol160–163. Therefore, the two master regulators can no longer turn on lysosomal gene expression, which has negative impacts on autophagy. Another member of the MiT/TFE family, MITF, was able to induce autophagy via upregulation of microRNA 211 (miR211)164. Inhibition of mTORC1 by the Torin-1 compound induces nuclear translocation of MITF and triggers the expression of genes involved in autophagy.Table 1 mTOR-mediated regulation of autophagic processes.

Signaling	Phosphorylation action of mTOR	Reference	
Autophagy induction	ULK-ATG13L-FIP200 (ATG1-ATG13-ATG17 in yeast)	- ATG13L (Ser258) and ULK1 (Ser637): Suppresses ULK1 kinase activity	148–150	
- ULK1 (Ser757): Disrupts the interaction between ULK1 and AMPK	143,204	
PIK3C3/VPS34 complexes	- ATG14 (Ser3, Ser223, Thr233, Ser383, Ser440): Inhibits PIK3C3 kinase activity of ATG14-containing PIK3C3	152	
AMBRA1	- AMBRA1 (Ser52): Inhibits the interaction with E3-ligase TRAF6, which stabilizes ULK1 self-association	151	
NRBF2	- NRBF2 (Ser113, Ser120): Blocks Ptdlns3K lipid kinase activity and autophagic process.	153	
Autophagy elongation	WIPI2	- WIPI2 (Ser395): Prevents autophagosome formation by directing WIPI2 to ubiquitination	154	
p300	- P300 (Ser2271, Ser2279, Ser2291, Ser2375): Disrupts intramolecular inhibition of p300, which in turn decreases acetylation of LC3 and loss of the LC3-Atg7 interaction	155	
Autophagy Maturation	UVRAG	- UVRAG (Ser498): Positively regulates the association of UVRAG with RUBICON, thereby inhibiting UVRAG-mediated autophagosome maturation	156	
- UVRAG (Ser550, Ser571): Activates UVRAG/VPS34 leading to decreased lysosomal tubulation upon prolonged starvation	205	
Pacer	- Pacer (Ser157): Disrupts the association of Pacer with Stx17 and HOPS complex, which inhibits autophagosome maturation	157	
TFEB/TFE3	- Phosphorylates TFEB (Ser211, Ser122, Ser142) and TFE3 (Ser321) to promote the binding of TFEB and 14-3-3 protein and retention in the cytosol.	160–162,206,207	
- Phosphorylates TFEB (Ser138 and Ser142) to facilitate the nuclear export of TFEB	163,208	
MITF	- mTORC1 inhibition induces MITF translocation, which enhances lysosomal biogenesis	164	

By functioning at different autophagic steps, mTORC1 is recognized as the master regulator of autophagy. Under nutrient-rich conditions, mTORC1 is known to inhibit the process at various steps of autophagy. Intriguingly, autophagy and mTORC1 have been shown to reciprocally regulate each other. During starvation, mTORC1 is inhibited, which is needed for the induction of autophagy. However, prolonged starvation leads to the reactivation of mTORC1 to restore the lysosomal system to maintain homeostasis165. Thus, there is an evolutionary cycle between autophagy and the master regulator mTORC1 to maintain the balance of the intracellular system, so any factor disrupting the regulatory cycle would lead to pathologic conditions and eventually the development of diseases.

Role of AMPK in autophagy regulation

Activation of AMPK signaling

AMPK, similar to mTOR signaling, is also an evolutionarily conserved key sensor and master regulator of metabolism. Under physiological or pathological conditions such as exercise, starvation, hypoxia, and shock, AMPK is activated through phosphorylation of upstream kinases related to a high AMP/ATP ratio or Ca2+ signaling. Activation of AMPK promotes catabolic processes such as glycolysis, fatty acid oxidation, and autophagic degradation and suppresses anabolic processes including synthesis of proteins, fatty acids, or cholesterol166. Mammalian AMPK is a heterotrimeric complex composed of three subunits: an α subunit harboring a protein kinase catalytic domain and noncatalytic β and γ regulatory subunits (Fig. 5). A serine/threonine kinase domain exists in the amino-terminal region of AMPKα, which contains the activation loop, playing a pivotal role in its regulation. In particular, phosphorylation of Thr172 in the activation loop is needed for maximal activities of AMPK167.Fig. 5 Upstream and downstream regulators of AMPK signaling related to autophagy and metabolism.

Lowered ATP, increased Ca2+, TNF-related apoptosis-inducing ligand (TRAIL), and DNA damage can trigger AMPK activation mediated by liver kinase B1 (LKB1), calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKKII), transforming growth factor-β activating kinase (TAK1), and sestrins, respectively. AMPK promotes autophagy by directly activating ULK1 and beclin1 as well as suppressing mTOR activity. AMPK increases mitochondrial biogenesis with activation of proliferator-activated receptor-γ coactivator 1α (PGC1α). AMPK decreases lipogenesis and cholesterol synthesis by inhibiting acetyl-CoA carboxylase (ACC) and HMG-CoA reductase (HMGR). Conversely, AMPK activates adipose triglyceride lipase (ATGL) for lipolysis and facilitates mitochondrial fatty acid uptake for β-oxidation by reducing an allosteric inhibitor, malonyl-CoA. MLCK Myosin light-chain kinase.

Three upstream kinases have been known to phosphorylate Thr172 in AMPK: liver kinase B1 (LKB1), calcium/CaM-dependent protein kinase kinase 2 (CaMKK2 or CaMKKβ), and transforming growth factor-β activating kinase (TAK1)168–170. First, the tumor suppressor LKB1 is a serine/threonine kinase functioning as a heterotrimer with two other subunits, the STE20-related adapter protein (STRAD) and the scaffolding mouse protein 25 (MO25)171. Genetic deletion of Lkb1 abrogates the activation of AMPK by agonists such as aminoimidazole-4-carboxamide ribonucleoside (AICAR), metformin or phenformin or in response to energy stress, revealing that LKB1 is responsible for the majority of AMPK activation172. Increases in AMP or ADP activate AMPK mainly by promoting the phosphorylation of LKB1 but also by inhibiting dephosphorylation by protein phosphatases or directly activating AMPK173.

Second, activation of CaMKK2 by intracellular Ca2+ phosphorylates Thr172 in AMPK, thus linking Ca2+ signaling to the regulation of energy metabolism169. Upstream signals for Ca2+ elevation, such as adiponectin receptor activation or muscle contraction (exercise), can increase Ca2+-bound CaM and activate CaMKK2 and AMPK174. As previously mentioned, the ER maintains cellular Ca2+ homeostasis and generates signals by releasing Ca2+. A relationship between AMPK and the ER-localized protein stromal interaction molecule 2 (STIM2) has been demonstrated175. The interaction among STIM2, CaMKK2, and AMPK could be promoted by the release of ER Ca2+ and store-operated Ca2+ entry.

TAK1 is a serine/threonine protein kinase of the mitogen-activated protein kinase (MAPKK) family, proposed as an alternative third AMPK kinase. TAK1 is induced in response to TNF-related apoptosis-inducing ligand (TRAIL), resulting in a cytoprotective autophagic response mediated by AMPK176. Stress-inducible proteins, sestrins, were identified to mediate DNA damage-induced AMPK activation. Sestrin1/2 induces AMPK phosphorylation at T172 and enhances AMPK-mediated mTOR suppression177.

The lysosome is known as a center of amino acid sensing and regulation of mTOR signaling178. Recently, the lysosome has emerged as a regulatory and functional site for AMPK, promoting the idea of lysosomes as hubs of cellular metabolic regulation179. Nutrient deprivation, such as glucose deprivation, which is not associated with gross changes in cellular ATP levels, promotes the formation of an LKB1-AMPK complex at the lysosomal surface180. AMPK not only localizes to the endolysosomal compartment but also mediates lysosomal biogenesis by regulating the nuclear translocation of TFEB/TFE3181. The target genes for TFEB/TFE3 carry a common genetic motif, coordinated lysosomal expression and regulation (CLEAR), which induces the expression of a network of lysosomal hydrolases, lysosomal membrane proteins, and autophagy-related proteins in response to pathways sensing lysosomal stress158. TEFB/TFE3 also induces TRPML1, which can regulate lysosomal membrane fusion with the plasma membrane, eliciting lysosomal exocytosis or autophagosome formation of autophagolysosomes and autophagic degradation. In addition to transcriptional upregulation of autophagy-related proteins, AMPK activation is involved in the initiation, maturation, and processing of autophagy in the endolysosome system.

Regulatory actions of AMPK on metabolism and autophagy

Upon energetic crisis, AMPK reprograms metabolic activity by covalent modifications, transcriptional regulation, altered substrate utilization, and ultimate restoration of cellular and whole organismal homeostasis182. AMPK reduces lipid storage and promotes mitochondrial fatty acid oxidation. The downstream enzymes inhibited by AMPK include mTOR, acetyl-CoA carboxylase (ACC), HMG-CoA reductase (HMGR), and fatty acid synthase (Fig. 5). AMPK phosphorylates and inhibits ACC, the enzyme catalyzing the conversion of acetyl-CoA into malonyl-CoA, the first step in fatty acid synthesis. AMPK also inhibits HMGR, which is the rate-limiting enzyme of cholesterol synthesis182. The phosphorylation of SREBP1c and SREBP2 by AMPK inhibits their activities, but there are additional indirect mechanisms involving the regulation of SREBP-mediated lipid synthesis by AMPK. AMPK activates adipose triglyceride lipase (ATGL), catalyzing lipolysis and facilitating mitochondrial fatty acid uptake for β-oxidation by reducing an allosteric inhibitor, malonyl-CoA.

Accumulating evidence suggests that AMPK increases mitochondrial mass (biogenesis) by phosphorylation and activation of proliferator-activated receptor-γ coactivator 1α (PGC1α)183. TFEB activated by AMPK also directly binds to and activates the promoter of the gene encoding PGC1α184. The consequences of AMPK activation on mitochondria are not restricted to biogenesis but also include mitochondrial dynamics (fission) and mitophagy. Mitochondrial fission is mediated by dynamin-related protein 1 (DRP1), which is recruited to the outer mitochondrial membrane by mitochondrial fission factor (MFF). AMPK phosphorylates two serines on a core component of MFF and activates its action on mitochondrial fission185.

AMPK activates the autophagic process, including mitophagy, by two main mechanisms: i) direct activation of ULK1 and ii) inhibition of the mTOR complex. AMPK binds to and phosphorylates ULK1 on multiple residues: Ser317, Ser467, Ser555, Thr574, Ser637 and Ser777143,186. Activated ULK1 in turn phosphorylates class III PI3K complex I, composed of VPS34, ATG14L and Beclin1. This complex generates phosphatidylinositol-3-phosphate as a key signal for the formation of a mature phagophore, encapsulating cytosolic constituents and delivering them to the lysosome187. Cells expressing nonphosphorylatable ULK1 mutants accumulate defective mitochondria, supporting the importance of the AMPK-ULK1 axis for the selective removal of damaged mitochondria via mitophagy186.

To maintain energy and nutrient homeostasis, cells must balance anabolic and catabolic inputs. Antagonistic cross-inhibition exists between mTORC1 and AMPK signaling in energy metabolism and autophagic processes. For the inhibition of the mTORC1 complex, AMPK phosphorylates TSC2 at its Thr1127 and Ser1345 sites, which promotes the GTPase activity of the TSC1/TSC2 complexes. Rheb-GTP is transformed into an inactive Rheb-GDP state, and mTORC1 activity is turned off188. AMPK also directly phosphorylates the Ser772 and Ser792 sites of Raptor, increasing 14-3-3 protein binding to Raptor, hindering the binding of Raptor to mTOR or mTOR substrates, and subsequently resulting in inhibition of the mTOR signaling pathway189.

Autophagic regulation on the lysosomal membrane

Activation of mTOR and AMPK on the lysosomal membrane

As described above, the autophagosome is degraded through autophagosome-lysosome fusion and digestion by lysosomal hydrolases. This membrane fusion has been hypothesized to involve soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins due to their known actions in vesicle fusion190. The proposed molecular mechanism suggests that syntaxin 17 tethered onto the autophagosome membrane recruits synaptosomal-associated protein 29 (SNAP29) to stabilize this complex with ATG14 and vesicle-associated membrane protein 8 (VAMP8). VAMP8 is localized on endosomes and lysosomes and forms a syntaxin17-SNAP29-VAMP8 complex allowing autophagolysosomal fusion191. There is crosstalk between different signaling pathways, including mTORC1 and AMPK, in which the lysosome plays an important role in autophagic flux.

The localization of mTORC1 to Rab7-containing lysosomes is needed for its function, implying that the lysosome is the site of mTOR activation115. mTORC1 localization to the lysosome is dependent on Rag GTPases and the Ragulator complex composed of late endosomal/lysosomal adapter and MAPK and mTOR activator 1-5 (LAMTOR1-5)116. Rag GTPases are a Ras superfamily of small GTPases of large molecular weight lacking the post-translational modification needed for membrane localization. Instead, the Ragulator complex, which acts as a guanine nucleotide exchange factor (GEF) for RagA/B, is the anchoring site for Rag GTPases to lysosomal membrane192. The active conformation of Rag GTPases (RagA/B-GTP and RagC/D-GDP) with the Ragulator complex can recruit Raptor and mTORC1 to the lysosomal membrane. Activation of Rag GTPases by amino acids and glucose abundance determines lysosomal localization of mTORC1, which can be regulated by a range of GEF and GTPase-activating proteins. In addition, mTORC1 on the lysosome brings it in close proximity to its regulator, Rheb, residing on the lysosome. Thus, the lysosome provides a nutrient signaling hub that tightly controls mTORC1 activation.

AMPK is known to be present in the nucleus and cytoplasm but was reported to be a residential protein of the late endosome/lysosome193. AXIN, being a scaffold protein for AMPK and its upstream regulator LKB1, interacts with a lysosome-anchoring protein, LAMTOR1, leading to the formation of an LKB1-AMPK- AXIN-LAMTOR complex on the lysosomal membrane116. Starvation or the presence of AMP enhances LKB1-AMPK-AXIN binding to LAMTOR1 and accelerates AMPK activation on the lysosome (Fig. 6). Furthermore, phospho-AMPK was found exclusively on the lysosomal membrane, suggesting that LKB1 phosphorylates AMPK on this membrane surface. In addition, the energy sensor vATPase regulates lysosomal localization of the LKB1-AMPK-AXIN-LAMTOR1 complex not only for turning on catabolic processes under glucose-starved conditions but also for turning off anabolic metabolism through lysosomal association and dissociation of mTORC1193.Fig. 6 Activation of mTOR and AMPK on the lysosomal membrane.

The antagonistic signals for anabolism and catabolism, mTOR and AMPK, reside on the lysosome upon activating cues. Perilysosomal Ca2+ elevation can simultaneously activate both mTOR and AMPK signals mediated by the Ca2+-calmodulin (CaM) complex and calcium/CaM-dependent protein kinase kinase 2 (CaMKKII), respectively. LKB1 liver kinase B1, Rheb Ras homolog enriched in brain, TSC Tuberous sclerosis complex.

The critical role of the perilysosomal LKB1-AMPK-AXIN-LAMTOR1-vATPase axis in regulating mTORC1 and AMPK signaling was further demonstrated by its action on the metformin-induced beneficial effects on metabolism. Metformin promotes the formation of the AMPK-AXIN-LAMTOR1-vATPase complex and activates AMPK on the lysosomal membrane. In parallel, metformin treatment results in dissociation of mTORC1 from the Ragulator-vATPase complex and inhibits mTORC1 signaling194,195. Another cross-regulation of mTORC1 and AMPKα2 on the lysosome is mediated by sestrin2, which interacts with TSC1/2 and AMPK. Their binding inhibits Rheb-GTP loading by TSC2 phosphorylation, thus decreasing mTORC1 activity and stimulating AMPK activity177. Increased AMPK and suppressed mTORC1 on the lysosomal membrane can accelerate autophagic initiation, maturation, and degradation, leading to beneficial metabolic consequences.

Perilysosomal regulation of autophagy in stressed β-cells

In most cells, Ca2+ signaling has been implicated in cell fate decisions, including proliferation, differentiation, migration, and cell death. In addition, Ca2+ regulates the autophagic process from initiation to lysosome fusion and degradation. As described above, lysosomal ion channels and transporters participate in Ca2+ release into perilysosomal microdomains, which can trigger important regulatory signaling in autophagy: (1) CaM-dependent AMPK and mTOR activation and (2) calcineurin-mediated TFEB/TFE3 translocation to the nucleus.

Increases in cytosolic Ca2+ levels, particularly near the lysosomal membrane, result in efficient phosphorylation and activation of AMPK by CaMKK2. Ca2+-induced activation of AMPK triggers autophagy induction by ULK1/2 phosphorylation and mTORC1 inhibition. AMPK suppresses mTORC1 via phosphorylation of TSC2 and Raptor. At the same time, elevation of perilysosomal Ca2+ can also activate mTORC1, which is an autophagic suppressor signal (Fig. 6). Hence, activation of mTORC1 could be prevented by Ca2+ chelators or TRPML1 depletion122. Blocking the interaction between mTOR and CaM by a CaM antagonist prevents mTORC1 activation, confirming the Ca2+/CaM-dependent mechanism. Thus, perilysosomal Ca2+ elevation activates both AMPK and mTORC1 signals mediated by CaM and CaM-dependent kinases. However, it is unclear how the activities of AMPK and mTORC1 are finely regulated through antagonistic suppression by each other under the same perilysosomal Ca2+ control.

As previously mentioned, lysosomal Ca2+ release can induce the activation of calcineurin, triggering TFEB/TFE3 dephosphorylation and nuclear translocation. TFEB and TFE3, as transcription factors and master regulators for lysosomal proteins, strongly upregulate autophagy-related gene expression and lysosomal biogenesis196. This signal effectively removes dysfunctional and ROS-producing mitochondria via mitophagy, since ROS trigger TRPML1-mediated Ca2+ release from the lysosome88. This mechanism is critical for β-cell function because insulin secretion relies on the synthesis of ATP and other coupling factors from mitochondrial metabolism197,198.

Mitochondrial stressors such as rotenone or oligomycin/antimycin increase mitophagy as a compensatory and protective process for survival. This phenomenon could be, in part, mediated by nuclear translocation of TFEB, which is stimulated by calcineurin or AMPK but inhibited by mTORC1. All these signals can be activated by Ca2+ increase, while BAPTA-AM, a membrane-permeable Ca2+ chelator, abrogates mitochondrial stressor-triggered TFEB translocation and mitophagy activation in β-cells199. A calcineurin inhibitor suppresses TFEB translocation and mitophagy and aggravates mitochondrial dysfunction induced by mitochondrial stressors200. Inhibition of TRPML1 decreases lysosomal Ca2+ release and mitophagy by mitochondrial stressors. Scavenging of mitochondrial superoxide also prevents mitochondrial stressor-mediated Ca2+ elevation and mitophagy, demonstrating the role of ROS in lysosomal Ca2+ release via TRPML1 in pancreatic β-cells.

Future strategies related to perilysosomal calcium regulation against beta cell lipotoxicity

In the pathogenic progression of type 2 diabetes, the most important determining step could be the β-cell failure to compensate for the elevated insulin need. Notably, high glucose and fatty acids produce noxious oxidative stress and deleterious consequences leading to β-cell death. Accumulated evidence suggests that oxidative stress from persistent elevated fatty acids induces ER Ca2+ dysregulation closely connected to mitochondrial dysfunction and defective lysosomal degradative capacity. Lysosomal function is critical for autophagic flux, including mitophagy, which is an essential quality control mechanism required for cell survival against mitochondrial or metabolic stresses in β-cell lipotoxicity.

Lysosomal biogenesis and autophagic activity are stimulated by TFEB/TFE3, as demonstrated by genetic ablation of TFEB in β-cells, which causes marked aggravation of glucose intolerance and impaired insulin secretion by a high-fat diet199. Potentiation of perilysosomal Ca2+-mediated TFEB activation could be a promising therapeutic strategy to augment autophagy, counteracting organellar dysfunction due to lipotoxic stress in β-cells. It is noteworthy that the whole process of autophagy is oppositely regulated by AMPK and mTOR at the lysosomal membrane, both of which are key sensors for bioenergetic and nutritional status. Until now, the molecular mechanism governing the predominance of signaling between AMPK and mTOR has not been elucidated. Different environmental factors, including the amount and duration of cytosolic Ca2+ rise, crosstalk with other cell signals, or accompanying oxidative stress, can affect this predominance and autophagic outcomes. Interestingly, reducing cellular Ca2+ overload by Ca2+ channel blockers (CCB) increases autophagic activity and cell survival against lipotoxicity in HepG2 cells as well as β-cells54,201. Consistently, verapamil, as a frequently prescribed CCB for hypertension, promotes endogenous β-cell function and therapeutic effects in patients with recent-onset type 1 diabetes202. Recently, we demonstrated that the small molecule ER Ca2+ pump activator CDN1163 increases ER Ca2+ content, improves mitochondrial function, and protects against palmitate-induced lipotoxicity in pancreatic β-cells203. We propose that therapeutic strategies to activate ER Ca2+ pumps could correct perilysosomal Ca2+ overload and mitigate autophagic defects induced by sustained Ca2+ elevations. In addition, recovering mitochondrial function by promoting organellar Ca2+ uptake could be another effective strategy to improve autophagy and protect β-cells from lipotoxicity.

Acknowledgements

This work was supported by the Myung Sun Kim Memorial Foundation (2016), Yonsei University, Korea. We would like to thank Luong Dai Ly, Da Dat Ly, and Pedro Gabriel Coffler Zorzal for their valuable support of this manuscript.

Author contributions

Conceptualized the study: Ha Thu Nguyen, Claes B. Wollheim, and Kyu-Sang Park; Drafted the manuscript: Ha Thu Nguyen and Kyu-Sang Park; Edited and revised manuscript: Andreas Wiederkehr, Claes B. Wollheim, and Kyu-Sang Park.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Weir GC Butler PC Bonner-Weir S The β-cell glucose toxicity hypothesis: attractive but difficult to prove Metabolism 2021 124 154870 10.1016/j.metabol.2021.154870 34480921
2. Prentki M Peyot ML Masiello P Madiraju SRM Nutrient-induced metabolic stress, adaptation, detoxification, and toxicity in the pancreatic β-cell Diabetes 2020 69 279 290 10.2337/dbi19-0014 32079704
3. Guest PC Rhodes CJ Hutton JC Regulation of the biosynthesis of insulin-secretory-granule proteins. Co-ordinate translational control is exerted on some, but not all, granule matrix constituents Biochem. J. 1989 257 431 437 10.1042/bj2570431 2649074
4. Steiner DF Adventures with insulin in the islets of Langerhans J. Biol. Chem. 2011 286 17399 17421 10.1074/jbc.X111.244764 21454641
5. Liu, M. et al. Normal and defective pathways in biogenesis and maintenance of the insulin storage pool. J. Clin. Invest. 13110.1172/jci142240 (2021).
6. Lenzen S Chemistry and biology of reactive species with special reference to the antioxidative defence status in pancreatic β-cells Biochim. Biophys. Acta Gen. Subj. 2017 1861 1929 1942 10.1016/j.bbagen.2017.05.013 28527893
7. Christensen KA Myers JT Swanson JA pH-dependent regulation of lysosomal calcium in macrophages J. Cell Sci. 2002 115 599 607 10.1242/jcs.115.3.599 11861766
8. Garrity, A. G. et al. The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes. Elife 510.7554/eLife.15887 (2016).
9. Ly LD Oxidative stress and calcium dysregulation by palmitate in type 2 diabetes Exp. Mol. Med. 2017 49 e291 10.1038/emm.2016.157 28154371
10. Cunha DA Initiation and execution of lipotoxic ER stress in pancreatic β-cells J. Cell Sci. 2008 121 2308 2318 10.1242/jcs.026062 18559892
11. Li G Role of ERO1-alpha-mediated stimulation of inositol 1,4,5-triphosphate receptor activity in endoplasmic reticulum stress-induced apoptosis J. Cell Biol. 2009 186 783 792 10.1083/jcb.200904060 19752026
12. Cooper LL Redox modification of ryanodine receptors by mitochondria-derived reactive oxygen species contributes to aberrant Ca2+ handling in ageing rabbit hearts J. Physiol. 2013 591 5895 5911 10.1113/jphysiol.2013.260521 24042501
13. Hamilton S Increased RyR2 activity is exacerbated by calcium leak-induced mitochondrial ROS Basic Res. Cardiol. 2020 115 38 10.1007/s00395-020-0797-z 32444920
14. Vangheluwe P Raeymaekers L Dode L Wuytack F Modulating sarco(endo)plasmic reticulum Ca2+ ATPase 2 (SERCA2) activity: cell biological implications Cell Calcium 2005 38 291 302 10.1016/j.ceca.2005.06.033 16105684
15. Chipurupalli, S., Samavedam, U. & Robinson, N. Crosstalk between ER stress, autophagy and inflammation. Front. Med. 810.3389/fmed.2021.758311 (2021).
16. Maechler P Secretagogues modulate the calcium concentration in the endoplasmic reticulum of insulin-secreting cells. Studies in aequorin-expressing intact and permeabilized ins-1 cells J. Biol. Chem. 1999 274 12583 12592 10.1074/jbc.274.18.12583 10212237
17. Carreras-Sureda A Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics Nat. Cell Biol. 2019 21 755 767 10.1038/s41556-019-0329-y 31110288
18. Tiwary S Nandwani A Khan R Datta M GRP75 mediates endoplasmic reticulum-mitochondria coupling during palmitate-induced pancreatic β-cell apoptosis J. Biol. Chem. 2021 297 101368 10.1016/j.jbc.2021.101368 34756890
19. Corazzari M Oncogenic BRAF induces chronic ER stress condition resulting in increased basal autophagy and apoptotic resistance of cutaneous melanoma Cell Death Differ. 2015 22 946 958 10.1038/cdd.2014.183 25361077
20. B’chir W The eIF2α/ATF4 pathway is essential for stress-induced autophagy gene expression Nucleic Acids Res. 2013 41 7683 7699 10.1093/nar/gkt563 23804767
21. Takeshige K Baba M Tsuboi S Noda T Ohsumi Y Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction J. Cell Biol. 1992 119 301 311 10.1083/jcb.119.2.301 1400575
22. Mizushima N Autophagy: process and function Genes Dev. 2007 21 2861 2873 10.1101/gad.1599207 18006683
23. Levine B Kroemer G Autophagy in the pathogenesis of disease Cell 2008 132 27 42 10.1016/j.cell.2007.12.018 18191218
24. Schnell AH Swenne I Borg LA Lysosomes and pancreatic islet function. A quantitative estimation of crinophagy in the mouse pancreatic B-cell Cell Tissue Res. 1988 252 9 15 10.1007/BF00213820 3288350
25. Bommer G Schäfer HJ Klöppel G Morphologic effects of diazoxide and diphenylhydantoin on insulin secretion and biosynthesis in B cells of mice Virchows Arch. A Pathol. Anat. Histol. 1976 371 227 241 10.1007/BF00433070 823696
26. Hayat, M. A. in Autophagy: Cancer, Other Pathologies, Inflammation, Immunity, Infection, and Aging (ed. Hayat, M. A.) 1–122 (Academic Press, 2017).
27. Kaniuk NA Ubiquitinated-protein aggregates form in pancreatic beta-cells during diabetes-induced oxidative stress and are regulated by autophagy Diabetes 2007 56 930 939 10.2337/db06-1160 17395740
28. Ebato C Autophagy is important in islet homeostasis and compensatory increase of beta cell mass in response to high-fat diet Cell Metab. 2008 8 325 332 10.1016/j.cmet.2008.08.009 18840363
29. Jung HS Loss of autophagy diminishes pancreatic beta cell mass and function with resultant hyperglycemia Cell Metab. 2008 8 318 324 10.1016/j.cmet.2008.08.013 18840362
30. Ren L Autophagy is essential for the differentiation of porcine PSCs into insulin-producing cells Biochem. Biophys. Res. Commun. 2017 488 471 476 10.1016/j.bbrc.2017.05.058 28501624
31. Riahi Y Autophagy is a major regulator of beta cell insulin homeostasis Diabetologia 2016 59 1480 1491 10.1007/s00125-016-3868-9 26831301
32. Twig G Fission and selective fusion govern mitochondrial segregation and elimination by autophagy EMBO J. 2008 27 433 446 10.1038/sj.emboj.7601963 18200046
33. Lamb CA Yoshimori T Tooze SA The autophagosome: origins unknown, biogenesis complex Nat. Rev. Mol. Cell Biol. 2013 14 759 774 10.1038/nrm3696 24201109
34. Schuck S Gallagher CM Walter P ER-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery J. Cell Sci. 2014 127 4078 4088 25052096
35. Tanemura M Rapamycin causes upregulation of autophagy and impairs islets function both in vitro and in vivo Am. J. Transpl. 2012 12 102 114 10.1111/j.1600-6143.2011.03771.x
36. Israeli T The nutrient sensor mTORC1 regulates insulin secretion by modulating ß-cell autophagy Diabetes 2021 71 453 469 10.2337/db21-0281
37. Himuro M Cellular autophagy in α cells plays a role in the maintenance of islet architecture J. Endocr. Soc. 2019 3 1979 1992 31620668
38. Rajak S MTORC1 inhibition drives crinophagic degradation of glucagon Mol. Metab. 2021 53 101286 10.1016/j.molmet.2021.101286 34224916
39. Marroqui L Pancreatic α cells are resistant to metabolic stress-induced apoptosis in type 2 diabetes EBioMedicine 2015 2 378 385 10.1016/j.ebiom.2015.03.012 26137583
40. Lytrivi M Castell AL Poitout V Cnop M Recent insights into mechanisms of beta-cell lipo- and glucolipotoxicity in type 2 diabetes J. Mol. Biol. 2020 432 1514 1534 10.1016/j.jmb.2019.09.016 31628942
41. Masini M Autophagy in human type 2 diabetes pancreatic beta cells Diabetologia 2009 52 1083 1086 10.1007/s00125-009-1347-2 19367387
42. Lim SW Jin L Jin J Yang CW Effect of exendin-4 on autophagy clearance in beta cell of rats with tacrolimus-induced diabetes mellitus Sci. Rep. 2016 6 29921 10.1038/srep29921 27436514
43. Komiya K Free fatty acids stimulate autophagy in pancreatic beta-cells via JNK pathway Biochem. Biophys. Res. Commun. 2010 401 561 567 10.1016/j.bbrc.2010.09.101 20888798
44. Martino L Palmitate activates autophagy in INS-1E beta-cells and in isolated rat and human pancreatic islets PLoS ONE 2012 7 e36188 10.1371/journal.pone.0036188 22563482
45. Choi SE Protective role of autophagy in palmitate-induced INS-1 beta-cell death Endocrinology 2009 150 126 134 10.1210/en.2008-0483 18772242
46. Las G Serada SB Wikstrom JD Twig G Shirihai OS Fatty acids suppress autophagic turnover in beta-cells J. Biol. Chem. 2011 286 42534 42544 10.1074/jbc.M111.242412 21859708
47. Mir SU Inhibition of autophagic turnover in beta-cells by fatty acids and glucose leads to apoptotic cell death J. Biol. Chem. 2015 290 6071 6085 10.1074/jbc.M114.605345 25548282
48. Guo, W., Zhong, W., Hao, L., Sun, X. & Zhou, Z. Activation of mTORC1 by free fatty acids suppresses LAMP2 and autophagy function via ER stress in alcohol-related liver disease. Cells 1010.3390/cells10102730 (2021).
49. Li S Sirtuin 3 acts as a negative regulator of autophagy dictating hepatocyte susceptibility to lipotoxicity Hepatology 2017 66 936 952 10.1002/hep.29229 28437863
50. Wen H Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling Nat. Immunol. 2011 12 408 415 10.1038/ni.2022 21478880
51. Yang L Li P Fu S Calay ES Hotamisligil GS Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance Cell Metab. 2010 11 467 478 10.1016/j.cmet.2010.04.005 20519119
52. Yamamoto T High-fat diet-induced lysosomal dysfunction and impaired autophagic flux contribute to lipotoxicity in the kidney J. Am. Soc. Nephrol. 2017 28 1534 1551 10.1681/ASN.2016070731 27932476
53. Assali EA Nanoparticle-mediated lysosomal reacidification restores mitochondrial turnover and function in β cells under lipotoxicity FASEB J. 2019 33 4154 4165 10.1096/fj.201801292R 30550357
54. Park HW Pharmacological correction of obesity-induced autophagy arrest using calcium channel blockers Nat. Commun. 2014 5 4834 10.1038/ncomms5834 25189398
55. Zhang Y Urolithin A suppresses glucolipotoxicity-induced ER stress and TXNIP/NLRP3/IL-1β inflammation signal in pancreatic β cells by regulating AMPK and autophagy Phytomedicine 2021 93 153741 10.1016/j.phymed.2021.153741 34656886
56. Xu G Chen J Jing G Shalev A Preventing β-cell loss and diabetes with calcium channel blockers Diabetes 2012 61 848 856 10.2337/db11-0955 22442301
57. Jiang Y Metformin plays a dual role in MIN6 pancreatic beta cell function through AMPK-dependent autophagy Int. J. Biol. Sci. 2014 10 268 277 10.7150/ijbs.7929 24644425
58. Chen ZF Liraglutide prevents high glucose level induced insulinoma cells apoptosis by targeting autophagy Chin. Med. J. 2013 126 937 941 10.3760/cma.j.issn.0366-6999.20122331 23489805
59. Lupi R Lipotoxicity in human pancreatic islets and the protective effect of metformin Diabetes 2002 51 S134 S137 10.2337/diabetes.51.2007.S134 11815472
60. Zhang E Preserving insulin secretion in diabetes by inhibiting VDAC1 overexpression and surface translocation in β cells Cell Metab. 2019 29 64 77.e66 10.1016/j.cmet.2018.09.008 30293774
61. Xi X Pioglitazone protects tubular cells against hypoxia/reoxygenation injury through enhancing autophagy via AMPK-mTOR signaling pathway Eur. J. Pharm. 2019 863 172695 10.1016/j.ejphar.2019.172695
62. Cunha DA Glucagon-like peptide-1 agonists protect pancreatic beta-cells from lipotoxic endoplasmic reticulum stress through upregulation of BiP and JunB Diabetes 2009 58 2851 2862 10.2337/db09-0685 19720788
63. Natalicchio A Exendin-4 protects pancreatic beta cells from palmitate-induced apoptosis by interfering with GPR40 and the MKK4/7 stress kinase signalling pathway Diabetologia 2013 56 2456 2466 10.1007/s00125-013-3028-4 23995397
64. Zummo FP Glucagon-like peptide 1 protects pancreatic β-cells from death by increasing autophagic flux and restoring lysosomal function Diabetes 2017 66 1272 1285 10.2337/db16-1009 28232493
65. Wang J Liraglutide protects pancreatic β-cells against free fatty acids in vitro and affects glucolipid metabolism in apolipoprotein E‑/‑mice by activating autophagy Mol. Med. Rep. 2015 12 4210 4218 10.3892/mmr.2015.3944 26080706
66. Liu L Liu J Yu X Dipeptidyl peptidase-4 inhibitor MK-626 restores insulin secretion through enhancing autophagy in high fat diet-induced mice Biochem. Biophys. Res. Commun. 2016 470 516 520 10.1016/j.bbrc.2016.01.116 26802468
67. Abe H Exendin-4 improves β-cell function in autophagy-deficient β-cells Endocrinology 2013 154 4512 4524 10.1210/en.2013-1578 24105478
68. Korbut, A. I. et al. SGLT2 inhibitor empagliflozin and DPP4 inhibitor linagliptin reactivate glomerular autophagy in db/db mice, a model of type 2 diabetes. Int. J. Mol. Sci. 2110.3390/ijms21082987 (2020).
69. Lee YH Empagliflozin attenuates diabetic tubulopathy by improving mitochondrial fragmentation and autophagy Am. J. Physiol. Ren. Physiol. 2019 317 F767 F780 10.1152/ajprenal.00565.2018
70. Xu, J., Kitada, M., Ogura, Y., Liu, H. & Koya, D. Dapagliflozin restores impaired autophagy and suppresses inflammation in high glucose-treated HK-2 cells. Cells 1010.3390/cells10061457 (2021).
71. Packer M Critical reanalysis of the mechanisms underlying the cardiorenal benefits of SGLT2 inhibitors and reaffirmation of the nutrient deprivation signaling/autophagy hypothesis Circulation 2022 146 1383 1405 10.1161/CIRCULATIONAHA.122.061732 36315602
72. Rivera JF Costes S Gurlo T Glabe CG Butler PC Autophagy defends pancreatic beta cells from human islet amyloid polypeptide-induced toxicity J. Clin. Invest. 2014 124 3489 3500 10.1172/JCI71981 25036708
73. Morita S Autophagy protects against human islet amyloid polypeptide-associated apoptosis J. Diabetes Investig. 2011 2 48 55 10.1111/j.2040-1124.2010.00065.x 24843461
74. de Duve C The lysosome turns fifty Nat. Cell Biol. 2005 7 847 849 10.1038/ncb0905-847 16136179
75. Ballabio A Bonifacino JS Lysosomes as dynamic regulators of cell and organismal homeostasis Nat. Rev. Mol. Cell Biol. 2020 21 101 118 10.1038/s41580-019-0185-4 31768005
76. Perera RM Zoncu R The lysosome as a regulatory hub Annu. Rev. Cell Dev. Biol. 2016 32 223 253 10.1146/annurev-cellbio-111315-125125 27501449
77. Li P Gu M Xu H Lysosomal ion channels as decoders of cellular signals Trends Biochem. Sci. 2019 44 110 124 10.1016/j.tibs.2018.10.006 30424907
78. Forgac M Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology Nat. Rev. Mol. Cell Biol. 2007 8 917 929 10.1038/nrm2272 17912264
79. Jung Y Kim W Shin NK Bae YM Wie J Unveiling the impact of lysosomal ion channels: balancing ion signaling and disease pathogenesis Korean J. Physiol. Pharm. 2023 27 311 323 10.4196/kjpp.2023.27.4.311
80. Xu H Ren D Lysosomal physiology Annu. Rev. Physiol. 2015 77 57 80 10.1146/annurev-physiol-021014-071649 25668017
81. Lloyd-Evans E Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium Nat. Med. 2008 14 1247 1255 10.1038/nm.1876 18953351
82. Dickson EJ Duman JG Moody MW Chen L Hille B Orai-STIM–mediated Ca2+ release from secretory granules revealed by a targeted Ca2+ and pH probe Proc. Natl Acad. Sci. 2012 109 E3539 E3548 10.1073/pnas.1218247109 23184982
83. Calcraft PJ NAADP mobilizes calcium from acidic organelles through two-pore channels Nature 2009 459 596 600 10.1038/nature08030 19387438
84. Lee HC Aarhus R A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADP-ribose (∗) J. Biol. Chem. 1995 270 2152 2157 10.1074/jbc.270.5.2152 7836444
85. Arredouani A Nicotinic acid adenine dinucleotide phosphate (NAADP) and endolysosomal two-pore channels modulate membrane excitability and stimulus-secretion coupling in mouse pancreatic β cells J. Biol. Chem. 2015 290 21376 21392 10.1074/jbc.M115.671248 26152717
86. Kilpatrick BS An endosomal NAADP-sensitive two-pore Ca(2+) channel regulates ER-endosome membrane contact sites to control growth factor signaling Cell Rep. 2017 18 1636 1645 10.1016/j.celrep.2017.01.052 28199837
87. Galione A A primer of NAADP-mediated Ca(2+) signalling: from sea urchin eggs to mammalian cells Cell Calcium 2015 58 27 47 10.1016/j.ceca.2014.09.010 25449298
88. Zhang X MCOLN1 is a ROS sensor in lysosomes that regulates autophagy Nat. Commun. 2016 7 12109 10.1038/ncomms12109 27357649
89. Wang W Up-regulation of lysosomal TRPML1 channels is essential for lysosomal adaptation to nutrient starvation Proc. Natl Acad. Sci. USA 2015 112 E1373 E1381 25733853
90. Dong X-p PI(3,5)P(2) controls membrane trafficking by direct activation of mucolipin Ca(2+) release channels in the endolysosome Nat. Commun. 2010 1 38 38 10.1038/ncomms1037 20802798
91. Höglinger D Intracellular sphingosine releases calcium from lysosomes Elife 2015 4 e10616 10.7554/eLife.10616 26613410
92. Patel S Churchill GC Galione A Coordination of Ca2+ signalling by NAADP Trends Biochem. Sci. 2001 26 482 489 10.1016/S0968-0004(01)01896-5 11504624
93. Heister PM Powell T Galione A Glucose and NAADP trigger elementary intracellular β-cell Ca(2+) signals Sci. Rep. 2021 11 10714 10.1038/s41598-021-88906-0 34021189
94. Hamilton A Adrenaline stimulates glucagon secretion by Tpc2-dependent Ca(2+) mobilization from acidic stores in pancreatic α-cells Diabetes 2018 67 1128 1139 10.2337/db17-1102 29563152
95. Cane MC Parrington J Rorsman P Galione A Rutter GA The two pore channel TPC2 is dispensable in pancreatic β-cells for normal Ca2+ dynamics and insulin secretion Cell Calcium 2016 59 32 40 10.1016/j.ceca.2015.12.004 26769314
96. Peng W Wong YC Krainc D Mitochondria-lysosome contacts regulate mitochondrial Ca(2+) dynamics via lysosomal TRPML1 Proc. Natl Acad. Sci. USA 2020 117 19266 19275 10.1073/pnas.2003236117 32703809
97. Mc Donald JM Krainc D Lysosomal proteins as a therapeutic target in neurodegeneration Annu. Rev. Med. 2017 68 445 458 10.1146/annurev-med-050715-104432 28099085
98. Plotegher N Duchen MR Mitochondrial dysfunction and neurodegeneration in lysosomal storage disorders Trends Mol. Med. 2017 23 116 134 10.1016/j.molmed.2016.12.003 28111024
99. Cheng X Shen D Samie M Xu H Mucolipins: intracellular TRPML1-3 channels FEBS Lett. 2010 584 2013 2021 10.1016/j.febslet.2009.12.056 20074572
100. LaPlante JM Identification and characterization of the single channel function of human mucolipin-1 implicated in mucolipidosis type IV, a disorder affecting the lysosomal pathway FEBS Lett. 2002 532 183 187 10.1016/S0014-5793(02)03670-0 12459486
101. Vergarajauregui S Connelly PS Daniels MP Puertollano R Autophagic dysfunction in mucolipidosis type IV patients Hum. Mol. Genet. 2008 17 2723 2737 10.1093/hmg/ddn174 18550655
102. Curcio-Morelli C Macroautophagy is defective in mucolipin-1-deficient mouse neurons Neurobiol. Dis. 2010 40 370 377 10.1016/j.nbd.2010.06.010 20600908
103. Medina DL Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB Nat. Cell Biol. 2015 17 288 299 10.1038/ncb3114 25720963
104. Kim HJ Soyombo AA Tjon-Kon-Sang S So I Muallem S The Ca(2+) channel TRPML3 regulates membrane trafficking and autophagy Traffic 2009 10 1157 1167 10.1111/j.1600-0854.2009.00924.x 19522758
105. Choi S Kim HJ The Ca2+ channel TRPML3 specifically interacts with the mammalian ATG8 homologue GATE16 to regulate autophagy Biochem. Biophys. Res. Commun. 2014 443 56 61 10.1016/j.bbrc.2013.11.044 24269818
106. Zeevi DA Lev S Frumkin A Minke B Bach G Heteromultimeric TRPML channel assemblies play a crucial role in the regulation of cell viability models and starvation-induced autophagy J. Cell Sci. 2010 123 3112 3124 10.1242/jcs.067330 20736310
107. Brailoiu E Essential requirement for two-pore channel 1 in NAADP-mediated calcium signaling J. Cell Biol. 2009 186 201 209 10.1083/jcb.200904073 19620632
108. Pereira GJ Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates autophagy in cultured astrocytes J. Biol. Chem. 2011 286 27875 27881 10.1074/jbc.C110.216580 21610076
109. Gómez-Suaga P Leucine-rich repeat kinase 2 regulates autophagy through a calcium-dependent pathway involving NAADP Hum. Mol. Genet. 2012 21 511 525 10.1093/hmg/ddr481 22012985
110. García-Rúa V Endolysosomal two-pore channels regulate autophagy in cardiomyocytes J. Physiol. 2016 594 3061 3077 10.1113/JP271332 26757341
111. Grimm C Chen CC Wahl-Schott C Biel M Two-pore channels: catalyzers of endolysosomal transport and function Front. Pharm. 2017 8 45 10.3389/fphar.2017.00045
112. Lu Y Two pore channel 2 (TPC2) inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH J. Biol. Chem. 2013 288 24247 24263 10.1074/jbc.M113.484253 23836916
113. Cao Q Calcium release through P2X4 activates calmodulin to promote endolysosomal membrane fusion J. Cell Biol. 2015 209 879 894 10.1083/jcb.201409071 26101220
114. Heitman J Movva NR Hall MN Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast Science 1991 253 905 909 10.1126/science.1715094 1715094
115. Sancak Y The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1 Science 2008 320 1496 1501 10.1126/science.1157535 18497260
116. Sancak Y Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids Cell 2010 141 290 303 10.1016/j.cell.2010.02.024 20381137
117. Mutvei AP Rap1-GTPases control mTORC1 activity by coordinating lysosome organization with amino acid availability Nat. Commun. 2020 11 1416 10.1038/s41467-020-15156-5 32184389
118. Graves LM An intracellular calcium signal activates p70 but not p90 ribosomal S6 kinase in liver epithelial cells J. Biol. Chem. 1997 272 1920 1928 10.1074/jbc.272.3.1920 8999881
119. Hannan KM Thomas G Pearson RB Activation of S6K1 (p70 ribosomal protein S6 kinase 1) requires an initial calcium-dependent priming event involving formation of a high-molecular-mass signalling complex Biochem. J. 2003 370 469 477 10.1042/bj20021709 12429015
120. Conus NM Hemmings BA Pearson RB Differential regulation by calcium reveals distinct signaling requirements for the activation of Akt and p70S6k J. Biol. Chem. 1998 273 4776 4782 10.1074/jbc.273.8.4776 9468542
121. Gulati P Amino acids activate mTOR complex 1 via Ca2+/CaM signaling to hVps34 Cell Metab. 2008 7 456 465 10.1016/j.cmet.2008.03.002 18460336
122. Li R-J Regulation of mTORC1 by lysosomal calcium and calmodulin Elife 2016 5 e19360 10.7554/eLife.19360 27787197
123. Amemiya, Y. et al. Amino acid-mediated intracellular Ca(2+) rise modulates mTORC1 by regulating the TSC2-Rheb axis through Ca(2+)/calmodulin. Int. J. Mol. Sci. 2210.3390/ijms22136897 (2021).
124. Wong CO Li R Montell C Venkatachalam K Drosophila TRPML is required for TORC1 activation Curr. Biol. 2012 22 1616 1621 10.1016/j.cub.2012.06.055 22863314
125. Venkatachalam K Wong CO Montell C Feast or famine: role of TRPML in preventing cellular amino acid starvation Autophagy 2013 9 98 100 10.4161/auto.22260 23047439
126. Sun X A negative feedback regulation of MTORC1 activity by the lysosomal Ca(2+) channel MCOLN1 (mucolipin 1) using a CALM (calmodulin)-dependent mechanism Autophagy 2018 14 38 52 10.1080/15548627.2017.1389822 29460684
127. Polak P Adipose-specific knockout of raptor results in lean mice with enhanced mitochondrial respiration Cell Metab. 2008 8 399 410 10.1016/j.cmet.2008.09.003 19046571
128. Cota D Hypothalamic mTOR signaling regulates food intake Science 2006 312 927 930 10.1126/science.1124147 16690869
129. Bentzinger CF Skeletal muscle-specific ablation of raptor, but not of rictor, causes metabolic changes and results in muscle dystrophy Cell Metab. 2008 8 411 424 10.1016/j.cmet.2008.10.002 19046572
130. Balcazar N mTORC1 activation regulates β-cell mass and proliferation by modulation of cyclin D2 synthesis and stability J. Biol. Chem. 2009 284 7832 7842 10.1074/jbc.M807458200 19144649
131. George NM Boerner BP Mir SU Guinn Z Sarvetnick NE Exploiting expression of hippo effector, yap, for expansion of functional islet mass Mol. Endocrinol. 2015 29 1594 1607 10.1210/me.2014-1375 26378466
132. Lakshmipathi J PKCζ is essential for pancreatic β-cell replication during insulin resistance by regulating mTOR and cyclin-D2 Diabetes 2016 65 1283 1296 10.2337/db15-1398 26868297
133. Zarrouki B Epidermal growth factor receptor signaling promotes pancreatic β-cell proliferation in response to nutrient excess in rats through mTOR and FOXM1 Diabetes 2014 63 982 993 10.2337/db13-0425 24194502
134. Nie J SAD-A kinase controls islet β-cell size and function as a mediator of mTORC1 signaling Proc. Natl Acad. Sci. USA 2013 110 13857 13862 10.1073/pnas.1307698110 23922392
135. Blandino-Rosano M Loss of mTORC1 signalling impairs β-cell homeostasis and insulin processing Nat. Commun. 2017 8 16014 10.1038/ncomms16014 28699639
136. Hamada S Upregulation of the mammalian target of rapamycin complex 1 pathway by Ras homolog enriched in brain in pancreatic β-cells leads to increased β-cell mass and prevention of hyperglycemia Diabetes 2009 58 1321 1332 10.2337/db08-0519 19258434
137. Gyurus E Kaposztas Z Kahan BD Sirolimus therapy predisposes to new-onset diabetes mellitus after renal transplantation: a long-term analysis of various treatment regimens Transpl. Proc. 2011 43 1583 1592 10.1016/j.transproceed.2011.05.001
138. Shigeyama Y Biphasic response of pancreatic β-cell mass to ablation of tuberous sclerosis complex 2 in mice Mol. Cell. Biol. 2008 28 2971 2979 10.1128/MCB.01695-07 18316403
139. Yuan T Reciprocal regulation of mTOR complexes in pancreatic islets from humans with type 2 diabetes Diabetologia 2017 60 668 678 10.1007/s00125-016-4188-9 28004151
140. Ardestani A Lupse B Kido Y Leibowitz G Maedler K mTORC1 signaling: a double-edged sword in diabetic β cells Cell Metab. 2018 27 314 331 10.1016/j.cmet.2017.11.004 29275961
141. Mir SU Inhibition of autophagic turnover in β-cells by fatty acids and glucose leads to apoptotic cell death J. Biol. Chem. 2015 290 6071 6085 10.1074/jbc.M114.605345 25548282
142. Bartolomé A Pancreatic β-cell failure mediated by mTORC1 hyperactivity and autophagic impairment Diabetes 2014 63 2996 3008 10.2337/db13-0970 24740570
143. Kim J Kundu M Viollet B Guan KL AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1 Nat. Cell Biol. 2011 13 132 141 10.1038/ncb2152 21258367
144. Hatanaka M Palmitate induces mRNA translation and increases ER protein load in islet β-cells via activation of the mammalian target of rapamycin pathway Diabetes 2014 63 3404 3415 10.2337/db14-0105 24834975
145. Bachar E Glucose amplifies fatty acid-induced endoplasmic reticulum stress in pancreatic beta-cells via activation of mTORC1 PLoS ONE 2009 4 e4954 e4954 10.1371/journal.pone.0004954 19305497
146. Al-Bari MAA Xu P Molecular regulation of autophagy machinery by mTOR-dependent and -independent pathways Ann. N. Y. Acad. Sci. 2020 1467 3 20 10.1111/nyas.14305 31985829
147. Noda T Ohsumi Y Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast J. Biol. Chem. 1998 273 3963 3966 10.1074/jbc.273.7.3963 9461583
148. Ganley IG ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy J. Biol. Chem. 2009 284 12297 12305 10.1074/jbc.M900573200 19258318
149. Jung CH ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery Mol. Biol. Cell 2009 20 1992 2003 10.1091/mbc.e08-12-1249 19225151
150. Hosokawa N Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy Mol. Biol. Cell 2009 20 1981 1991 10.1091/mbc.e08-12-1248 19211835
151. Nazio F mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6 Nat. Cell Biol. 2013 15 406 416 10.1038/ncb2708 23524951
152. Yuan HX Russell RC Guan KL Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy Autophagy 2013 9 1983 1995 10.4161/auto.26058 24013218
153. Ma X MTORC1-mediated NRBF2 phosphorylation functions as a switch for the class III PtdIns3K and autophagy Autophagy 2017 13 592 607 10.1080/15548627.2016.1269988 28059666
154. Wan W mTORC1-regulated and HUWE1-mediated WIPI2 degradation controls autophagy flux Mol. Cell 2018 72 303 315.e6 10.1016/j.molcel.2018.09.017 30340022
155. Wan W mTORC1 phosphorylates acetyltransferase p300 to regulate autophagy and lipogenesis Mol. Cell 2017 68 323 335.e6 10.1016/j.molcel.2017.09.020 29033323
156. Kim YM mTORC1 phosphorylates UVRAG to negatively regulate autophagosome and endosome maturation Mol. Cell 2015 57 207 218 10.1016/j.molcel.2014.11.013 25533187
157. Cheng X Pacer is a mediator of mTORC1 and GSK3-TIP60 signaling in regulation of autophagosome maturation and lipid metabolism Mol. Cell 2019 73 788 802.e7 10.1016/j.molcel.2018.12.017 30704899
158. Sardiello M A gene network regulating lysosomal biogenesis and function Science 2009 325 473 477 10.1126/science.1174447 19556463
159. Martina JA The nutrient-responsive transcription factor TFE3 promotes autophagy, lysosomal biogenesis, and clearance of cellular debris Sci. Signal 2014 7 ra9 10.1126/scisignal.2004754 24448649
160. Roczniak-Ferguson A The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis Sci. Signal 2012 5 ra42 10.1126/scisignal.2002790 22692423
161. Vega-Rubin-de-Celis S Peña-Llopis S Konda M Brugarolas J Multistep regulation of TFEB by MTORC1 Autophagy 2017 13 464 472 10.1080/15548627.2016.1271514 28055300
162. Napolitano G Ballabio A TFEB at a glance J. Cell Sci. 2016 129 2475 2481 27252382
163. Napolitano G mTOR-dependent phosphorylation controls TFEB nuclear export Nat. Commun. 2018 9 3312 10.1038/s41467-018-05862-6 30120233
164. Ozturk DG MITF-MIR211 axis is a novel autophagy amplifier system during cellular stress Autophagy 2019 15 375 390 10.1080/15548627.2018.1531197 30290719
165. Yu L Termination of autophagy and reformation of lysosomes regulated by mTOR Nature 2010 465 942 946 10.1038/nature09076 20526321
166. Hardie DG Schaffer BE Brunet A AMPK: an energy-sensing pathway with multiple inputs and outputs Trends Cell Biol. 2016 26 190 201 10.1016/j.tcb.2015.10.013 26616193
167. Suter M Dissecting the role of 5′-AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase* J. Biol. Chem. 2006 281 32207 32216 10.1074/jbc.M606357200 16943194
168. Woods A LKB1 is the upstream kinase in the AMP-activated protein kinase cascade Curr. Biol. 2003 13 2004 2008 10.1016/j.cub.2003.10.031 14614828
169. Hawley SA Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase Cell Metab. 2005 2 9 19 10.1016/j.cmet.2005.05.009 16054095
170. Momcilovic M Hong SP Carlson M Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro J. Biol. Chem. 2006 281 25336 25343 10.1074/jbc.M604399200 16835226
171. Boudeau J MO25alpha/beta interact with STRADalpha/beta enhancing their ability to bind, activate and localize LKB1 in the cytoplasm EMBO J. 2003 22 5102 5114 10.1093/emboj/cdg490 14517248
172. Hawley SA Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade J. Biol. 2003 2 28 10.1186/1475-4924-2-28 14511394
173. Gowans GJ Hawley SA Ross FA Hardie DG AMP is a true physiological regulator of AMP-activated protein kinase by both allosteric activation and enhancing net phosphorylation Cell Metab. 2013 18 556 566 10.1016/j.cmet.2013.08.019 24093679
174. Iwabu M Adiponectin and AdipoR1 regulate PGC-1alpha and mitochondria by Ca(2+) and AMPK/SIRT1 Nature 2010 464 1313 1319 10.1038/nature08991 20357764
175. Nelson ME Phosphoproteomics reveals conserved exercise‐stimulated signaling and AMPK regulation of store‐operated calcium entry EMBO J. 2019 38 e102578 10.15252/embj.2019102578 31381180
176. Herrero‐Martín G TAK1 activates AMPK‐dependent cytoprotective autophagy in TRAIL‐treated epithelial cells EMBO J. 2009 28 677 685 10.1038/emboj.2009.8 19197243
177. Budanov AV Karin M p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling Cell 2008 134 451 460 10.1016/j.cell.2008.06.028 18692468
178. Saxton RA Sabatini DM mTOR signaling in growth, metabolism, and disease Cell 2017 168 960 976 10.1016/j.cell.2017.02.004 28283069
179. Lamming DW Bar‐Peled L Lysosome: the metabolic signaling hub Traffic 2019 20 27 38 10.1111/tra.12617 30306667
180. Li M Transient receptor potential V channels are essential for glucose sensing by aldolase and AMPK Cell Metab. 2019 30 508 524.e12 10.1016/j.cmet.2019.05.018 31204282
181. Paquette M AMPK-dependent phosphorylation is required for transcriptional activation of TFEB and TFE3 Autophagy 2021 17 3957 3975 10.1080/15548627.2021.1898748 33734022
182. Steinberg GR Carling D AMP-activated protein kinase: the current landscape for drug development Nat. Rev. Drug Discov. 2019 18 527 551 10.1038/s41573-019-0019-2 30867601
183. Jäger S Handschin C St.-Pierre J Spiegelman BM AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1α Proc. Natl Acad. Sci. 2007 104 12017 12022 10.1073/pnas.0705070104 17609368
184. Settembre C TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop Nat. Cell Biol. 2013 15 647 658 10.1038/ncb2718 23604321
185. Toyama EQ AMP-activated protein kinase mediates mitochondrial fission in response to energy stress Science 2016 351 275 281 10.1126/science.aab4138 26816379
186. Egan DF Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy Science 2011 331 456 461 10.1126/science.1196371 21205641
187. Herzig S Shaw RJ AMPK: guardian of metabolism and mitochondrial homeostasis Nat. Rev. Mol. Cell Biol. 2018 19 121 135 10.1038/nrm.2017.95 28974774
188. Inoki K Zhu T Guan KL TSC2 mediates cellular energy response to control cell growth and survival Cell 2003 115 577 590 10.1016/S0092-8674(03)00929-2 14651849
189. Gwinn DM AMPK phosphorylation of raptor mediates a metabolic checkpoint Mol. Cell 2008 30 214 226 10.1016/j.molcel.2008.03.003 18439900
190. Furuta N Fujita N Noda T Yoshimori T Amano A Combinational soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins VAMP8 and Vti1b mediate fusion of antimicrobial and canonical autophagosomes with lysosomes Mol. Biol. Cell 2010 21 1001 1010 10.1091/mbc.e09-08-0693 20089838
191. Itakura E Kishi-Itakura C Mizushima N The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes Cell 2012 151 1256 1269 10.1016/j.cell.2012.11.001 23217709
192. Bar-Peled L Schweitzer LD Zoncu R Sabatini DM Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1 Cell 2012 150 1196 1208 10.1016/j.cell.2012.07.032 22980980
193. Zhang CS The lysosomal v-ATPase-Ragulator complex is a common activator for AMPK and mTORC1, acting as a switch between catabolism and anabolism Cell Metab. 2014 20 526 540 10.1016/j.cmet.2014.06.014 25002183
194. Howell JJ Metformin inhibits hepatic mTORC1 signaling via dose-dependent mechanisms involving AMPK and the TSC complex Cell Metab. 2017 25 463 471 10.1016/j.cmet.2016.12.009 28089566
195. Zhang CS Metformin activates AMPK through the lysosomal pathway Cell Metab. 2016 24 521 522 10.1016/j.cmet.2016.09.003 27732831
196. Settembre C TFEB links autophagy to lysosomal biogenesis Science 2011 332 1429 1433 10.1126/science.1204592 21617040
197. Wollheim CB Maechler P Beta-cell mitochondria and insulin secretion: messenger role of nucleotides and metabolites Diabetes 2002 51 S37 S42 10.2337/diabetes.51.2007.S37 11815456
198. Rutter GA Georgiadou E Martinez-Sanchez A Pullen TJ Metabolic and functional specialisations of the pancreatic beta cell: gene disallowance, mitochondrial metabolism and intercellular connectivity Diabetologia 2020 63 1990 1998 10.1007/s00125-020-05205-5 32894309
199. Park K Lysosomal Ca(2+)-mediated TFEB activation modulates mitophagy and functional adaptation of pancreatic β-cells to metabolic stress Nat. Commun. 2022 13 1300 10.1038/s41467-022-28874-9 35288580
200. Park, K. et al. Impaired TFEB activation and mitophagy as a cause of PPP3/calcineurin inhibitor-induced pancreatic β-cell dysfunction. Autophagy 1–15 10.1080/15548627.2022.2132686 (2022).
201. Ly LD Mitochondrial Ca(2+) uptake relieves palmitate-induced cytosolic Ca(2+) overload in MIN6 cells Mol. Cells 2020 43 66 75 31931552
202. Ovalle F Verapamil and beta cell function in adults with recent-onset type 1 diabetes Nat. Med. 2018 24 1108 1112 10.1038/s41591-018-0089-4 29988125
203. Nguyen HT Noriega Polo C Wiederkehr A Wollheim CB Park KS CDN1163, an activator of sarco/endoplasmic reticulum Ca(2+) ATPase, up-regulates mitochondrial functions and protects against lipotoxicity in pancreatic β-cells Br. J. Pharm. 2023 180 2762 2776 10.1111/bph.16160
204. Dorsey FC Mapping the phosphorylation sites of Ulk1 J. Proteome Res. 2009 8 5253 5263 10.1021/pr900583m 19807128
205. Munson MJ mTOR activates the VPS34-UVRAG complex to regulate autolysosomal tubulation and cell survival EMBO J. 2015 34 2272 2290 10.15252/embj.201590992 26139536
206. Martina JA Chen Y Gucek M Puertollano R MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB Autophagy 2012 8 903 914 10.4161/auto.19653 22576015
207. Settembre C A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB EMBO J. 2012 31 1095 1108 10.1038/emboj.2012.32 22343943
208. Li L A TFEB nuclear export signal integrates amino acid supply and glucose availability Nat. Commun. 2018 9 2685 10.1038/s41467-018-04849-7 29992949
