Lysosomal physiology and pancreatic lysosomal stress in diabetes mellitus

The identity of lysosomes

Discovered by Christian de Duve in the 1950s,1 lysosomes are dynamic and heterogeneous organelles enclosed by a single lipid bilayer membrane. Lysosomes are found in all eukaryotic cells. However, their size, number, distribution, morphology and enzyme context vary greatly depending on different species, cell types and cell status. Ranging from 100 nm to 500 nm in diameter, on nutrient starvation, the size of the lysosome increases to 500–1500 nm as a result of membrane fusion.2–5 In each mammalian cell, there are generally several hundred lysosomes, dramatically reduced to <50 per cell on acute nutrient starvation. However, when exposed to prolonged starvation, the lysosome number recovers.3 6 Overall, lysosomes comprise about 0.5%–5% of the cell’s mass. In phagocytes like the mammalian macrophage, lysosomes collectively take up 2%–10% of cell volume.7

There are four types of lysosomes depending on their morphology and function: primary, secondary, residual and auto-lysosomes.8 Primary lysosomes are homologous, small, newly formed vesicles that originate from the Golgi apparatus which contain >60 acid hydrolytic enzymes but no substrate to act on. The secondary lysosomes contain large amounts of cargoes and substrates, which are subjected to digestion by lysosomal enzymes.8 Residual lysosomes, also known as telolysosomes, contain indigestible substances that eventually move outwards and fuse with the plasma membrane in order to transport debris into the extracellular environment by exocytosis. Lastly, auto-lysosomes are created by the fusion of several primary lysosomes to remove intracellular cargoes, also known as autophagy.8

Lysosomal biogenesis

Lysosomal biogenesis is one of the most important mechanisms by which cells adapt to extracellular and intracellular demands. During starvation-induced autophagy, lysosome numbers increase. Furthermore, lysosomal reformation involves cargo-mediated degradation of lysosomes, resulting in regeneration and maintenance of the cellular lysosome pool. The process of lysosomal biogenesis requires mechanisms that rely on both lysosomal protein biosynthesis and endosome-lysosome trafficking. Lysosomal enzymes are synthesised and tagged with mannose-6-phosphate residues in the rough endoplasmic reticulum (RER), modified in the Golgi apparatus and delivered to the lysosomes.9 10 Formation of lysosomal vesicles results from the fusion between vesicles originating from post-Golgi transport and those filled with cargo along the endocytic pathway.11 For example, endocytic vesicles (EVs) from the plasma membrane mature into late endosomes that eventually become lysosomes. Lysosomes could also be formed through vesicular transport, ‘kiss and run’ events and fusion-fission processes.12–15 Lastly, the intermediate forms of hybrid lysosomes are also observed in diverse sets of cells—for example, endocytic lysosome, autophagic lysosome and phagocytic lysosome reformation, which have been reviewed by Yang and Wang.15

Lysosome localisation and motility

Intracellularly, lysosomes can be found in a relatively immobile perinuclear cluster and a set of peripheral pools.16 Lysosomes move bi-directionally in a stop-and-go manner due to the alternating activities of kinesin and dynein motor proteins.16 Depending on the cellular metabolic status or specific stimuli, lysosomes move to either the cell periphery or to the perinuclear region. The positioning of lysosomes is intimately associated with the activity of the mechanistic target of rapamycin complex 1 (mTORC1) and nutrient levels. For example, mTORC1 activation under nutrient-rich conditions causes scattering of lysosomes to the periphery.16 During starvation, peripheral lysosomes move towards the perinuclear region where autophagosome-lysosome fusion occurs. Functionally, peripheral lysosomes are less acidic and have reduced vacuolar-type ATPase activity compared with perinuclear localised lysosomes.17 18

Catabolic role of the lysosome

Lysosomes are known for their terminal degradation function in eukaryotic cells. Their primary function is maintaining cellular health by processing and removing toxic components, damaged organelles and excess metabolites. Moreover, the lysosome plays a crucial role in breaking down and recycling a variety of biological macromolecules, such as proteins, lipids, carbohydrates and nucleic acids. To accomplish its functions, lysosomes use various enzymatic reactions that are activated in an acidic environment.19 There are over 60 resident acid hydrolases, which are classified by substrate preference, including proteases, lipases, nucleases, sulfatases, phosphatases and glycosidases.11 The end-products of lysosome-mediated digestion are then transported to the cytoplasm via specific lysosomal integral membrane proteins. These products play a vital role in various biosynthetic reactions essential for the cell’s growth and development.12

The autophagy-lysosomal pathway

Autophagy, a fundamental cellular degradation process, serves a vital housekeeping function in maintaining cellular homeostasis. During the autophagic process, the autophagic cargoes are delivered to lysosomes for degradation and finally recycled to provide nutrients and building blocks for cells. Basal levels of autophagy occur naturally in most tissues to control protein and organelle quality.20 21 Whereas, in response to various stimuli such as nutritional/cellular stress or infection, autophagy is induced above baseline levels to serve as an adaptive response to re-establish organ/tissue homeostasis and immunity.22 23 Dysregulation of adaptive autophagy has been linked to the pathogenesis of many human diseases, including cancer, degenerative and immune disorders, metabolic disease and ageing.24

Role of the lysosome in ionic balancing

Lysosomes contain ions and harbour ion channels, which exert indispensable roles in regulating lysosomal pH and function.25 Specifically, the lysosomal lumen comprises various ions including Fe²⁺, Na⁺, K⁺, Zn²⁺, H⁺ and Ca²⁺ (which is ~0.5 mM and 5000 times higher than cytosolic Ca²⁺ stores). Furthermore, the lysosome membrane contains more than six types of ion channels to balance the lysosomal ionic gradient.26 These lysosomal ion channels and transporters mediate ion flux to regulate lysosomal ion homeostasis, membrane potential, catabolite export, membrane trafficking and nutrient sensing. Dysregulation of lysosomal channels has been involved in the pathogenesis of many diseases, such as lysosomal storage diseases, metabolic diseases and neurodegenerative diseases,3 27 all of which have been reviewed in detail elsewhere.3 12

The role of lysosome in immunity

The lysosome’s function is not limited to cellular waste disposal and contributes significantly to the immune system.28 The lysosome serves as a central hub for both innate and adaptive immunity, aiding the host’s defence against harmful intruders such as bacteria and viruses.28 In response to a diverse range of stimuli, dendritic cells (DCs) enhance the ability to form peptide-major histocompatibility complex (MHC) II class complexes in lysosomes to initiate antigen-specific immune responses.29 30 Regarding T cells, virus-induced autophagy plays a critical role in MHC class antigen presentation and CD8⁺ T cell responses.31 Due to their phagocytic nature, macrophages are abundant in lysosomes for degrading dead cells and foreign substances.32 On infection, macrophages undergo significant alterations in the quantity and composition of the lysosomal proteome, respectively.33 In addition to local lysosomal events, the immune cells’ autophagy-lysosome pathway aids pathogen detection, thus enhancing the immune system’s effectiveness.31 34 Lastly, autophagy regulators mediate innate immunity by directly modulating inflammasomes in macrophages.35

The metabolic function of lysosome

The lysosome modulates extracellular nutritional sensing by degrading or recycling plasma membrane receptors, such as transferrin (Tf)-Tf receptor complex and low-density lipoprotein (LDL) receptor.36–40 Motile and primary cilia are microtubule-based structures located at the cell surface which modulate cellular functions ranging from motility to sensing nutritional signalling from the environment.41 Notably, recent studies highlight the interplay between autophagy and ciliogenesis during nutritional stress.42 43 Intracellularly, in mammalian and yeast cells, activation of the mTORC1 protein kinase and AMP-activated protein kinase (AMPK), two essential anabolic and catabolic regulators, respectively, occur at the lysosome.44–47

In addition to sensing nutritional status, lysosomes play a crucial role in maintaining intracellular metabolic balancing by degrading both internal and external substances for cellular energy fuel resources.3 11 48 49 Furthermore, lysosome-mediated signalling pathways and transcriptional programmes are responsible for controlling the switch between anabolism and catabolism. This is achieved by regulating the transcription factor EB (TFEB)-mediated lysosomal biogenesis and the autophagy programme.50 51 Lysosomes also control intracellular metabolic status by modulating other organelles’ functions through interorganelle communication. This organellar direct communication is crucial for lysosomal function, positioning and repair of damaged lysosomal membranes,52 and for fine-tuning intracellular calcium transfer, lipid transport, signal transduction, glucose metabolism and mitochondrial respiration.12 53–60 Together, the lysosome plays a vital role in detecting nutrients, transmitting metabolic signals and regulating both cellular anabolic and catabolic processes. The functions of the lysosome are summarised in figure 1.

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Physiological pancreatic lysosomal function

The pancreas consists of endocrine and exocrine glands. The exocrine compartment of pancreas consists of acinar and ductal cells to produce and drain the digestive enzymes. The endocrine compartment, islets of Langerhans, include α, β, δ, ε and pancreatic polypeptide (PP) cells which secrete glucagon, insulin, somatostatin, ghrelin and pancreatic polypeptide, respectively.74 Mounting studies have demonstrated that β-cell lysosome-autophagy process plays an important role in insulin biosynthesis, secretion and degradation.75–78 Moreover, β-cell autophagy plays an important role in fine-tuning organelle homeostasis, such as the ER function, to ensure a functional β-cell.79 In addition to serving as the terminal site of the autophagy pathway, lysosomes are responsible for monitoring and sensing nutritional flux in β-cells. It has been demonstrated that β-cell mTORC1 signalling controls systemic glucose homeostasis by regulating β-cell mass, proliferation, apoptosis, insulin secretion and insulin secretory granule degradation.80 Moreover, the inactivation of β-cell AMPK elevates insulin secretion while the overactivation of β-cell AMPK prevents insulin secretion.67 81 82 Taken together, these studies demonstrated that basal lysosome-autophagy process is essential for maintaining β-cell cellular and insulin homeostasis (figure 2). However, thus far there are limited studies investigating lysosomal function in other cell types of the pancreas.

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Pancreatic β-cell lysosomal stress in type 1 diabetes and type 2 diabetes

Diabetes mellitus (DM) is a common disease that is increasing worldwide. Type 2 diabetes (T2D) is characterised by hyperglycaemia due to insulin resistance and impaired insulin secretion.83 Mounting studies have shown that autophagy is essential for maintaining the structure, mass, survival and cellular homeostasis of β-cells in response to cellular stress,84–87 and dysregulation of autophagy in β-cells has been linked to an increased incidence of T2D.86 88 89 Although numerous studies have demonstrated the pathophysiological relevance of dysfunctional autophagy in T2D, less evidence has been provided to support this link in the context of type 1 diabetes (T1D). T1D is an autoimmune disease attributed to the autoimmune mechanism-induced destruction of β-cells in pancreatic islets.90 Of note, several T1D susceptibility genes are known to play critical roles in autophagic degradation pathways. For example, in mouse models of T1D, Clec16a, a T1D disease susceptibility gene, has been demonstrated to play a critical role in regulating mitophagy, glucose-stimulated insulin secretion91 and protection of β-cells against cytokine-induced apoptosis.92 93 Recently, a study by Muralidharan et al further supported the translational relevance of lysosomal dysfunction in T1D pathogenesis, demonstrating a blocked autophagic flux in the islets of non-obese diabetic(NOD) mice as well as in residual β cells of human donors with T1D.94

Cathepsins (CTS) are the most abundant lysosomal proteases with a broad spectrum of functions, such as intracellular protein degradation, energy metabolism and immune response.95 Increasing evidence suggests that dysregulation of several lysosomal CTS are genetically associated with T1D. For example, CTSC has been identified as a causal risk gene in T1D,96 and CTSC has been shown to positively regulate cytokine-induced β-cell apoptosis in the context of T1D.97 Moreover, it has been demonstrated that deletion of CTSS, CTSB and CTSL ameliorates T1D development and diabetes incidence in mice.98 In terms of T2D, reduced transcription of CTSB and CTSD has been linked to β-cell dysfunction and cell death in patients with T2D.99 Lastly, abnormal lysosomal glycohydrolase activities, such as β-N-acetylhexosaminidase, β-galactosidase and α-glucosidase, have been revealed in patients with juvenile DM.100

In addition to serving as a central site of proteostasis, the lysosome plays an important role in modulating intracellular calcium flux.101 For example, lysosomal calcium release via transient receptor potential mucolipin 1 (TRPML1) is known to play a key role in inducing the nuclear translocation of TFEB, autophagosome fusion, mitophagy and lysosomal adaptation during starvation.102 Recent studies have begun to reveal the functional relevance of these lysosomal calcium channels in disease progression.103 104 Of note, Park et al identified a lysosomal Ca²⁺-mediated TFEB activation in mitophagy and functional adaptation in pancreatic β-cell adaptation to metabolic stress.105 106

Lysosome stress of pancreatic α cells in DM

It is proposed that one key cause of DM’s hyperglycaemia is the excessive production of glucagon (or hyperglucagonaemia), which reflects dysregulated glucagon secretion from pancreatic α cells.107 Impaired intracellular trafficking of glucagon has been recognised as a mechanism underlying defective glucagon secretion in diabetes.108 Notably, altered lysosomal trafficking of glucagon has been proposed as a new pathway for glucagon hypersecretion in diabetes.109 110 This is caused by a switch from trafficking lysosome (Lamp 2A+) to autophagic lysosomes to secretory lysosomes (Lamp 1+), as well as an increase in glucagon trafficking through secretory granules.109

Lysosome stress of immune cells in DM

The pancreas’s endocrine unit comprises a diverse cell population including immune cells.111 During T1D progression, immune cells infiltrate the pancreas creating an inflammatory environment characteristic of insulitis. In turn, insulitis accelerates T1D development by increasing exposure of islet antigens and attacking β-cell.112 113 Both adaptive and innate immune systems are involved in the onset and progression of T1D.114 115 Although the lysosome-autophagy axis plays a crucial role in modulating immunity,116 the immune cell-lysosomal function in T1D is poorly studied. It has been shown that autophagy regulates T cell metabolic flexibility and survival to prevent autoimmune attacks on pancreatic β-cells.94 Furthermore, autophagy-deficient DCs display accelerated expression of MHC I leading to CD8⁺ T cell activation.117 It is proposed that B cells autophagy might impact intracellular MHC I presentation by reducing the amount of neoantigens formed in β-cells.118 At the site of the lysosome, it is reported that T1D elevates CTSL expression in peripheral CD8⁺ T in NOD mice.119 Moreover, it is shown that the CTSL-processed granule proteins create chimeric epitopes for diabetogenic CD4⁺ T cells augmenting impairment of peripheral self-tolerance in T1D.120

Another key feature of T1D and T2D immunity is the elevation of islet macrophage infiltration. It has been shown that in mice with STZ-induced T1D diabetes, macrophages make up to 0.9% of total islet cells compared with 0.5% in normal mice.121 In T2D, it is estimated that the average number of islet macrophages is around 1.34 macrophage/islet compared with 0.52 macrophage/islet under normal condition.121 In mice with diabetes, the elevation of mitochondrial reactive oxygen species (ROS) promotes macrophage polarisation towards the proinflammatory M1 phenotype.122 Of note, this process is mediated by impairing lysosomal function and autophagic flux in peritoneal macrophages of mice with diabetes.122 Sterile stimuli, such as damage-associated molecular patterns, sterile particulates and intracellular cytokines released from necrotic cells can trigger sterile inflammation by activating the host immune system.123 124 It is well recognised that the NLR family pyrin domain containing 3 inflammasomes is the key regulator for sterile inflammatory responses.125–127 Aberrant macrophage polarisation and inflammasome activation coexist in the pathogenesis and progression of diabetes and its complications.128–130 Notably, the lysosome plays a crucial role in both the priming and assembly phases of the lipotoxic inflammasome in peritoneal macrophages.131 Although it is largely unknown to what extent islet macrophage lysosome-modulated inflammasome activity contributes to DM pathologies, lysosomes may be potential targets for therapies to address the hyperinflammatory phenotypes observed in patients with diabetes.131

Functional relevance of lysosomal stress in other types of DM

Gestational diabetes mellitus (GDM) occurs when the body cannot respond properly to pregnancy-related metabolic challenges and insulin resistance.132 After giving birth, blood glucose levels return to normal in most individuals with gestational diabetes. However, these patients have a considerable risk of developing T2D in the future.133 Of note, a recent human study has identified an inversed correlation between fetal pancreas-autophagic markers and GDM-associated maternal metabolic risk.134 Furthermore, using a humanised islet amyloid polypeptide transgenic mouse line, Gurlo et al found a compromised β-cell autophagy in female mice with GDM.135 However, the pathophysiological relevance of pancreatic lysosomal function in the context of GDM has not been explored. Another distinct type of DM is cystic fibrosis-related diabetes (CFRD), which is characterised by decreased islet mass, β-cell dysfunction, hypoinsulinaemia and systemic insulin resistance.136–138 Thus far, there are only a few published studies on lysosomal stress in CFRD. High-mobility group box 1 protein (HMGB1) is a non-histone nuclear factor which regulates diverse biological processes depending on its subcellular or extracellular localization.139 During inflammation, HMGB1 is actively secreted into the extracellular space by lysosome-mediated exocytosis.140 Notably, patients with CFRD have elevated circulating levels of HMGB1 due to loss of function of cystic fibrosis transmembrane conductance regulator.141 However, what are the cell resources for the lysosome-mediated HMGB1 secretion and how lysosome stress affects the CFRD pathogenesis remain largely unknown.