RISING STARS: Evidence for established and emerging forms of β-cell death

in Journal of Endocrinology
Authors:
Kaitlyn A Colglazier Lilly Diabetes Center of Excellence, Indiana Biosciences Research Institute, Indianapolis, Indiana, USA

Search for other papers by Kaitlyn A Colglazier in
Current site
Google Scholar
PubMed
Close
,
Noyonika Mukherjee Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA

Search for other papers by Noyonika Mukherjee in
Current site
Google Scholar
PubMed
Close
,
Christopher J Contreras Division of Endocrinology, Department of Medicine, Roudebush VA Medical Center and Indiana University School of Medicine, Indianapolis, Indiana, USA

Search for other papers by Christopher J Contreras in
Current site
Google Scholar
PubMed
Close
, and
Andrew T Templin Lilly Diabetes Center of Excellence, Indiana Biosciences Research Institute, Indianapolis, Indiana, USA
Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
Division of Endocrinology, Department of Medicine, Roudebush VA Medical Center and Indiana University School of Medicine, Indianapolis, Indiana, USA
Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, Indiana, USA

Search for other papers by Andrew T Templin in
Current site
Google Scholar
PubMed
Close
https://orcid.org/0000-0002-6241-9304

Correspondence should be addressed to A T Templin: templin@iu.edu

This paper is part of a collection of articles highlighting the breadth and depth of research being undertaken across the field of basic endocrinology by early- and mid-career researchers. The collection is published across the Journal of Endocrinology and the Journal of Molecular Endocrinology.

Open access

Sign up for journal news

β-Cell death contributes to β-cell loss and insulin insufficiency in type 1 diabetes (T1D), and this β-cell demise has been attributed to apoptosis and necrosis. Apoptosis has been viewed as the lone form of programmed β-cell death, and evidence indicates that β-cells also undergo necrosis, regarded as an unregulated or accidental form of cell demise. More recently, studies in non-islet cell types have identified and characterized novel forms of cell death that are biochemically and morphologically distinct from apoptosis and necrosis. Several of these mechanisms of cell death have been categorized as forms of regulated necrosis and linked to inflammation and disease pathogenesis. In this review, we revisit discoveries of β-cell death in humans with diabetes and describe studies characterizing β-cell apoptosis and necrosis. We explore literature on mechanisms of regulated necrosis including necroptosis, ferroptosis and pyroptosis, review emerging literature on the significance of these mechanisms in β-cells, and discuss experimental approaches to differentiate between various mechanisms of β-cell death. Our review of the literature leads us to conclude that more detailed experimental characterization of the mechanisms of β-cell death is warranted, along with studies to better understand the impact of various forms of β-cell demise on islet inflammation and β-cell autoimmunity in pathophysiologically relevant models. Such studies will provide insight into the mechanisms of β-cell loss in T1D and may shed light on new therapeutic approaches to protect β-cells in this disease.

Abstract

β-Cell death contributes to β-cell loss and insulin insufficiency in type 1 diabetes (T1D), and this β-cell demise has been attributed to apoptosis and necrosis. Apoptosis has been viewed as the lone form of programmed β-cell death, and evidence indicates that β-cells also undergo necrosis, regarded as an unregulated or accidental form of cell demise. More recently, studies in non-islet cell types have identified and characterized novel forms of cell death that are biochemically and morphologically distinct from apoptosis and necrosis. Several of these mechanisms of cell death have been categorized as forms of regulated necrosis and linked to inflammation and disease pathogenesis. In this review, we revisit discoveries of β-cell death in humans with diabetes and describe studies characterizing β-cell apoptosis and necrosis. We explore literature on mechanisms of regulated necrosis including necroptosis, ferroptosis and pyroptosis, review emerging literature on the significance of these mechanisms in β-cells, and discuss experimental approaches to differentiate between various mechanisms of β-cell death. Our review of the literature leads us to conclude that more detailed experimental characterization of the mechanisms of β-cell death is warranted, along with studies to better understand the impact of various forms of β-cell demise on islet inflammation and β-cell autoimmunity in pathophysiologically relevant models. Such studies will provide insight into the mechanisms of β-cell loss in T1D and may shed light on new therapeutic approaches to protect β-cells in this disease.

Invited Author’s profile

Andrew T Templin, PhD, is a β-cell biologist and diabetologist who holds positions at Indiana University School of Medicine, Indiana Biosciences Research Institute, and the Roudebush VA Medical Center. Research in the Templin Lab focuses on understanding the relationship between islet immune responses and β-cell dysfunction and death in the setting of both major forms of diabetes. Emphasis is placed on the concept that β-cell intrinsic properties are drivers of islet inflammation and immune responses, and together these promote a system of β-cell dysfunction and loss that leads to diabetes. In this context, the Templin Lab investigates underappreciated mechanisms of islet inflammation and β-cell cytotoxicity, and the relationship of these to diabetogenic β-cell loss. Through this work, the Templin Lab aims to advance our understanding of β-cell demise and improve the health of individuals with diabetes. Andrew is a native Hoosier who enjoys travel, soccer, the Chicago Cubs, Golden Retrievers, and spending time with family.

Introduction

β-Cell death contributes to β-cell loss, insufficient insulin secretion, and hyperglycemia in the pathogenesis of type 1 diabetes (T1D) (Mandrup-Poulsen 2001, Mathis et al. 2001, Cnop et al. 2005, Rhodes 2005), and studies of the cellular and molecular mechanisms that mediate β-cell death have focused principally on β-cell apoptosis (Augstein et al. 1998, Mandrup-Poulsen 2001, Butler et al. 2003, 2007) and necrosis (Hoorens et al. 2001, Scarim et al. 2001, Fehsel et al. 2003). Apoptosis is triggered by intrinsic or extrinsic signals and has classically been considered the lone form of programmed cell death (PCD) (Alberts et al. 2002), whereas necrosis has been regarded as an unprogrammed or accidental form of cell death that results from chemical or physical injury (Buja et al. 1993, Majno & Joris 1995). More recent studies in non-islet cell types have identified and characterized several additional mechanisms of PCD (Proskuryakov et al. 2003, Berghe et al. 2014, Conrad et al. 2016) that are molecularly, morphologically, and functionally distinct from apoptosis (Linkermann & Green 2014, Man et al. 2017, Zheng & Conrad 2020, Bertheloot et al. 2021). Although there is growing evidence that β-cells are susceptible to these emerging forms of PCD (Bruni et al. 2018, Yang et al. 2020, Tonnus et al. 2021, Contreras et al. 2022), the roles of these forms of β-cell demise in diabetogenic β-cell loss have not been well established. In this review, we discuss our current understanding of β-cell death in human diabetes, review literature on mechanisms of regulated necrosis in non-islet cell types, examine the relevance of emerging forms of cell death to diabetogenic β-cell loss, explore heterogeneity of β-cell death responses, and describe experimental approaches to differentiate between various mechanisms of cell death. We begin by examining evidence for β-cell death as a factor in the pathogenesis of human T1D.

β-Cell death in the pathogenesis of human T1D

Interest in understanding and targeting mechanisms of β-cell death to combat T1D arises from the extensive literature linking β-cell cytotoxicity to insulin insufficiency and hyperglycemia in this disease. Although the decreased abundance of insulin-producing β-cells was observed in T1D as early as the 1950s (Maclean & Ogilvie 1959), such observations were not linked to increased rates of β-cell death until decades later. Methods developed in the 1990s facilitated the detection and quantification of dead cells in tissue sections, including terminal deoxy-nucleotidyl transferase dUTP nick end labeling (TUNEL) (Gorczyca et al. 1992), annexin V staining (Vermes et al. 1995), and propidium iodide staining (Douglas et al. 1995). Utilizing these techniques, studies were conducted to quantify β-cell death in human pancreas sections from non-diabetic and T1D organ donors, and evidence emerged that β-cell death is elevated in individuals with T1D. For example, two early studies across 23 non-diabetic and 51 T1D pancreas samples revealed that β-cell death was increased approximately two-fold in those with T1D (Meier et al. 2005, Butler et al. 2007). Although these studies observed that increased rates of β-cell death contribute to β-cell loss and insulin insufficiency in individuals with T1D, they also provoked several questions and led to additional work to better understand the role of β-cell death in T1D disease pathogenesis.

Several factors must be considered when evaluating β-cell death in human pancreas sections and interpreting relevance to T1D disease pathogenesis. One important consideration is that human pancreas samples are typically donated years or decades after diagnosis with T1D. Thus, the rate of β-cell death quantified in these samples probably underestimates that which occurs during the period of active β-cell demise, which is thought to begin years prior to the clinical onset of the disease. Substantial loss of β-cell mass at the time of T1D diagnosis likely reduces the potential to observe ongoing β-cell death, and remaining β-cells may be those least susceptible to cytotoxic insults (Rui et al. 2017). Indeed, pancreas samples from organ donors with T1D exhibit only modestly increased rates of β-cell death, with studies typically finding ~1 dead β-cell in every two to three diabetic islets (Meier et al. 2005, Butler et al. 2007), and it has been debated whether this low rate of β-cell death could account for the diminished β-cell mass observed in T1D. We feel it is essential to consider the timing of active β-cell demise in T1D disease pathogenesis and the fact that dead islet cells exist for only a finite period of time before being cleared by islet macrophages in situ. Thus, establishing the true contribution of β-cell death to β-cell loss in T1D requires both capturing the active period of β-cell demise and determining how accumulation of cell death translates to diminished β-cell mass over time. Given that human pancreas tissue is collected at a single time point (post mortem), our ability to determine the contribution of β-cell death to β-cell loss in human T1D pancreas samples is limited.

Biomarkers of β-cell death in human T1D

To address this limitation, studies have aimed to identify and measure biomarkers of β-cell death in living humans earlier in disease progression. For example, methodologies have emerged that facilitate the analysis of β-cell death in situ using the quantification of circulating unmethylated insulin or amylin promoter DNA from blood samples. These particular types of DNA are found exclusively in β-cells (Akirav et al. 2011, Lehmann-Werman et al. 2016, Olsen et al. 2016) and their release into circulation provides specific biomarkers of β-cell death in vivo. Using this approach, studies determined that unmethylated insulin and amylin DNA are increased in serum from individuals with newly diagnosed T1D and that it remains elevated eight weeks after disease diagnosis (Akirav et al. 2011, Fisher et al. 2013, Olsen et al. 2016, Neyman et al. 2019). Other work failed to identify increased unmethylated insulin DNA in those with recent onset T1D (Neiman et al. 2020), suggesting that heterogeneity in the timeline (relapsing-remitting) or mode of active β-cell demise may exist in late T1D pathogenesis. The use of this technique also revealed that unmethylated insulin DNA is elevated following islet transplantation, indicative of β-cell death in this setting (Husseiny et al. 2014, Neiman et al. 2020). Although additional work is needed to understand β-cell death in early T1D pathogenesis, these studies reinforce findings from pancreas sections that show increased rates of β-cell death in T1D and support a model wherein β-cell death contributes to β-cell loss, insulin insufficiency, and hyperglycemia in this disease.

Approaches to identify biomarkers of β-cell demise early in T1D pathogenesis may also provide great clinical and therapeutic value. Such biomarkers could be used to identify individuals at risk for T1D and allow for initiation of β-cell protective therapies at a time when significant β-cell mass still exists. In this context, the presence of islet autoantibodies including insulin (IAA), glutamic acid decarboxylase (GAD), protein tyrosine phosphatase (IA2), and zinc transporter 8 (ZnT8) are being used as biomarkers for T1D disease progression (Ziegler et al. 2013). In a study of non-diabetic individuals positive for at least 2 islet autoantibodies, a 14-day treatment with an anti-CD3 monoclonal antibody significantly prolonged the time to T1D onset (Sims et al. 2021), highlighting the therapeutic utility of early detection of β-cell demise. In addition to islet autoantibodies, other biomarkers of β-cell failure including unmethylated insulin and amylin promoter DNA, proinsulin:C-peptide ratio, and miRNAs (miR375, miR21, miR34a, miR146a) are being investigated toward the development of reliable biomarker panels for the detection of β-cell demise in early T1D (Roggli et al. 2010, Tersey et al. 2012, Erener et al. 2013, Mirmira et al. 2016). Moreover, techniques to quantify cell mass and cell death in vivo are emerging (Cordeiro et al. 2010, Khanna et al. 2010, Yamazaki et al. 2020, Lehrstrand et al. 2024), and future studies might apply these in vivo imaging methodologies to better understand the timing and extent of β-cell death in early T1D.

Mechanisms of β-cell death

Given the current limitations of studying β-cell death in human disease, much of our understanding of β-cell demise in T1D is derived from experimental models. Numerous in vitro and in vivo studies have shed light on the occurrence and mechanisms of β-cell death in T1D (as reviewed here), as well as intrinsic properties of β-cells (such as high metabolic demand, high secretory demand, and limited antioxidant capacity) that make them susceptible to cytotoxic insults (Grankvist et al. 1981, Kulkarni et al. 2022). Many β-cell cytotoxic stimuli have been identified, including proinflammatory cytokines, autoimmunity, glucotoxicity, oxidative stress, and ER stress. Here, we focus on the various cell-intrinsic mechanisms of β-cell death that are activated in response to such stimuli. With respect to β-cell-intrinsic death pathways, most work has focused on apoptosis and necrosis, which we categorize as established mechanisms of β-cell death herein. In addition, several mechanisms of programmed cell death (PCD) that are distinct from apoptosis and necrosis have emerged in the last two decades, but the significance of these forms of cell demise to diabetogenic β-cell death is unclear. In this section, we examine the literature that underlies our current understanding of established forms of cell death such as apoptosis and necrosis, as well as emerging forms of cell death such as necroptosis, ferroptosis, and pyroptosis. We also explore literature that suggests these types of cell death are germane to β-cells and discuss considerations for accurate experimental characterization of various forms of cell death.

Established mechanisms of β-cell death

Apoptosis

Apoptosis is the most well-known and well-characterized form of PCD (see Table 1 for a list of abbreviations). It is tightly regulated by cell-intrinsic and cell-extrinsic signaling mechanisms (Elmore 2007), and it is required for several physiological processes including development, tumor suppression, and removal of damaged or unwanted cells during normal cellular turnover (Kerr et al. 1972, Vaux & Korsmeyer 1999, Norbury & Hickson 2001). Apoptotic programs are critically dependent on caspases, a family of cysteine-aspartic proteases that are activated in response to specific stimuli (Chen & Wang 2002). With respect to apoptosis signaling, caspases can be categorized as initiator caspases (caspase 2, 8, 9, and 10) or executioner caspases (caspase 3, 6, and 7) (Cohen 1997, Chen & Wang 2002). Upon appropriate stimulation, initiator caspases are activated by autoproteolysis; these initiator caspases activate executioner caspases via proteolysis, and executioner caspases then initiate apoptotic programs via proteolysis of key structural, cell cycle, and signal transduction proteins (Parrish et al. 2013). Cells undergoing apoptosis exhibit cytoplasmic shrinkage, chromatin condensation, DNA fragmentation, and plasma membrane blebbing (Elmore 2007). These membrane blebs encompass cellular contents to form apoptotic bodies that are then taken up by phagocytes through efferocytosis; this feature of apoptosis is critical in that it allows apoptotic cells to be removed in a non-inflammatory manner (Boada-Romero et al. 2020).

Table 1

Cell death-related abbreviations.

Abbreviationa Expanded form
PCD Programmed cell death
T1D Type 1 diabetes
T2D Type 2 diabetes
TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling
STZ Streptozotocin
HFD High fat diet
IL Interleukin
TNFα Tumor necrosis factor alpha
TNFR Tumor necrosis factor receptor
IFNγ Interferon gamma
RIPK1 Receptor interacting protein kinase 1
RIPK3 Receptor interacting protein kinase 3
MLKL Mixed lineage kinase domain-like pseudokinase
DAMPs Danger-associated molecular patterns
PAMPs Pathogen-associated molecular patterns
TRADD Tumor necrosis factor receptor type 1-associated death domain
cIAP1/2 Cellular inhibitors of apoptosis proteins
LUBAC Linear ubiquitin chain assembly complex
IFNGR Interferon gamma receptor
TLR Toll-like receptors
TRAILR TNF-related apoptosis-inducing ligand receptor
RHIM RIP homotypic interaction motif
Nec-1 Necrostatin-1
NFκB Nuclear factor κB
GSH Glutathione
GPX4 Glutathione peroxidase 4
NADPH Nicotinamide adenine dinucleotide phosphate
ROS Reactive oxygen species
LDH Lactate dehydrogenase
Fer-1 Ferrostatin
GSDM Gasdermin
HMGB1 High mobility group box 1
NLRP3 NLR family pyrin domain containing 3
PRR Pattern-recognition receptors
NLR NOD-like receptor
Poly I:C Polyinosinic:polycytidylic acid

aKey abbreviations mentioned within the review in the order mentioned.

There is a great deal of evidence that β-cells undergo apoptosis in response to diabetes-relevant cell death stimuli (Mandrup-Poulsen 2001, Hui et al. 2004, Thomas et al. 2009). Studies performed by Yamada et al. in 1999 found that Fas signaling in β-cells results in cell death characterized by chromatin condensation, nucleolar disintegration, DNA fragmentation, and annexin V positivity, hallmarks of apoptosis (Yamada et al. 1999). In 2005, Liadis and colleagues found that caspase 3-deficient mice were protected from hyperglycemia following multiple low-dose streptozotocin (STZ) treatment, and this was related to reduced β-cell loss (Liadis et al. 2005). Similarly, a study utilizing caspase 3-deficient islets or a small molecule caspase 3 inhibitor found that caspase 3 activity mediates amyloid-induced β-cell death (Li et al. 2008). Studies of proinflammatory cytokine-induced β-cell death have also identified apoptosis as a key mediator of β-cell cytotoxicity, with studies finding that a cytokine cocktail (IL-1β + IFNγ + TNFα) elicits β-cell death in association with caspase 3 activation (Grunnet et al. 2009), and that suppressor of cytokine signaling-1 (SOCS-1) mediates IL-1β + IFNγ + TNFα-induced β-cell death in a caspase 3-dependent fashion (Zaitseva et al. 2009). In a study of β-cell-specific loss of caspase 8, islets were protected from Fas ligand and ceramide-induced cell death in vitro, and mice were protected from STZ-induced hyperglycemia in vivo (Liadis et al. 2007). Notably, this study also showed that the loss of caspase 8 in β-cells results in elevated rates of islet cell death and glucose intolerance in aged chow-fed mice in vivo, indicating distinct roles for β-cell caspase 8 under these conditions (Liadis et al. 2007).

Apoptosis has also been identified as a key contributor to β-cell demise in the context of islet transplantation (Emamaullee et al. 2007, McCall & Shapiro 2012, Pepper et al. 2017). For example, treatment of isolated mouse islets with a small molecule pan-caspase inhibitor (zVAD) prior to transplantation combined with 5 days of zVAD treatment after transplant resulted in significantly improved rates of euglycemia following islet transplantation, and the glycemic benefit of zVAD-FMK treatment remained 1 year after islet transplantation (Emamaullee et al. 2007). Similarly, Pepper and colleagues cultured human islets with a pan-caspase inhibitor (F573) for 24 hours prior to transplantation, then administered F573 for 5 days post transplantation in immunodeficient mice. This approach resulted in improved blood glucose during an intraperitoneal glucose tolerance test (IPGTT) for up to 100 days post transplant and was accompanied by the preservation of β-cell mass and viability (Pepper et al. 2017). Other studies using small molecule caspase inhibitors to protect transplanted islets have observed similar results (Emamaullee et al. 2008, McCall et al. 2011). In addition, several studies found that the blockade of IL-1β and TNFα, classical apoptotic stimuli, significantly improves islet transplant outcomes (Bellin et al. 2008, Matsumoto et al. 2011, Onaca et al. 2020).

These and other studies demonstrate that β-cells undergo apoptosis in response to diabetes-relevant cell death stimuli.

Necrosis

β-Cells are also susceptible to non-apoptotic forms of cell death such as necrosis (Hoorens et al. 2001, Fehsel et al. 2003). Necrosis was first described by Rudolf Virchow in 1858 and is considered distinct from mechanisms of regulated cell death or PCD (Majno & Joris 1995). Necrosis is typically understood as a form of unprogrammed or accidental cell death that results from cellular damage, infection, or trauma (Buja et al. 1993). In contrast to apoptosis, necrosis is characterized by lytic loss of membrane integrity that leads to the unregulated release of cell contents (Buja et al. 1993, Majno & Joris 1995). Given that these cell contents are not packaged in apoptotic bodies or membrane blebs, this release of cell contents is inflammatory in nature (Davidovich et al. 2014, Rock & Kono 2008). Thus, necrosis can be characterized by the release of cellular factors such as high mobility group box 1 (HMGB1), a nuclear-localized protein that is normally associated with chromatin (Raucci et al. 2007). In addition, morphological hallmarks of necrotic cell death such as karyolysis, karyorrhexis, and pyknosis, all of which relate to altered morphology of nuclear DNA within the dying cell body, can be used to identify necrotic cells (Anthony 1990). Such changes are not observed in apoptosis, where proteases and nucleases break down nuclear contents into apoptotic bodies (Anthony 1990).

Several studies have found that necrosis contributes to β-cell loss in diabetes. Notably, studies by Fehsel et al. examined STZ-treated mouse islets in vitro and prediabetic diabetes-prone BB rat pancreas sections ex vivo using electron microscopy, quantification of DNA damage, and annexin V staining (Fehsel et al. 2003). These studies identified only a small number of cells expressing markers of apoptosis, but β-cells with characteristics of necrosis were significantly more abundant (Fehsel et al. 2003). When evaluating mechanisms of IL-1β-mediated cell death in rat β-cells, Steer and colleagues found evidence of necrosis, including loss of viability, a lack of caspase 3 activity, annexin V positivity, and robust HMGB1 release, whereas the apoptosis inducer camptothecin strongly induced caspase 3 activity and annexin V positivity, but not HMGB1 release (Steer et al. 2006). In addition, two studies using a rat β-cell line and rat islets found that IL-1β + IFNγ-induced β-cell cytotoxicity was not associated with increased caspase 3 activity or annexin V positivity (Collier et al. 2011, 2006). Similarly, TNFα + IFNγ treatment was observed to induce mouse islet cell death in a caspase 3-independent manner (Irawaty et al. 2002), and IFNγ + synthetic double-stranded RNA (poly I:C) induces NO-dependent necrosis of rat islet cells (Scarim et al. 2001). Necrosis has also been identified as a mediator of β-cell cytotoxicity in islet transplantation, with studies observing characteristics of islet cell necrosis such as pyknotic nuclei and HMGB1 release following islet graft failure (Biarnés et al. 2002, Cheng et al. 2017, Gebe et al. 2018).

Together, these studies provide evidence that β-cells undergo necrosis in response to diabetogenic cell death stimuli.

Emerging mechanisms of β-cell death

Although necrosis has traditionally been viewed as an unregulated, accidental form of cell death, many of the studies described above identified programmed β-cell death that is morphologically consistent with necrosis. How might this occur when all mechanisms of PCD have customarily been considered apoptosis (Alberts et al. 2002, Elmore 2007)? An explanation has surfaced from studies that identified and characterized mechanisms of regulated necrosis which occur downstream of programmed signaling events, are distinct from apoptosis, and constitute novel forms of PCD (Proskuryakov et al. 2003, Linkermann & Green 2014, Berghe et al. 2014, Conrad et al. 2016). These discoveries have been aided by improved definitions of distinct cell death signaling pathways (Galluzzi et al. 2009) and have led to renewed interest in understanding molecular mechanisms of PCD and their roles in human disease, including diabetes (Bruni et al. 2018, Yang et al. 2020, Tonnus et al. 2021, Contreras et al. 2022). We propose that novel mechanisms of PCD, such as necroptosis, ferroptosis, and pyroptosis, may contribute to T1D pathogenesis, not only as end-stage mechanisms of β-cell loss, but potentially as early-stage mechanisms of islet inflammation and β-cell autoimmunity. In the following section, we review literature on basic mechanisms of regulated necrosis, including necroptosis, ferroptosis, and pyroptosis. We also examine recent studies linking these mechanisms of inflammatory PCD to β-cell cytotoxicity and discuss the need for additional research to examine the relevance of these mechanisms to diabetogenic β-cell death.

Necroptosis

Necroptosis is a regulated form of necrotic cell death that is mediated by receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and mixed lineage kinase domain-like pseudokinase (MLKL) (Linkermann & Green 2014) (Fig. 1). Necroptosis was first identified as a form of cell death that occurs downstream of tumor necrosis factor receptor 1 (TNFR1) signaling when caspase activity is inhibited (Vercammen et al. 1998, Liu et al. 2003), and the pathway is most well-defined in this context. In canonical necroptosis signaling, TNFα binds to TNFR1, and molecules including TNFR-associated death domain (TRADD), TNFR-associated factor 2 (TRAF2), cellular inhibitors of apoptosis proteins (cIAP1/2), linear ubiquitin chain assembly complex (LUBAC), and RIPK1 are recruited to form complex I (Wilson et al. 2009). RIPK1 undergoes extensive ubiquitination and deubiquitination in complex I, and this process regulates its pro-survival and pro-death functions (Brenner et al. 2015, Conrad et al. 2016, Li et al. 2020). In its pro-survival role, linear ubiquitination of RIPK1 in complex I is crucial for NF-κB activation, leading to the upregulation of pro-survival gene expression (Gerlach et al. 2011, Berghe et al. 2014). Deubiquitination of RIPK1 facilitates the formation of complex II with caspase-8 and Fas-associated death domain (FADD)(Brenner et al. 2015). When caspases are active, caspase 8 in complex IIa or IIb inactivates RIPK1 (Lin et al. 1999) and RIPK3 (Feng et al. 2007), eliciting caspase 3/7 activation and apoptosis (Brenner et al. 2015). When caspases are inactive, however, complex IIc (the necrosome) forms via phosphorylation and interaction of RIPK1 and RIPK3, leading to the recruitment and phosphorylation of MLKL, the terminal effector of necroptosis (Cai et al. 2014, Dondelinger et al. 2014). This RIPK1–RIPK3–MLKL phosphorylation cascade leads to a conformational change in MLKL that exposes its four-helix bundle domain and leads to its oligomerization, membrane translocation, and loss of cell membrane integrity (Hildebrand et al. 2014). Although the precise mechanisms of necroptosis execution downstream of MLKL oligomerization are not fully established, membrane-localized MLKL pore formation results in cell swelling and lysis (Cai et al. 2014). This lysis releases cell contents including danger-associated molecular patterns (DAMPs) that promote inflammation and immune responses (Kaczmarek et al. 2013, Murai et al. 2018). In addition to TNFR1, several other receptors including interferon-gamma receptor (IFNGR), toll-like receptors (TLRs), Fas receptor, and TNF-related apoptosis-inducing ligand receptor (TRAILR) have been implicated in necroptosis signaling and are thought to converge at the level of necrosome formation (Holler et al. 2000, Meurette et al. 2005, Thapa et al. 2013, Linkermann & Green 2014, Berghe et al. 2014).

Figure 1
Figure 1

Basic mechanisms of necroptosis signaling. The binding of TNFα to TNFR1 initiates the recruitment of TRAF2, TRADD, RIPK1, cIAP1/2, and LUBAC to form complex I at the receptor. Under appropriate stimulation, RIPK1 associates with pro-caspase 8, FADD, and RIPK3 to form complex IIb. When pro-caspase 8 is inactive, RIPK1 activates RIPK3 via phosphorylation, and activated RIPK3 recruits and phosphorylates MLKL in complex IIc (the necrosome). Phosphorylation of MLKL leads to its conformational change, oligomer formation, membrane translocation, and disruption of membrane integrity, resulting in cell lysis. Other receptors including IFNγR, TRAILR, FAS, and TLRs have also been found to contribute to necrosome formation and necroptosis.

Citation: Journal of Endocrinology 262, 2; 10.1530/JOE-23-0378

Stimuli known to induce necroptosis have previously been observed to elicit caspase-independent β-cell death. For example, TNFα + IFNγ (Irawaty et al. 2002), IL-1β + IFNγ (Collier et al. 2006, 2011), and IFNγ + double stranded RNA (Scarim et al. 2001) have been found to induce β-cell death with necrotic morphology in the absence of caspase activation, consistent with regulated necrosis. Although these studies did not examine whether such mechanisms of β-cell death are biochemically consistent with necroptosis, they provide early evidence that diabetes-relevant stimuli can trigger programmed β-cell death distinct from caspase-mediated apoptosis. In addition, recent biochemical and transcriptomics studies have identified RIPK1, RIPK3, and MLKL expression in rodent and human β-cells (Thompson et al. 2019, Yang et al. 2020, Contreras et al. 2022, Elgamal et al. 2023), again rationalizing the need for additional research into the role of necroptosis signaling in β-cell cytotoxicity.

RIPK1 is a key upstream regulator of necroptosis. It is a multifunctional protein with an N-terminal kinase domain, a C-terminal death domain, and a receptor-interacting protein (RIP) homotypic interaction motif (RHIM) that mediates interactions with RIPK3 and other RHIM-containing molecules (Li & Yuan 2023). Human Pancreas Analysis Program (HPAP) data indicate that Ripk1 gene expression is increased 2.7-fold in β-cells from individuals with T1D compared to those without diabetes (Kaestner et al. 2019), and several studies have investigated the role of RIPK1 kinase function in β-cell cytotoxicity using small molecule RIPK1 kinase inhibitors. One study evaluated the effects of necrostatin-1 (Nec-1) on human islets cultured in low-nutrient and low-oxygen conditions, finding that Nec-1 treatment reduced dsDNA and uric acid release from human islets, indicative of decreased lytic cell death (Paredes-Juarez et al. 2015). Another study in rodent β-cell lines and mouse islets found that inhibition of RIPK1 kinase function with Nec-1 protects from NO donor-induced β-cell cytotoxicity in vitro (Tamura et al. 2011). In a study of young porcine islets, Nec-1 was found to improve islet viability in response to NO or hypoxia in a dose-dependent manner in vitro (Lau et al. 2020) and another recent study found that Nec-1 treatment prevented β-cell loss in a zebrafish model of overnutrition and insulin resistance (Yang et al. 2020). Our recent study found that CRISPR-mediated Ripk1 gene editing protects NIT-1 β-cells from TNFα-induced cell death both in the presence and absence of caspase 3/7 activation (Contreras et al. 2022). In contrast to these studies, mice harboring a Ripk1S25D/S25D mutation that mimics inhibitory phosphorylation of RIPK1 were not protected from hyperglycemia following STZ or HFD (Takiishi et al. 2023). Considering that RIPK1 has several inhibitory and activating phosphorylation sites as well as actions in multiple tissues (Li & Yuan 2023), studies evaluating Ripk1 kinase dead (Ripk1D138N/D138N) or Ripk1 tissue-specific knockout (Ripk1flox/flox) mice may shed additional light on the role of RIPK1 in diabetogenic β-cell decline in vivo. In sum, these data indicate that RIPK1 plays a key role in regulating β-cell fate and suggest that inhibition of RIPK1 kinase activity could protect β-cells from diabetogenic insults.

RIPK3 is a major downstream phosphorylation target of RIPK1 that is known to promote inflammation and cell death signaling in non-islet cell types (Newton et al. 2014, Lawlor et al. 2015, Orozco & Oberst 2017). In comparison to RIPK1, RIPK3 retains an N-terminal kinase domain and a RIP homotypic interaction motif (RHIM) but does not contain a death domain (Moriwaki & Chan 2017). Human Pancreas Analysis Program (HPAP) data indicate that Ripk3 gene expression is increased 2.2-fold in β-cells from individuals with T1D compared to those without (Kaestner et al. 2019), and recent studies have begun to decipher the roles of RIPK3 in β-cell inflammation and cell death signaling. Our recent observations revealed that β-cell lines and isolated mouse islets express RIPK3 at levels similar to other necroptosis-susceptible cells (Contreras et al. 2022). This work also revealed that CRISPR-mediated gene editing of Ripk3 protects NIT-1 β-cells from TNFα-induced cell death independent of caspase 3/7 activation in vitro, and that RIPK3 knockout mice are protected from STZ-induced hyperglycemia in vivo (Contreras et al. 2022). These data suggest that TNFα elicits both caspase-mediated apoptosis and RIPK3-mediated necroptosis in NIT-1 β-cells, with necroptosis serving as a secondary form of cell death when caspase activation is insufficient to elicit apoptosis.

RIPK3 also promotes inflammation in a cell death-independent manner in non-islet cell types (Orozco & Oberst 2017), and a recent study found that RIPK3 contributes to inflammation in zebrafish, mouse, and human β-cells (Yang et al. 2020). This work found that endoplasmic reticulum (ER) stress activates RIPK3 to elicit NF-κB-mediated proinflammatory gene expression in cultured β-cells and that RIPK3 kinase inhibition protects mouse islets from palmitate-induced β-cell dysfunction in vitro (Yang et al. 2020). Islet inflammation, β-cell dysfunction, and β-cell loss were found to occur in a RIPK3-dependent manner in a zebrafish model of β-cell cytotoxicity, and human islets grafted in hyperglycemic mice underwent a marked increase in RIPK3 and NF-κB activation that was accompanied by increased islet macrophage infiltration (Yang et al. 2020). Moreover, RIPK3 was found to promote amyloid-associated islet inflammation and β-cell cytotoxicity in a humanized mouse model of islet amyloidosis (Mukherjee et al. 2024). Although further investigation is needed to clarify the specific roles of RIPK3 in islet inflammation and β-cell death, these data indicate that therapies targeting RIPK3 could protect β-cells from diabetes-relevant cytotoxic stimuli.

Necroptosis has also been identified as a mediator of graft failure following transplantation, including in the setting of heart, kidney, and lung transplants (Lau et al. 2013, Pavlosky et al. 2014, Kim et al., 2018). In a study of islet transplantation, porcine islets treated with the RIPK1 kinase inhibitor Nec-1 were found to exhibit increased insulin content and insulin secretion following glucose stimulation in vitro, and treatment of islets with Nec-1 prior to transplantation in diabetic athymic mice resulted in shorter times to normoglycemia and higher plasma insulin levels in vivo (Lau et al., 2021). In contrast, a study evaluating transplantation of RIPK3 deficient mouse islets found them to be normally susceptible to CD4+ T cell-mediated destruction in vivo (Zhao et al., 2015). Additional studies are warranted to understand the role of necroptosis in islet graft failure.

In sum, the literature reviewed here indicates that necroptosis signaling components such as RIPK1 and RIPK3 play important roles in regulating β-cell fate. However, additional in vitro and in vivo studies are needed to understand the mechanisms of β-cell necroptosis and its relevance to diabetes pathogenesis.

Ferroptosis

Ferroptosis is a non-apoptotic form of PCD linked to lethal lipid peroxidation from iron-dependent ROS accumulation (Xie et al. 2016) (Fig. 2). It is identified morphologically by a unique intact nucleus, reduced mitochondrial volume, increased lipid bilayer membrane density and rupture, and reduced mitochondrial cristae (Xie et al. 2016). Ferroptosis was identified in 2012 when Dixon et al. studied the mechanisms and morphology of cell death following treatment of fibrosarcoma cells with erastin (Dixon et al. 2012), a small molecule that prevents the synthesis of the antioxidant glutathione via inhibition of the cystine–glutamate antiporter system XC and reduced intracellular cysteine (Dixon et al. 2012, Sun et al. 2018). Cysteine is a rate-limiting substrate for the biosynthesis of glutathione (GSH), and GSH is a cofactor of glutathione peroxidase 4 (GPX4), a major lipid peroxide scavenger (Brigelius-Flohé & Maiorino 2013). Erastin itself was discovered by Dolma and colleagues while screening for compounds that could kill human tumor cells and was found to be lethal to tumor cells with small T oncoproteins and oncogenic alleles, although the dead cells did not exhibit hallmarks of apoptosis such as chromatin condensation or DNA fragmentation (Dolma et al. 2003). In line with these findings, Yang et al. found that erastin treatment decreased NADPH oxidation and GPX4 activity in human foreskin fibroblasts, consistent with cytotoxic lipid peroxidation (Yang et al. 2014). Moreover, inhibition of GPX4 activity, increased ROS generation, and increased lipid peroxidation were identified as common mechanisms elicited by several ferroptosis-inducing small molecules including erastin and RSL3, a ferroptosis inducer and potent inhibitor of GPX4 (Yang et al. 2014, Sui et al. 2018). In sum, the absence of sufficient GPX4 activity leads to iron-dependent ROS production, lipid peroxidation, and disruption of plasma membrane integrity, leading to ferroptotic cell death (Bruni et al. 2018).

Figure 2
Figure 2

Basic mechanisms of ferroptosis signaling. Ferroptosis is triggered by an imbalance of intracellular free iron and cell antioxidant capacity, eventually leading to excessive lipid peroxidation and cell lysis. Cystine is essential for glutathione (GSH) production, and GSH is a cofactor for glutathione peroxidase 4 (GPX4), a critical lipid peroxidase. Iron is taken up by the cell, and stable Fe3+ can be converted to free redox-active Fe2+. This transition facilitates the accumulation of reactive oxygen species (ROS) through the Fenton reaction with H2O2, and GPX4 is needed to counteract lipid peroxidation that arises through this process. Stimuli that increase intracellular free iron, reduce cellular cystine uptake, or decrease cellular antioxidant capacity can lead to ferroptosis.

Citation: Journal of Endocrinology 262, 2; 10.1530/JOE-23-0378

Studies to understand the role of ferroptosis in β-cell death are now emerging. β-cells naturally express low levels of antioxidant enzymes compared to other hormone-secreting cells (Grankvist et al. 1981, Lei & Vatamaniuk 2011), leaving them vulnerable to oxidative stress and, potentially, ferroptosis. Following treatment with erastin or RLS3, human islets exhibited decreased glucose-stimulated insulin release and viability (as measured by LDH release), indicating they are susceptible to ferroptosis (Bruni et al. 2018). Moreover, treatment with ferrostatin (Fer-1, a ferroptosis inhibitor) for 24 hours protected human islets from erastin-induced impairments in insulin secretion and viability (Bruni et al. 2018, Miotto et al. 2020). Another recent study evaluated the role of ferroptosis in high glucose-, proinflammatory cytokine-, hydrogen peroxide (H2O2)- and STZ-induced β-cell death in vitro (Stancic et al. 2022). The authors found that each of these stimuli increased cell death in RIN-5F rat insulinoma cells, and that this cytotoxicity occurred in conjunction with increased abundance of ROS, lipid peroxides, and iron as well as decreased GPX4 expression (Stancic et al. 2022). Cotreatment with Fer-1 rescued RIN-5F cell death caused by high glucose, H2O2, or STZ but failed to protect from proinflammatory cytokine-induced death (Stancic et al. 2022). With respect to in vivo studies, STZ treatment was found to increase islet lipid peroxidation and elicit β-cell loss in C57BL/6 mice, and these effects were associated with downregulation of GPX4 and NRF2, a transcription factor that regulates expression of antioxidant genes (Markelic et al. 2023). Treatment with Fer-1 was found to reduce lipid peroxidation, upregulate GPX4 and NRF2, and ameliorate β-cell loss in STZ-treated mice (Markelic et al. 2023). A separate study investigated the role of cystine import in β-cell cytotoxicity using a mouse model deficient for Slc7a11, a cystine/glutamate antiporter needed for GSH production and GPX4 activity (de Baat et al. 2023). Loss of Slc7a11 reduced levels of cystine and glutathione in mouse islets and led to reduced insulin secretion, downregulation of β-cell identity genes, and an increase in ER stress markers (de Baat et al. 2023). These observations appeared to be dependent on β-cell phenotypes, as myeloid-specific deletion of Slc7a11 did not elicit such changes (de Baat et al. 2023).

Iron homeostasis also plays a critical role in ferroptosis. Transferrin and transferrin receptors transport iron (Fe3+) into the cytosol for storage and controlled release by ferritin in a process that regulates iron balance (Plays et al. 2021, Ems et al. 2023). Excess free iron (Fe2+) reacts with hydrogen peroxide through the Fenton reaction, generating toxic hydroxyl radicals that can attack the lipid membrane (Xie et al. 2016), and several studies have linked elevated iron concentrations to β-cell cytotoxicity. Therefore, understanding β-cell iron homeostasis could lead to a greater appreciation of the mechanisms that elicit ferroptosis in β-cells. In 1994, a mouse model of hemochromatosis (Hfe −/−) was examined and found to have a 72% increase in islet iron content compared with wild-type mice, and this was associated with reduced Ins1 and Ins2 expression and diminished insulin content in vitro (Cooksey et al. 2004). At 6-8 months of age, Hfe −/− mice exhibited reduced islet size, increased islet caspase 3 activity, and increased islet TUNEL staining compared to wild-type mice (Cooksey et al. 2004). Masuda et al. found that exposure of rat islets to iron sucrose led to oxidative stress and pancreatic islet cell death in a dose-dependent manner (Masuda et al. 2013), and another study found that treatment of MIN6 cells with high iron concentrations resulted in a 15-fold increase in cellular iron content, elevated levels of lipid peroxidation, decreased insulin content and secretion, and reduced viability (Blesia et al. 2021).

The role of ferroptosis in islet transplantation has also been an area of research interest, but its role in islet graft failure remains unclear. Although Bruni et al. found that human islets are susceptible to ferroptosis in vitro, neither chemical induction (with erastin) nor inhibition (with Fer-1) of ferroptosis altered human islet engraftment following transplant in immunodeficient mice (Bruni et al., 2018). However, an early study found that desferrioxamine (DFO, an iron chelator) prevented islet allograft damage (Bradley et al., 1986). Although the mechanisms underlying this protection were not determined, the results are suggestive of a role of ferroptosis (Bradley et al., 1986). In more recent studies, encapsulated human islets pretreated with DFO were found to have increased insulin release following transplant in STZ-treated mice (Vaithilingam et al., 2010), and bilirubin was found to decrease islet graft ferroptosis via effects on GPX4 expression, NRF2 expression, and iron chelation (Yao et al., 2020).

Together, these studies have advanced our understanding of β-cell ferroptosis. However, additional studies using physiologically relevant models of diabetes are needed to clarify the importance of β-cell ferroptosis in diabetes pathogenesis.

Pyroptosis

Pyroptosis is an inflammatory form of lytic PCD that occurs in response to infection and generates an immune response (Bergsbaken et al. 2009). A central molecular player in pyroptosis is gasdermin D (GSDMD), a protein that contains an N-terminal pore-forming domain and a C-terminal repressor domain separated by a linker region (Cookson & Brennan 2001). Activation of inflammatory caspases (caspase 1, 4, 5, and 11) elicits cleavage of the GSDMD linker domain, releasing N-terminal GSDMD (GSDMD-N) that facilitates membrane pore formation, allowing release of inflammatory molecules including HMGB1 and IL-1β, and promoting cell death (Kuang et al. 2017, Volchuk et al. 2020, Phulphagar et al. 2021) (Fig. 3). Pyroptosis has been studied in the context of immune responses to infection (Lara-Tejero et al. 2006) and inflammatory disease pathogenesis (Wu et al. 2022). Like apoptosis, pyroptosis exhibits DNA damage, chromatin condensation, and formation of membrane blebs (Bergsbaken et al. 2009). However, pyroptosis is a distinct form of lytic cell death associated with membrane leakage, flattening of the cytoplasm, and inflammation (Chen et al. 2016).

Figure 3
Figure 3

Basic mechanisms of canonical pyroptosis signaling. In canonical pyroptosis, extracellular signals such as danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) elicit priming via upregulation of the NLRP3 inflammasome, pro-IL-1β, and pro-IL-18. During the activation phase, stimuli trigger the NLRP3 inflammasome leading to caspase 1 activation. Caspase 1 then cleaves gasdermin D (GSDMD), resulting in the formation of GSDMD-N, which forms pores in the cell membrane. Simultaneously, caspase 1 generates mature IL-1β and IL-18, which are released through GSDMD-N pores, culminating in pyroptosis.

Citation: Journal of Endocrinology 262, 2; 10.1530/JOE-23-0378

In canonical pyroptosis signaling, PAMPs and DAMPs are recognized extracellularly by pattern-recognition receptors (PRRs) such as TLRs. In this priming phase of pyroptosis, extracellular stimulation of PRRs upregulates the expression of NLR family pyrin domain containing 3 (NLRP3) and cytokines in the cell. In the activation phase, cytosolic PRRs such as NOD-like receptors (NLRs) are activated by stimuli such as ATP, nucleic acids, crystalline substances, and intracellular DAMPs and PAMPs, leading to NLR-directed assembly of the NLRP3 inflammasome (Song et al. 2022). The relationship between caspase 1 activity and pyroptosis was uncovered when Salmonella-induced cell death was found to be blocked by a small molecule caspase 1 inhibitor (Brennan & Cookson 2000). It was later revealed that a complex including caspase 1, caspase 5, PYCARD, and NALP1 controls the activation of inflammatory caspases, and this complex was termed the inflammasome (Martinon et al. 2002). Depletion of PYCARD or NALP1 was found to decrease caspase 1 and caspase 5 activation and to reduce pro-IL-1β processing (Martinon et al. 2002). Agostini and colleagues showed that NLRP3 contributes to inflammasome assembly, with NLRP3 associating with PYCARD, CARD8, and caspase 1 to generate an inflammasome complex with high pro-caspase 1 and pro-IL-1β processing activity (Agostini et al. 2004). Activated caspase 1 cleaves GSDMD, promoting GSDMD-N pore formation in the plasma membrane (Bergsbaken et al. 2009). The importance of GSDMD in pyroptosis was identified using CRISPR screens (Shi et al. 2015) and GSDMD-null mice (Russo et al. 2016). In both cases, Gsdmd deficient BMDMs exhibited reduced IL-1β release and cytolysis following treatment with known inducers of pyroptosis (Shi et al. 2015, Russo et al. 2016). Once GSDMD-N pores are formed on the plasma membrane, inflammatory mediators including HMGB1, IL-1β, and IL-18 are released to elicit immune responses (Fink & Cookson 2006, Russo et al. 2016).

The growing understanding of pyroptosis has led to several studies of this form of cell death in β-cells. Human Pancreas Analysis Program (HPAP) data indicates that GSDMD gene expression is increased 2.2-fold in β-cells from individuals with T1D compared to non-diabetic individuals (Kaestner et al. 2019), suggesting a role for GSDMD in β-cell cytotoxicity in this disease. In line with these findings, a recent study found that GSDMD is upregulated at both the RNA and protein levels in response to proinflammatory cytokines (IL-1β+IFNγ+TNFα) in human β-cells (EndoC-βH5) and human islets (Frørup et al. 2024). In addition, a 2021 study investigating the effect of miR-17-5p on β-cell cytotoxicity found that miR-17-5p improved glucose homeostasis in C57B/L6 mice subjected to HFD and STZ in vivo, and this phenotype was associated with increased pancreas insulin area, decreased caspase 1 activation, and reduced GSDMD-N expression ( Liu et al. 2021b ). Irisin, a hormone released from skeletal muscle that mediates beneficial effects of exercise (Vaughan et al. 2014), was found to reduce caspase 1 activity as well as GSDMD-N, IL-1β, and IL-18 protein expression, indicative of protection from pyroptosis signaling (Tan et al. 2023). Other molecules, including the natural products salidroside and emodin, have also been shown to decrease GSDMD expression in INS-1 cells following exposure to high glucose (Xing et al. 2022, Zhou et al. 2023), and the sodium/glucose cotransporter 2 inhibitor (SGLT2i) empagliflozin was shown to reduce NLRP3, caspase 1, and GSDMD expression in mouse β-cells (βTC-6) in response to high glucose, an effect that was also observed in islets of empagliflozin-treated db/db mice (Liu et al. 2021a ).

Although direct links between pyroptosis and islet graft failure have yet to be established, evidence indicates pyroptosis-relevant molecules such as the NLRP3 inflammasome and IL-1β are involved in islet transplant demise. For example, rat islets transplanted under the kidney capsule of immunodeficient mice exhibited significant increases in NLRP3 and IL-1β expression 2 days post transplant (Lavallard et al. 2020), and transplantation of either Nlrp3−/− or Casp1−/− islets in STZ-treated mice accelerated the restoration of normoglycemia and improved glucose tolerance compared to WT islets (Wrublewsky et al. 2022).

Together, these studies suggest that pyroptosis contributes to diabetes-relevant β-cell cytotoxicity. Additional studies in disease-relevant models are needed to understand the importance of pyroptosis in diabetogenic β-cell demise.

Heterogeneity of β-cell death responses

In this review, we’ve examined studies of established and emerging mechanisms of cell death and their relevance to β-cell cytotoxicity. Although one may assume that a cell death stimulus elicits a single mode of death, evidence indicates that cell death responses are heterogeneous within a cell population. Indeed, several studies have observed that a specific cell death stimulus induces both apoptosis and necrosis concurrently within a population of cells. For example, glutamate was found to elicit early necrosis in a subset of neurons (characterized by nuclear swelling and release of cellular debris), and cells spared from early necrosis later underwent apoptosis, as evidenced by the formation of apoptotic bodies and chromatin degradation (Ankarcrona et al. 1995). Acetaminophen overdose causes cell death in mouse hepatocytes with hallmarks of both necrosis and apoptosis including TUNEL positivity, membrane permeabilization, and caspase 3 activation (Kon et al. 2004). Tanshinone IIA, a natural product of Salvia miltiorrhiza, was found to simultaneously elicit apoptosis and necroptosis in HepG2 cells, and this apoptosis could be converted to necroptosis via treatment with a small molecule pan-caspase inhibitor (Lin et al. 2016). Preedy and colleagues recently observed heterogeneity in TNFα/TNFR1 signaling within a population of mouse fibroblasts, leading them to advocate for the use of novel live-cell imaging techniques to understand cell fate at the level of single cells (Preedy et al. 2024). Such heterogeneity in cell death responses may be influenced by different transcriptional or mitochondrial states of single cells at the time of exposure to a cell death stimulus.

Heterogeneity of cell death responses has also been observed in β-cells. For example, Saini et al. found that STZ treatment elicited both apoptosis and necrosis in INS-1 cells in vitro, with necrosis being more common than apoptosis at high STZ concentrations (Saini et al. 1996). β-cells isolated from Wistar rats were found to undergo both necrosis and apoptosis following oleate and palmitate treatment, as determined by neutral red and PI staining (Cnop et al. 2001). Saldeen and colleagues showed that treatment with IL-1β, IFNγ, and TNFα increased both necrosis (17% of cells) and apoptosis (5% of cells) in isolated rat islets via a Bcl-2-inhibitable pathway (Saldeen 2000). Moreover, IFNγ and double-stranded RNA (Poly I:C) treatment was found to stimulate a five-fold increase in necrosis and a seven-fold increase in apoptosis after 48 hours in Sprague-Dawley rat islets, as determined by acridine orange and ethidium bromide staining (Scarim et al. 2001). In addition, islet graft failure was found to be associated with both apoptosis and necrosis following transplantation in STZ-treated C57BL/6 mice (Biarnés et al. 2002). Although transcriptional and functional heterogeneity within β-cell populations is well recognized, these findings indicate that heterogeneity in the context of β-cell death deserves further consideration.

We performed studies to quantify apoptotic and non-apoptotic cell death in NIT-1 β-cells derived from the NOD mouse model of T1D at the single-cell level. NIT-1 β-cells are known to be sensitive to TNFα- and IFNγ-induced cytotoxicity (Hamaguchi et al. 1991, Stephens et al. 1999, Contreras et al. 2022), and this has been attributed primarily to apoptosis. Here, we treated NIT-1 β-cells with TNFα+IFNγ and evaluated cell death using Cytotox Red Dye, a membrane-impermeable DNA binding dye that enters cells with the loss of membrane integrity, and we concurrently monitored caspase 3/7 activation using Caspase 3/7 Green Dye, a membrane-permeable dye that is non-fluorescent until cleaved by caspase 3 or 7, leading to the release of the DNA binding dye and nuclear staining (Fig. 4). This approach allows for hourly quantification of Cytotox Red-positive cells, Caspase 3/7 Green-positive cells, and cells positive for both at the single-cell level. Consistent with previous findings, we found that TNFα + IFNγ treatment provokes early non-apoptotic cell death (Fig. 4B and E, Cytotox Red+, Caspase 3/7 Green−, 2–4 h) followed by the induction of caspase 3/7 activation and apoptotic cell death (Fig. 4CE, Cytotox Red+, Caspase 3/7 Green+, 4–6 h) in NIT-1 β-cells. We believe the use of novel methodologies such as these will enable a greater understanding of the mechanisms that underlie diabetogenic β-cell death.

Figure 4
Figure 4

Heterogeneity of β-cell death responses. A Sartorius Incucyte S3 live cell imaging and analysis instrument was used to monitor cell death (Cytotox Red, Sartorius, 250 nM) and caspase 3/7 activation in real-time (Caspase 3/7 Green, Sartorius, 5 µM). NIT-1 β-cells were plated and treated with vehicle (circles) or TNFα (40 µg/mL) + IFNγ (100 µg/mL) (squares). Cytotox Red−, Caspase 3/7 Green+ (caspase 3/7 activation), Cytotox Red+, Caspase 3/7 Green− (dead, non-apoptotic), and Cytotox Red+; Caspase 3/7 Green+ (dead, apoptotic) objects were monitored hourly over 6 h and quantified at 0, 2, 4, and 6 h post treatment. Total cell count was determined using AI-mediated cell-by-cell phase contrast analysis, and data is represented as a percent of total cells. (A) Percent live cells (dark gray: vehicle, light gray: TNFα+IFNγ), (B) percent non-apoptotic dead cells (Cytotox Red+, Caspase 3/7 Green−; gray: vehicle, red: TNFα+IFNγ), (C) percent apoptotic dead cells (Cytotox Red+; Caspase 3/7 Green+; gray: vehicle, yellow: TNFα+IFNγ), and (D) percent caspase 3/7 activated live cells (Cytotox Red−, Caspase 3/7 Green+; gray: vehicle, green: TNFα+IFNγ) were quantified. (E) Representative images of Cytotox Red and Caspase 3/7 Green-positive NIT-1 cells 0, 2, 4, and 6 h post TNFα+IFNγ treatment (10× magnification). Data are presented as mean ± s.e.m . and were analyzed by two-way ANOVA with Holm–Sidák multiple comparisons correction. ns, not significant; *P < 0.05; ****P < 0.0001, P > 0.05.

Citation: Journal of Endocrinology 262, 2; 10.1530/JOE-23-0378

Characterization of distinct mechanisms of PCD

Given the variety of cell death signaling mechanisms that β-cells may employ, methodologies to identify specific forms of cell death are needed. β-cell death has often been labeled as apoptosis without attendant evidence of biochemical or morphological markers of apoptosis such as caspase activation, DNA laddering, or the presence of apoptotic bodies. In recent years, improved understanding of the processes underlying cell death and advances in methods to monitor it have allowed more specific characterization of mechanisms thereof. In light of this growing understanding, guidelines for appropriate classification of various forms of cell death have been established by the Nomenclature Committee on Cell Death (Kroemer et al. 2009). With the publication of “Classification of cell death: recommendations of the Nomenclature Committee on Cell Death” (Kroemer et al. 2009), “Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes” (Galluzzi et al. 2009) and subsequent updates (Galluzzi et al. 2012, 2018) the Committee has provided detailed information on biochemical, morphological, and functional characterization of cell death, and we suggest interested readers refer directly to these works.

Characterizing the specific mode of death a cell undergoes requires a high degree of specificity. Several DNA-binding dyes are used to identify dead cells, and they are often described as specifically detecting apoptosis. For example, TUNEL is commonly marketed as an apoptosis-specific marker and is widely employed to identify apoptotic cells (Butler et al. 2003, Meier et al. 2005, Jurgens et al. 2011). However, studies indicate that TUNEL staining identifies dead cells of both apoptotic and necrotic origin (Grasl-Kraupp et al. 1995, Kelly et al. 2003), and the DNA fragmentation identified with TUNEL is a known feature of various forms of cell death (Higuchi 2003). Given that altered membrane composition is also a common feature of many forms of PCD, methods to identify dead cells that rely on changes in membrane integrity, including DNA-binding dyes such as SYTOX (De Schutter et al. 2021) and propidium iodide (PI) (Kelly et al. 2003) and phosphatidylserine detection with annexin V (Crowley et al. 2016), have similar limitations in their ability to discriminate between distinct mechanisms of cell death. Therefore, the use of multiple methods to characterize cell death phenotypes is helpful in distinguishing between distinct forms of cell death. For example, luminogenic and fluorogenic caspase substrates can be employed to monitor caspase activity in either static or real-time assays (Shi et al. 2012), and genetically encoded caspase activity biosensors have also been developed (Zhang et al. 2013). As described above, morphological characterization of dead or dying cells (e.g., identification of apoptotic bodies, organelle swelling, or plasma membrane rupture) can also be employed to differentiate between mechanisms of cell death. Specific biochemical characteristics of apoptosis, necroptosis, ferroptosis, and pyroptosis have been described, and identification of these biochemical signatures can be used to distinguish these forms of cell death. In addition, methodologies that allow single cell analysis of cell death responses enable a better understanding of the mechanisms of cell death that arise in response to a specific stimulus. We have provided a brief overview of cell death pathways, their morphological features, and methods of detection in Table 2. The selection presented in Table 2 reflects methodologies discussed within the context of this review. In addition, “Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes” provides a more comprehensive classification of distinct forms of cell death and describes tools to accurately characterize mechanisms of cell death (Galluzzi et al. 2009, 2018). We believe a more robust understanding of β-cell death signaling pathways will accelerate efforts to identify β-cell protective therapeutics for use in diabetes.

Table 2

Morphological features, detection techniques, and biomarkers of cell death.

Cell death mechanism Morphology Detection techniques
Apoptosis Cytoplasmic shrinkage; nuclear fragmentation; plasma membrane blebbing;

chromatin condensation;

DNA fragmentation
Caspase 3/7 activation; DNA laddering
Necrosis Organelle swelling; plasma membrane rupture; karyolysis; karyorrhexis; pyknosis HMBG1 release,a LDH releasea
Necroptosis Plasma membrane rupture, intracellular leakage, cell swelling, and an intact nucleus RIPK1, RIPK3, MLKL phosphorylation

Ferroptosis Plasma membrane rupture;

intracellular leakage; reduced mitochondrial volume and cristae; intact nucleus
Fe2+ release;

GPX4, GSH expression;

lipid peroxidation detection
Pyroptosis Membrane pore formation; plasma membrane blebbing, intracellular leakage; cytoplasm flattening; chromatin condensation; DNA damage Caspase 1 activation;

cleavage of GSDMD;

IL-1β, IL-18 release

aTechniques that can be applied to other forms of lytic cell death.

Conclusion

β-Cell death is an important contributor to β-cell loss, insulin insufficiency, and hyperglycemia in T1D. Several factors may trigger β-cell cytotoxicity in the pathogenesis of T1D, including metabolic stress, ER stress, inflammation, oxidative stress, and autoimmune attack. Given that identification of individuals at risk for T1D prior to the clinical onset of the disease is now possible, it is imperative that we identify novel therapeutics to protect β-cells in these individuals. To do so, we must better understand the multitude of signaling pathways that underlie programmed β-cell death, how these pathways are triggered in the pathogenesis of T1D, and how they relate to the loss of β-cell mass and the induction of islet inflammation and β-cell autoimmunity over time. Given the current evidence for novel forms of PCD such as necroptosis, ferroptosis, and pyroptosis in β-cells, we propose that such mechanisms may contribute to T1D pathogenesis both as early-stage mechanisms that elicit islet inflammation and β-cell autoimmunity and as late-stage mechanisms of β-cell loss. However, additional studies in disease-relevant models are needed to establish the importance of these forms of cell death to β-cell demise in T1D. Unraveling the pathways involved in β-cell death signaling more clearly may lead to novel approaches to prevent β-cell death, promote β-cell survival, and maintain insulin production and glucose homeostasis in individuals with T1D.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported in part by funding from the U.S. Department of Veterans Affairs (IK2 BX004659 to ATT), the Richard L. Roudebush VA Medical Center, the National Institutes of Health (P30 DK097512 to Indiana University Center for Diabetes and Metabolic Diseases, and T32 DK064466 to Indiana University Diabetes and Obesity Research Training Program), and the Ralph W. and Grace M. Showalter Research Trust (080657-00002B to ATT).

Author contribution statement

K.C., N.M., and C.J.C. performed research, analyzed and interpreted literature, and wrote the manuscript. A.T.T. performed research, analyzed and interpreted literature, and wrote and revised the manuscript.

References

  • Agostini L, Martinon F, Burns K, McDermott MF, Hawkins PN & & Tschopp J 2004 NALP3 forms an IL-1beta-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder. Immunity 20 319325. (https://doi.org/10.1016/s1074-7613(0400046-9)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Akirav EM, Lebastchi J, Galvan EM, Henegariu O, Akirav M, Ablamunits V, Lizardi PM & & Herold KC 2011 Detection of β cell death in diabetes using differentially methylated circulating DNA. Proceedings of the National Academy of Sciences of the United States of America 108 1901819023. (https://doi.org/10.1073/pnas.1111008108)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Alberts B, Johnson A, Lewis J, Raff M, Roberts K & & Walter P 2002 Programmed cell death (apoptosis). In Molecular Biology of the Cell,4th ed. New York, NY, USA: Garland Science.

    • PubMed
    • Export Citation
  • Ankarcrona M, Dypbukt JM, Bonfoco E, Zhivotovsky B, Orrenius S, Lipton SA & & Nicotera P 1995 Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15 961973. (https://doi.org/10.1016/0896-6273(9590186-8)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Anthony P 1990 Robbins’ pathologic basis of disease. Journal of Clinical Pathology 43 176.117176. (https://doi.org/10.1136/jcp.43.2.176-a)

  • Augstein P, Elefanty AG, Allison J & & Harrison LC 1998 Apoptosis and beta-cell destruction in pancreatic islets of NOD mice with spontaneous and cyclophosphamide-accelerated diabetes. Diabetologia 41 13811388. (https://doi.org/10.1007/s001250051080)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Bellin MD, Kandaswamy R, Parkey J, Zhang HJ, Liu B, Ihm SH, Ansite JD, Witson J, Bansal-Pakala P, Balamurugan AN, et al.2008 Prolonged insulin independence after islet allotransplants in recipients with type 1 diabetes. American Journal of Transplantation 8 24632470. (https://doi.org/10.1111/j.1600-6143.2008.02404.x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Berghe TV, Linkermann A, Jouan-Lanhouet S, Walczak H & & Vandenabeele P 2014 Regulated necrosis: the expanding network of non-apoptotic cell death pathways. Nature Reviews. Molecular Cell Biology 15 135147. (https://doi.org/10.1038/nrm3737)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Bergsbaken T, Fink SL & & Cookson BT 2009 Pyroptosis: host cell death and inflammation. Nature Reviews. Microbiology 7 99109. (https://doi.org/10.1038/nrmicro2070)

  • Bertheloot D, Latz E & & Franklin BS 2021 Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. Cellular and Molecular Immunology 18 11061121. (https://doi.org/10.1038/s41423-020-00630-3)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Biarnés M, Montolio M, Nacher V, Raurell M, Soler J & & Montanya E 2002 β-cell death and mass in syngeneically transplanted islets exposed to short- and long-term hyperglycemia. Diabetes 51 6672. (https://doi.org/10.2337/diabetes.51.1.66)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Blesia V, Patel VB, Al-Obaidi H, Renshaw D & & Zariwala MG 2021 Excessive iron induces oxidative stress promoting cellular perturbations and insulin secretory dysfunction in min6 beta cells. Cells 10 1141. (https://doi.org/10.3390/cells10051141)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Boada-Romero E, Martinez J, Heckmann BL & & Green DR 2020 The clearance of dead cells by efferocytosis. Nature Reviews. Molecular Cell Biology 21 398414. (https://doi.org/10.1038/s41580-020-0232-1)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Bradley B, Prowse SJ, Bauling P & & Lafferty KJ 1986 Desferrioxamine treatment prevents chronic islet allograft damage. Diabetes 35 550555. (https://doi.org/10.2337/diab.35.5.550)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Brennan MA & & Cookson BT 2000 Salmonella induces macrophage death by caspase-1-dependent necrosis. Molecular Microbiology 38 3140. (https://doi.org/10.1046/j.1365-2958.2000.02103.x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Brenner D, Blaser H & & Mak TW 2015 Regulation of tumour necrosis factor signalling: live or let die. Nature Reviews. Immunology 15 362374. (https://doi.org/10.1038/nri3834)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Brigelius-Flohé R & & Maiorino M 2013 Glutathione peroxidases. Biochimica et Biophysica Acta 1830 32893303. (https://doi.org/10.1016/j.bbagen.2012.11.020)

  • Bruni A, Pepper AR, Pawlick RL, Gala-Lopez B, Gamble AF, Kin T, Seeberger K, Korbutt GS, Bornstein SR, Linkermann A, et al.2018 Ferroptosis-inducing agents compromise in vitro human islet viability and function. Cell Death and Disease 9 595. (https://doi.org/10.1038/s41419-018-0506-0)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Buja LM, Eigenbrodt ML & & Eigenbrodt EH 1993 Apoptosis and necrosis. Basic types and mechanisms of cell death. Archives of Pathology and Laboratory Medicine 117 12081214.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Butler AE, Galasso R, Meier JJ, Basu R, Rizza RA & & Butler PC 2007 Modestly increased beta cell apoptosis but no increased beta cell replication in recent-onset type 1 diabetic patients who died of diabetic ketoacidosis. Diabetologia 50 23232331. (https://doi.org/10.1007/s00125-007-0794-x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA & & Butler PC 2003 β-Cell deficit and increased β-cell apoptosis in humans with type 2 diabetes. Diabetes 52 102110. (https://doi.org/10.2337/diabetes.52.1.102)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cai Z, Jitkaew S, Zhao J, Chiang H-C, Choksi S, Liu J, Ward Y, Wu L-G & & Liu Z-G 2014 Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nature Cell Biology 16 5565. (https://doi.org/10.1038/ncb2883)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chen M & & Wang J 2002 Initiator caspases in apoptosis signaling pathways. Apoptosis 7 313319. (https://doi.org/10.1023/A:1016167228059)

  • Chen X, He WT, Hu L, Li J, Fang Y, Wang X, Xu X, Wang Z, Huang K & & Han J 2016 Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis. Cell Research 26 10071020. (https://doi.org/10.1038/cr.2016.100)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cheng Y, Xiong J, Chen Q, Xia J, Zhang Y, Yang X, Tao K, Zhang S & & He S 2017 Hypoxia/reoxygenation-induced HMGB1 translocation and release promotes islet proinflammatory cytokine production and early islet graft failure through TLRs signaling. Biochimica et Biophysica Acta. Molecular Basis of Disease 1863 354364. (https://doi.org/10.1016/j.bbadis.2016.11.012)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cnop M, Hannaert JC, Hoorens A, Eizirik DL & & Pipeleers DG 2001 Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation. Diabetes 50 17711777. (https://doi.org/10.2337/diabetes.50.8.1771)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cnop M, Welsh N, Jonas J-C, Jörns A, Lenzen S & & Eizirik DL 2005 Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities. Diabetes 54(Supplement 2) S97S107. (https://doi.org/10.2337/diabetes.54.suppl_2.s97)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cohen GM 1997 Caspases: the executioners of apoptosis. Biochemical Journal 326 116. (https://doi.org/10.1042/bj3260001)

  • Collier JJ, Burke SJ, Eisenhauer ME, Lu D, Sapp RC, Frydman CJ & & Campagna SR 2011 Pancreatic β-cell death in response to pro-inflammatory cytokines is distinct from genuine apoptosis. PLoS One 6 e22485. (https://doi.org/10.1371/journal.pone.0022485)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Collier JJ, Fueger PT, Hohmeier HE & & Newgard CB 2006 Pro- and antiapoptotic proteins regulate apoptosis but do not protect against cytokine-mediated cytotoxicity in rat islets and beta-cell lines. Diabetes 55 13981406. (https://doi.org/10.2337/db05-1000)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Conrad M, Angeli JPF, Vandenabeele P & & Stockwell BR 2016 Regulated necrosis: disease relevance and therapeutic opportunities. Nature Reviews. Drug Discovery 15 348366. (https://doi.org/10.1038/nrd.2015.6)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Contreras CJ, Mukherjee N, Branco RCS, Lin L, Hogan MF, Cai EP, Oberst AA, Kahn SE & & Templin AT 2022 RIPK1 and RIPK3 regulate TNFα-induced β-cell death in concert with caspase activity. Molecular Metabolism 65 101582. (https://doi.org/10.1016/j.molmet.2022.101582)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cooksey RC, Jouihan HA, Ajioka RS, Hazel MW, Jones DL, Kushner JP & & McClain DA 2004 Oxidative stress, beta-cell apoptosis, and decreased insulin secretory capacity in mouse models of hemochromatosis. Endocrinology 145 53055312. (https://doi.org/10.1210/en.2004-0392)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cookson BT & & Brennan MA 2001 Pro-inflammatory programmed cell death. Trends in Microbiology 9 113114. (https://doi.org/10.1016/S0966-842X(0001936-3)

  • Cordeiro MF, Guo L, Coxon KM, Duggan J, Nizari S, Normando EM, Sensi SL, Sillito AM, Fitzke FW, Salt TE, et al.2010 Imaging multiple phases of neurodegeneration: a novel approach to assessing cell death in vivo. Cell Death and Disease 1 e3e3. (https://doi.org/10.1038/cddis.2009.3)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Crowley LC, Marfell BJ, Scott AP & & Waterhouse NJ 2016 Quantitation of apoptosis and necrosis by annexin V binding, propidium iodide uptake, and flow cytometry. Cold Spring Harbor Protocols 2016 pdb.prot087288. (https://doi.org/10.1101/pdb.prot087288)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Davidovich P, Kearney CJ & & Martin SJ 2014 Inflammatory outcomes of apoptosis, necrosis and necroptosis. Biological Chemistry 395 11631171. (https://doi.org/10.1515/hsz-2014-0164)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • de Baat A, Meier DT, Rachid L, Fontana A, Böni-Schnetzler M & & Donath MY 2023 Cystine/glutamate antiporter system xc deficiency impairs insulin secretion in mice. Diabetologia 66 20622074. (https://doi.org/10.1007/s00125-023-05993-6)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • De Schutter E, Cappe B, Wiernicki B, Vandenabeele P & & Riquet FB 2021 Plasma membrane permeabilization following cell death: many ways to dye! Cell Death Discovery 7 183. (https://doi.org/10.1038/s41420-021-00545-6)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, et al.2012 Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149 10601072. (https://doi.org/10.1016/j.cell.2012.03.042)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Dolma S, Lessnick SL, Hahn WC & & Stockwell BR 2003 Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells. Cancer Cell 3 285296. (https://doi.org/10.1016/s1535-6108(0300050-3)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Dondelinger Y, Declercq W, Montessuit S, Roelandt R, Goncalves A, Bruggeman I, Hulpiau P, Weber K, Sehon CA, Marquis RW, et al.2014 MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Reports 7 971981. (https://doi.org/10.1016/j.celrep.2014.04.026)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Douglas RS, Tarshis AD, Pletcher CH, Nowell PC & & Moore JS 1995 A simplified method for the coordinate examination of apoptosis and surface phenotype of murine lymphocytes. Journal of Immunological Methods 188 219228. (https://doi.org/10.1016/0022-1759(9500216-2)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Elgamal RM, Kudtarkar P, Melton RL, Mummey HM, Benaglio P, Okino M-L & & Gaulton KJ 2023 An integrated map of cell type-specific gene expression in pancreatic islets. Diabetes 72 17191728. (https://doi.org/10.2337/db23-0130)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Elmore S 2007 Apoptosis: a review of programmed cell death. Toxicologic Pathology 35 495516. (https://doi.org/10.1080/01926230701320337)

  • Emamaullee JA, Davis J, Pawlick R, Toso C, Merani S, Cai S-X, Tseng B & & Shapiro AMJ 2008 The caspase selective inhibitor EP1013 augments human islet graft function and longevity in marginal mass islet transplantation in mice. Diabetes 57 15561566. (https://doi.org/10.2337/db07-1452)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Emamaullee JA, Stanton L, Schur C & & Shapiro AMJ 2007 Caspase inhibitor therapy enhances marginal mass islet graft survival and preserves long-term function in islet transplantation. Diabetes 56 12891298. (https://doi.org/10.2337/db06-1653)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ems T, St Lucia K & Huecker MR 2023 Biochemistry, iron absorption. In StatPearls. Treasure Island, FL, USA: StatPearls Publishing. (available at: https://www.ncbi.nlm.nih.gov/books/NBK448204/)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Erener S, Mojibian M, Fox JK, Denroche HC & & Kieffer TJ 2013 Circulating miR-375 as a biomarker of β-cell death and diabetes in mice. Endocrinology 154 603608. (https://doi.org/10.1210/en.2012-1744)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Fehsel K, Kolb-Bachofen V & & Kröncke K-D 2003 Necrosis is the predominant type of islet cell death during development of insulin-dependent diabetes mellitus in BB rats. Laboratory Investigation 83 549559. (https://doi.org/10.1097/01.lab.0000063927.68605.ff)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Feng S, Yang Y, Mei Y, Ma L, Zhu DE, Hoti N, Castanares M & & Wu M 2007 Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain. Cellular Signalling 19 20562067. (https://doi.org/10.1016/j.cellsig.2007.05.016)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Fink SL & & Cookson BT 2006 Caspase-1-dependent pore formation during pyroptosis leads to osmotic lysis of infected host macrophages. Cellular Microbiology 8 18121825. (https://doi.org/10.1111/j.1462-5822.2006.00751.x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Fisher MM, Perez Chumbiauca CN, Mather KJ, Mirmira RG & & Tersey SA 2013 Detection of islet β-cell death in vivo by multiplex PCR analysis of differentially methylated DNA. Endocrinology 154 34763481. (https://doi.org/10.1210/en.2013-1223)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Frørup C, Svane CAS, Henriksen K, Kaur S & & Størling J 2024 Beta-cell pyroptosis – a burning flame in type 1 diabetes? bioRxiv [epub]. (https://doi.org/10.1101/2024.01.05.574294)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH, Bazan NG, Blagosklonny MV, Blomgren K, Borner C, et al.2009 Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death and Differentiation 16 10931107. (https://doi.org/10.1038/cdd.2009.44)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, Dawson TM, Dawson VL, El-Deiry WS, Fulda S, et al.2012 Molecular definitions of cell death subroutines: recommendations of the nomenclature committee on cell death 2012. Cell Death and Differentiation 19 107120. (https://doi.org/10.1038/cdd.2011.96)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, Alnemri ES, Altucci L, Amelio I, Andrews DW, et al.2018 Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death and Differentiation 25 486541. (https://doi.org/10.1038/s41418-017-0012-4)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gebe JA, Preisinger A, Gooden MD, D’Amico LA & & Vernon RB 2018 Local, controlled release in vivo of vascular endothelial growth factor within a subcutaneous scaffolded islet implant reduces early islet necrosis and improves performance of the graft. Cell Transplantation 27 531541. (https://doi.org/10.1177/0963689718754562)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gerlach B, Cordier SM, Schmukle AC, Emmerich CH, Rieser E, Haas TL, Webb AI, Rickard JA, Anderton H, Wong WW-L, et al.2011 Linear ubiquitination prevents inflammation and regulates immune signalling. Nature 471 591596. (https://doi.org/10.1038/nature09816)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gorczyca W, Bruno S, Darzynkiewicz R, Gong J & & Darzynkiewicz Z 1992 DNA strand breaks occurring during apoptosis - their early insitu detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. International Journal of Oncology 1 639648. (https://doi.org/10.3892/ijo.1.6.639)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grankvist K, Marklund SL & & Täljedal IB 1981 CuZn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase in pancreatic islets and other tissues in the mouse. Biochemical Journal 199 393398. (https://doi.org/10.1042/bj1990393)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H, Bukowska K, Bursch W & & Schulte-Hermann R 1995 In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note. Hepatology 21 14651468. (https://doi.org/10.1002/hep.1840210534)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grunnet LG, Aikin R, Tonnesen MF, Paraskevas S, Blaabjerg L, Størling J, Rosenberg L, Billestrup N, Maysinger D & & Mandrup-Poulsen T 2009 Proinflammatory cytokines activate the intrinsic apoptotic pathway in beta-cells. Diabetes 58 18071815. (https://doi.org/10.2337/db08-0178)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hamaguchi K, Gaskins HR & & Leiter EH 1991 NIT-1, a pancreatic beta-cell line established from a transgenic NOD/Lt mouse. Diabetes 40 842849. (https://doi.org/10.2337/diab.40.7.842)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Higuchi Y 2003 Chromosomal DNA fragmentation in apoptosis and necrosis induced by oxidative stress. Biochemical Pharmacology 66 15271535. (https://doi.org/10.1016/s0006-2952(0300508-2)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hildebrand JM, Tanzer MC, Lucet IS, Young SN, Spall SK, Sharma P, Pierotti C, Garnier J-M, Dobson RCJ, Webb AI, et al.2014 Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. Proceedings of the National Academy of Sciences of the United States of America 111 1507215077. (https://doi.org/10.1073/pnas.1408987111)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, Bodmer JL, Schneider P, Seed B & & Tschopp J 2000 Fas triggers an alternative, caspase-8–independent cell death pathway using the kinase RIP as effector molecule. Nature Immunology 1 489495. (https://doi.org/10.1038/82732)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hoorens A, Stangé G, Pavlovic D & & Pipeleers D 2001 Distinction between interleukin-1–induced necrosis and apoptosis of islet cells. Diabetes 50 551557. (https://doi.org/10.2337/diabetes.50.3.551)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hui H, Dotta F, Di Mario U & & Perfetti R 2004 Role of caspases in the regulation of apoptotic pancreatic islet beta-cells death. Journal of Cellular Physiology 200 177200. (https://doi.org/10.1002/jcp.20021)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Husseiny MI, Kaye A, Zebadua E, Kandeel F & & Ferreri K 2014 Tissue-specific methylation of human insulin gene and PCR assay for monitoring beta cell death. PLOS ONE 9 e94591. (https://doi.org/10.1371/journal.pone.0094591)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Irawaty W, Kay TWH & & Thomas HE 2002 Transmembrane TNF and IFNγ induce caspase-independent death of primary mouse pancreatic beta cells. Autoimmunity 35 369375. (https://doi.org/10.1080/0891693021000024834)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Jurgens CA, Toukatly MN, Fligner CL, Udayasankar J, Subramanian SL, Zraika S, Aston-Mourney K, Carr DB, Westermark P, Westermark GT, et al.2011 β-cell loss and β-cell apoptosis in human type 2 diabetes are related to islet amyloid deposition. American Journal of Pathology 178 26322640. (https://doi.org/10.1016/j.ajpath.2011.02.036)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kaczmarek A, Vandenabeele P & & Krysko DV 2013 Necroptosis: the release of damage-associated molecular patterns and its physiological relevance. Immunity 38 209223. (https://doi.org/10.1016/j.immuni.2013.02.003)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kaestner KH, Powers AC, Naji A, HPAP Consortium & Atkinson MA 2019 NIH initiative to improve understanding of the pancreas, islet, and autoimmunity in type 1 diabetes: the Human Pancreas Analysis Program (HPAP). Diabetes 68 13941402. (https://doi.org/10.2337/db19-0058)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kelly KJ, Sandoval RM, Dunn KW, Molitoris BA & & Dagher PC 2003 A novel method to determine specificity and sensitivity of the TUNEL reaction in the quantitation of apoptosis. American Journal of Physiology-Cell Physiology 284 C1309C1318. (https://doi.org/10.1152/ajpcell.00353.2002)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kerr JFR, Wyllie AH & & Currie AR 1972 Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer 26 239257. (https://doi.org/10.1038/bjc.1972.33)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Khanna D, Hamilton CA, Bhojani MS, Lee KC, Dlugosz A, Ross BD & & Rehemtulla A 2010 A transgenic mouse for imaging caspase-dependent apoptosis within the skin. Journal of Investigative Dermatology 130 17971806. (https://doi.org/10.1038/jid.2010.55)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kim H, Zamel R, Bai X-H, Lu C, Keshavjee S, Keshavjee S & & Liu M 2018 Ischemia-reperfusion induces death receptor-independent necroptosis via calpain-STAT3 activation in a lung transplant setting. American Journal of Physiology. Lung Cellular and Molecular Physiology 315 L595L608. (https://doi.org/10.1152/ajplung.00069.2018)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kon K, Kim J-S, Jaeschke H & & Lemasters JJ 2004 Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes. Hepatology 40 11701179. (https://doi.org/10.1002/hep.20437)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, Blagosklonny MV, El-Deiry WS, Golstein P, Green DR, et al.2009 Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death and Differentiation 16 311. (https://doi.org/10.1038/cdd.2008.150)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kuang S, Zheng J, Yang H, Li S, Duan S, Shen Y, Ji C, Gan J, Xu X-W & & Li J 2017 Structure insight of GSDMD reveals the basis of GSDMD autoinhibition in cell pyroptosis. Proceedings of the National Academy of Sciences of the United States of America 114 1064210647. (https://doi.org/10.1073/pnas.1708194114)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kulkarni A, Muralidharan C, May SC, Tersey SA & & Mirmira RG 2022 Inside the β cell: molecular stress response pathways in diabetes pathogenesis. Endocrinology 164 bqac184. (https://doi.org/10.1210/endocr/bqac184)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lara-Tejero M, Sutterwala FS, Ogura Y, Grant EP, Bertin J, Coyle AJ, Flavell RA & & Galán JE 2006 Role of the caspase-1 inflammasome in Salmonella typhimurium pathogenesis. Journal of Experimental Medicine 203 14071412. (https://doi.org/10.1084/jem.20060206)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lau A, Wang S, Jiang J, Haig A, Pavlosky A, Linkermann A, Zhang ZX & & Jevnikar AM 2013 RIPK3-mediated necroptosis promotes donor kidney inflammatory injury and reduces allograft survival. American Journal of Transplantation 13 28052818. (https://doi.org/10.1111/ajt.12447)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lau H, Corrales N, Rodriguez S, Luong C, Mohammadi M, Khosrawipour V, Li S, Alexander M, de Vos P & & Lakey JRT 2020 Dose-dependent effects of necrostatin-1 supplementation to tissue culture media of young porcine islets. PLoS One 15 e0243506. (https://doi.org/10.1371/journal.pone.0243506)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lau H, Li S, Corrales N, Rodriguez S, Mohammadi M, Alexander M, de Vos P & & Lakey JR 2021 Necrostatin-1 supplementation to islet tissue culture enhances the in-vitro development and graft function of young porcine islets. International Journal of Molecular Sciences 22 8367. (https://doi.org/10.3390/ijms22168367)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lavallard V, Cottet-Dumoulin D, Wassmer C-H, Rouget C, Parnaud G, Brioudes E, Lebreton F, Bellofatto K, Berishvili E, Berney T, et al.2020 NLRP3 inflammasome is activated in rat pancreatic islets by transplantation and hypoxia. Scientific Reports 10 7011. (https://doi.org/10.1038/s41598-020-64054-9)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lawlor KE, Khan N, Mildenhall A, Gerlic M, Croker BA, D’Cruz AA, Hall C, Kaur Spall S, Anderton H, Masters SL, et al.2015 RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL. Nature Communications 6 6282. (https://doi.org/10.1038/ncomms7282)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lehmann-Werman R, Neiman D, Zemmour H, Moss J, Magenheim J, Vaknin-Dembinsky A, Rubertsson S, Nellgård B, Blennow K, Zetterberg H, et al.2016 Identification of tissue-specific cell death using methylation patterns of circulating DNA. Proceedings of the National Academy of Sciences of the United States of America 113 E1826E1834. (https://doi.org/10.1073/pnas.1519286113)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lehrstrand J, Davies WIL, Hahn M, Korsgren O, Alanentalo T & & Ahlgren U 2024 Illuminating the complete ß-cell mass of the human pancreas- signifying a new view on the islets of Langerhans. Nature Communications 15 3318. (https://doi.org/10.1038/s41467-024-47686-7)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lei XG & & Vatamaniuk MZ 2011 Two tales of antioxidant enzymes on β cells and diabetes. Antioxidants and Redox Signaling 14 489503. (https://doi.org/10.1089/ars.2010.3416)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Li W & & Yuan J 2023 Targeting RIPK1 kinase for modulating inflammation in human diseases. Frontiers in Immunology 14 1159743. (https://doi.org/10.3389/fimmu.2023.1159743)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Li H, Khosrow-Khavar F, Speck M, Kieffer T, Woo M & & Marzban L 2008 Suppression of caspase-3 activation protects primary islet β-cells from the cytotoxic effects of human islet amyloid polypeptide. Canadian Journal of Diabetes 32 302. (https://doi.org/10.1016/S1499-2671(0824016-5)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Li X, Zhang M, Huang X, Liang W, Li G, Lu X, Li Y, Pan H, Shi L, Zhu H, et al.2020 Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners. Nature Communications 11 6364. (https://doi.org/10.1038/s41467-020-19935-y)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Liadis N, Murakami K, Eweida M, Elford AR, Sheu L, Gaisano HY, Hakem R, Ohashi PS & & Woo M 2005 Caspase-3-dependent β-cell apoptosis in the initiation of autoimmune diabetes mellitus. Molecular and Cellular Biology 25 36203629. (https://doi.org/10.1128/MCB.25.9.3620-3629.2005)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Liadis N, Salmena L, Kwan E, Tajmir P, Schroer SA, Radziszewska A, Li X, Sheu L, Eweida M, Xu S, et al.2007 Distinct in vivo roles of caspase-8 in β-cells in physiological and diabetes models. Diabetes 56 23022311. (https://doi.org/10.2337/db06-1771)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lin Y, Devin A, Rodriguez Y & & Liu ZG 1999 Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes and Development 13 25142526. (https://doi.org/10.1101/gad.13.19.2514)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lin CY, Chang TW, Hsieh WH, Hung MC, Lin IH, Lai SC & & Tzeng YJ 2016 Simultaneous induction of apoptosis and necroptosis by tanshinone IIA in human hepatocellular carcinoma HepG2 cells. Cell Death Discovery 2 16065. (https://doi.org/10.1038/cddiscovery.2016.65)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Linkermann A & & Green DR 2014 Necroptosis. New England Journal of Medicine 370 455465. (https://doi.org/10.1056/NEJMra1310050)

  • Liu C-Y, Takemasa A, Liles WC, Goodman RB, Jonas M, Rosen H, Chi E, Winn RK, Harlan JM & & Chuang PI 2003 Broad-spectrum caspase inhibition paradoxically augments cell death in TNF-α–stimulated neutrophils. Blood 101 295304. (https://doi.org/10.1182/blood-2001-12-0266)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Liu P, Zhang Z, Wang J, Zhang X, Yu X & & Li Y 2021a Empagliflozin protects diabetic pancreatic tissue from damage by inhibiting the activation of the NLRP3/caspase-1/GSDMD pathway in pancreatic β cells: in vitro and in vivo studies. Bioengineered 12 93569366. (https://doi.org/10.1080/21655979.2021.2001240)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Liu S, Tang G, Duan F, Zeng C, Gong J, Chen Y & & Tan H 2021b MiR-17-5p inhibits TXNIP/NLRP3 inflammasome pathway and suppresses pancreatic β-cell pyroptosis in diabetic mice. Frontiers in Cardiovascular Medicine 8 768029. (https://doi.org/10.3389/fcvm.2021.768029)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Maclean N & & Ogilvie RF 1959 Observations on the pancreatic islet tissue of young diabetic subjects. Diabetes 8 8391. (https://doi.org/10.2337/diab.8.2.83)

  • Majno G & & Joris I 1995 Apoptosis, oncosis, and necrosis. An overview of cell death. American Journal of Pathology 146 315.

  • Man SM, Karki R & & Kanneganti T-D 2017 Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases. Immunological Reviews 277 6175. (https://doi.org/10.1111/imr.12534)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mandrup-Poulsen T 2001 beta-cell apoptosis: stimuli and signaling. Diabetes 50(Supplement 1) S58S63. (https://doi.org/10.2337/diabetes.50.2007.S58)

  • Markelic M, Stancic A, Saksida T, Grigorov I, Micanovic D, Velickovic K, Martinovic V, Savic N, Gudelj A & & Otasevic V 2023 Defining the ferroptotic phenotype of beta cells in type 1 diabetes and its inhibition as a potential antidiabetic strategy. Frontiers in Endocrinology 14 1227498. (https://doi.org/10.3389/fendo.2023.1227498)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Martinon F, Burns K & & Tschopp J 2002 The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Molecular Cell 10 417426. (https://doi.org/10.1016/s1097-2765(0200599-3)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Masuda Y, Ichii H & & Vaziri ND 2013 At pharmacologically relevant concentrations intravenous iron preparations cause pancreatic beta cell death. American Journal of Translational Research 6 6470.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mathis D, Vence L & & Benoist C 2001 Beta-cell death during progression to diabetes. Nature 414 792798. (https://doi.org/10.1038/414792a)

  • Matsumoto S, Takita M, Chaussabel D, Noguchi H, Shimoda M, Sugimoto K, Itoh T, Chujo D, SoRelle J, Onaca N, et al.2011 Improving efficacy of clinical islet transplantation with iodixanol-based islet purification, thymoglobulin induction, and blockage of IL-1β and TNF-α. Cell Transplantation 20 16411647. (https://doi.org/10.3727/096368910X564058)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • McCall M & & Shapiro AMJ 2012 Update on islet transplantation. Cold Spring Harbor Perspectives in Medicine 2 a007823. (https://doi.org/10.1101/cshperspect.a007823)

  • McCall M, Toso C, Emamaullee J, Pawlick R, Edgar R, Davis J, Maciver A, Kin T, Arch R & & Shapiro AMJ 2011 The caspase inhibitor IDN-6556 (PF3491390) improves marginal mass engraftment after islet transplantation in mice. Surgery 150 4855. (https://doi.org/10.1016/j.surg.2011.02.023)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Meier JJ, Bhushan A, Butler AE, Rizza RA & & Butler PC 2005 Sustained beta cell apoptosis in patients with long-standing type 1 diabetes: indirect evidence for islet regeneration? Diabetologia 48 22212228. (https://doi.org/10.1007/s00125-005-1949-2)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Meurette O, Huc L, Rebillard A, Le Moigne G, Lagadic-Gossmann D & & Dimanche-Boitrel M-T 2005 TRAIL (TNF-related apoptosis-inducing ligand) induces necrosis-like cell death in tumor cells at acidic extracellular pH. Annals of the New York Academy of Sciences 1056 379387. (https://doi.org/10.1196/annals.1352.018)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Miotto G, Rossetto M, Di Paolo ML, Orian L, Venerando R, Roveri A, Vučković A-M, Bosello Travain V, Zaccarin M, Zennaro L, et al.2020 Insight into the mechanism of ferroptosis inhibition by ferrostatin-1. Redox Biology 28 101328. (https://doi.org/10.1016/j.redox.2019.101328)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mirmira RG, Sims EK, Syed F & & Evans-Molina C 2016 Biomarkers of β-cell stress and death in type 1 diabetes. Current Diabetes Reports 16 95. (https://doi.org/10.1007/s11892-016-0783-x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Moriwaki K & & Chan FK-M 2017 Chapter Seven - The inflammatory signal adaptor RIPK3: functions beyond necroptosis. International Review of Cell and Molecular Biology 328 253275. (https://doi.org/10.1016/bs.ircmb.2016.08.007)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mukherjee N, Contreras CJ, Lin L, Colglazier KA, Mather EG, Kalwat MA, Esser N, Kahn SE & & Templin AT 2024 RIPK3 promotes islet amyloid-induced β-cell loss and glucose intolerance in a humanized mouse model of type 2 diabetes. Molecular Metabolism 80 101877. (https://doi.org/10.1016/j.molmet.2024.101877)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Murai S, Yamaguchi Y, Shirasaki Y, Yamagishi M, Shindo R, Hildebrand JM, Miura R, Nakabayashi O, Totsuka M, Tomida T, et al.2018 A FRET biosensor for necroptosis uncovers two different modes of the release of DAMPs. Nature Communications 9 4457. (https://doi.org/10.1038/s41467-018-06985-6)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Neiman D, Gillis D, Piyanzin S, Cohen D, Fridlich O, Moss J, Zick A, Oron T, Sundberg F, Forsander G, et al.2020 Multiplexing DNA methylation markers to detect circulating cell-free DNA derived from human pancreatic β cells. JCI Insight 5 e136579 , 136579. (https://doi.org/10.1172/jci.insight.136579)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Newton K, Dugger DL, Wickliffe KE, Kapoor N, de Almagro MC, Vucic D, Komuves L, Ferrando RE, French DM, Webster J, et al.2014 Activity of protein kinase RIPK3 determines whether cells die by necroptosis or apoptosis. Science 343 13571360. (https://doi.org/10.1126/science.1249361)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Neyman A, Nelson J, Tersey SA, Mirmira RG, Evans-Molina C & & Sims EK 2019 Persistent elevations in circulating INS DNA among subjects with longstanding type 1 diabetes. Diabetes, Obesity and Metabolism 21 95102. (https://doi.org/10.1111/dom.13489)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Norbury CJ & & Hickson ID 2001 Cellular responses to DNA damage. Annual Review of Pharmacology and Toxicology 41 367401. (https://doi.org/10.1146/annurev.pharmtox.41.1.367)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Olsen JA, Kenna LA, Spelios MG, Hessner MJ & & Akirav EM 2016 Circulating differentially methylated amylin DNA as a biomarker of β-cell loss in type 1 diabetes. PLoS One 11 e0152662. (https://doi.org/10.1371/journal.pone.0152662)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Onaca N, Takita M, Levy MF & & Naziruddin B 2020 Anti-inflammatory approach with early double cytokine blockade (IL-1β and TNF-α) is safe and facilitates engraftment in islet allotransplantation. Transplantation Direct 6 e530. (https://doi.org/10.1097/TXD.0000000000000977)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Orozco S & & Oberst A 2017 RIPK3 in cell death and inflammation: the good, the bad, and the ugly. Immunological Reviews 277 102112. (https://doi.org/10.1111/imr.12536)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Paredes-Juarez GA, Sahasrabudhe NM, Tjoelker RS, de Haan BJ, Engelse MA, de Koning EJP, Faas MM & & de Vos P 2015 DAMP production by human islets under low oxygen and nutrients in the presence or absence of an immunoisolating-capsule and necrostatin-1. Scientific Reports 5 14623. (https://doi.org/10.1038/srep14623)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Parrish AB, Freel CD & & Kornbluth S 2013 Cellular mechanisms controlling caspase activation and function. Cold Spring Harbor Perspectives in Biology 5 a008672. (https://doi.org/10.1101/cshperspect.a008672)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Pavlosky A, Lau A, Su Y, Lian D, Huang X, Yin Z, Haig A, Jevnikar AM & & Zhang ZX 2014 RIPK3-mediated necroptosis regulates cardiac allograft rejection. American Journal of Transplantation 14 17781790. (https://doi.org/10.1111/ajt.12779)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Pepper AR, Bruni A, Pawlick R, Wink J, Rafiei Y, Gala-Lopez B, Bral M, Abualhassan N, Kin T & & Shapiro AMJ 2017 Engraftment site and effectiveness of the Pan-caspase inhibitor F573 to improve engraftment in mouse and human islet transplantation in mice. Transplantation 101 23212329. (https://doi.org/10.1097/TP.0000000000001638)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Phulphagar K, Kühn LI, Ebner S, Frauenstein A, Swietlik JJ, Rieckmann J & & Meissner F 2021 Proteomics reveals distinct mechanisms regulating the release of cytokines and alarmins during pyroptosis. Cell Reports 34 108826. (https://doi.org/10.1016/j.celrep.2021.108826)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Plays M, Müller S & & Rodriguez R 2021 Chemistry and biology of ferritin. Metallomics 13 mfab021. (https://doi.org/10.1093/mtomcs/mfab021)

  • Preedy MK, White MRH & & Tergaonkar V 2024 Cellular heterogeneity in TNF/TNFR1 signalling: live cell imaging of cell fate decisions in single cells. Cell Death and Disease 15 202. (https://doi.org/10.1038/s41419-024-06559-z)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Proskuryakov SY, Konoplyannikov AG & & Gabai VL 2003 Necrosis: a specific form of programmed cell death? Experimental Cell Research 283 116. (https://doi.org/10.1016/S0014-4827(0200027-7)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Raucci A, Palumbo R & & Bianchi ME 2007 HMGB1: a signal of necrosis. Autoimmunity 40 285289. (https://doi.org/10.1080/08916930701356978)

  • Rhodes CJ 2005 Type 2 diabetes-a matter of beta-cell life and death? Science 307 380384. (https://doi.org/10.1126/science.1104345)

  • Rock KL & & Kono H 2008 The inflammatory response to cell death. Annual Review of Pathology 3 99126. (https://doi.org/10.1146/annurev.pathmechdis.3.121806.151456)

  • Roggli E, Britan A, Gattesco S, Lin-Marq N, Abderrahmani A, Meda P & & Regazzi R 2010 Involvement of microRNAs in the cytotoxic effects exerted by proinflammatory cytokines on pancreatic beta-cells. Diabetes 59 978986. (https://doi.org/10.2337/db09-0881)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Rui J, Deng S, Arazi A, Perdigoto AL, Liu Z & & Herold KC 2017 β Cells that resist immunological attack develop during progression of autoimmune diabetes in NOD Mice. Cell Metabolism 25 727738. (https://doi.org/10.1016/j.cmet.2017.01.005)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Russo HM, Rathkey J, Boyd-Tressler A, Katsnelson MA, Abbott DW & & Dubyak GR 2016 Active caspase-1 induces plasma membrane pores that precede pyroptotic lysis and are blocked by lanthanides. Journal of Immunology 197 13531367. (https://doi.org/10.4049/jimmunol.1600699)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Saini KS, Thompson C, Winterford CM, Walker NI & & Cameron DP 1996 Streptozotocin at low doses induces apoptosis and at high doses causes necrosis in a murine pancreatic beta cell line, INS-1. Biochemistry and Molecular Biology International 39 12291236. (https://doi.org/10.1080/15216549600201422)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Saldeen J 2000 Cytokines induce both necrosis and apoptosis via a common Bcl-2-inhibitable pathway in rat insulin-producing cells. Endocrinology 141 20032010. (https://doi.org/10.1210/endo.141.6.7523)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Scarim AL, Arnush M, Blair LA, Concepcion J, Heitmeier MR, Scheuner D, Kaufman RJ, Ryerse J, Buller RM & & Corbett JA 2001 Mechanisms of β-cell death in response to double-stranded (ds) RNA and interferon-γ: dsRNA-dependent protein kinase apoptosis and nitric oxide-dependent necrosis. American Journal of Pathology 159 273283. (https://doi.org/10.1016/S0002-9440(1061693-8)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Shi H, Kwok RTK, Liu J, Xing B, Tang BZ & & Liu B 2012 Real-time monitoring of cell apoptosis and drug screening using fluorescent light-up probe with aggregation-induced emission characteristics. Journal of the American Chemical Society 134 1797217981. (https://doi.org/10.1021/ja3064588)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Shi J, Zhao Y, Wang K, Shi X, Wang Y, Huang H, Zhuang Y, Cai T, Wang F & & Shao F 2015 Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526 660665. (https://doi.org/10.1038/nature15514)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sims EK, Bundy BN, Stier K, Serti E, Lim N, Long SA, Geyer SM, Moran A, Greenbaum CJ, Evans-Molina C, et al.2021 Teplizumab improves and stabilizes beta cell function in antibody-positive high-risk individuals. Science Translational Medicine 13 eabc8980. (https://doi.org/10.1126/scitranslmed.abc8980)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Song H, Yang B, Li Y, Qian A, Kang Y & & Shan X 2022 Focus on the mechanisms and functions of pyroptosis, inflammasomes, and inflammatory caspases in infectious diseases. Oxidative Medicine and Cellular Longevity 2022 2501279. (https://doi.org/10.1155/2022/2501279)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Stancic A, Saksida T, Markelic M, Vucetic M, Grigorov I, Martinovic V, Gajic D, Ivanovic A, Velickovic K, Savic N, et al.2022 Ferroptosis as a novel determinant of β-cell death in diabetic conditions. Oxidative Medicine and Cellular Longevity 2022 3873420. (https://doi.org/10.1155/2022/3873420)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Steer SA, Scarim AL, Chambers KT & & Corbett JA 2006 Interleukin-1 stimulates beta-cell necrosis and release of the immunological adjuvant HMGB1. PLoS Medicine 3 e17. (https://doi.org/10.1371/journal.pmed.0030017)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Stephens LA, Thomas HE, Ming L, Grell M, Darwiche R, Volodin L & & Kay TW 1999 Tumor necrosis factor-α-activated cell death pathways in NIT-1 insulinoma cells and primary pancreatic β cells. Endocrinology 140 32193227. (https://doi.org/10.1210/endo.140.7.6873)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sui X, Zhang R, Liu S, Duan T, Zhai L, Zhang M, Han X, Xiang Y, Huang X, Lin H, et al.2018 RSL3 drives ferroptosis through GPX4 inactivation and ROS production in colorectal cancer. Frontiers in Pharmacology 9 1371. (https://doi.org/10.3389/fphar.2018.01371)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sun Y, Zheng Y, Wang C & & Liu Y 2018 Glutathione depletion induces ferroptosis, autophagy, and premature cell senescence in retinal pigment epithelial cells. Cell Death and Disease 9 753. (https://doi.org/10.1038/s41419-018-0794-4)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Takiishi T, Xiao P, Franchimont M, Gilglioni EH, Arroba EN, Gurzov EN, Bertrand MJ & & Cardozo AK 2023 Inhibition of RIPK1 kinase does not affect diabetes development: β-cells survive RIPK1 activation. Molecular Metabolism 69 101681. (https://doi.org/10.1016/j.molmet.2023.101681)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tamura Y, Chiba Y, Tanioka T, Shimizu N, Shinozaki S, Yamada M, Kaneki K, Mori S, Araki A, Ito H, et al.2011 NO donor induces Nec-1-inhibitable, but RIP1-independent, necrotic cell death in pancreatic β-cells. FEBS Letters 585 30583064. (https://doi.org/10.1016/j.febslet.2011.08.028)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tan A, Li T, Yang J, Yu J & & Chen H 2023 Irisin attenuates pyroptosis in high glucose-induced pancreatic beta cells via the miR-133a-3p/FOXO1 axis. Endokrynologia Polska 74 277284. (https://doi.org/10.5603/EP.a2023.0035)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tersey SA, Nishiki Y, Templin AT, Cabrera SM, Stull ND, Colvin SC, Evans-Molina C, Rickus JL, Maier B & & Mirmira RG 2012 Islet beta-cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model. Diabetes 61 818827. (https://doi.org/10.2337/db11-1293)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Thapa RJ, Nogusa S, Chen P, Maki JL, Lerro A, Andrake M, Rall GF, Degterev A & & Balachandran S 2013 Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases. Proceedings of the National Academy of Sciences of the United States of America 110 E3109E3118. (https://doi.org/10.1073/pnas.1301218110)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Thomas HE, McKenzie MD, Angstetra E, Campbell PD & & Kay TW 2009 Beta cell apoptosis in diabetes. Apoptosis 14 13891404. (https://doi.org/10.1007/s10495-009-0339-5)

  • Thompson PJ, Shah A, Ntranos V, Van Gool F, Atkinson M & & Bhushan A 2019 Targeted elimination of senescent beta cells prevents type 1 diabetes. Cell Metabolism 29 10451060.e10. (https://doi.org/10.1016/j.cmet.2019.01.021)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tonnus W, Belavgeni A, Beuschlein F, Eisenhofer G, Fassnacht M, Kroiss M, Krone NP, Reincke M, Bornstein SR & & Linkermann A 2021 The role of regulated necrosis in endocrine diseases. Nature Reviews. Endocrinology 17 497510. (https://doi.org/10.1038/s41574-021-00499-w)

    • PubMed