Caspases, a family of cysteine-dependent aspartate-speciﬁc proteases, are central to the maintenance of cellular and organismal homoeostasis by functioning as key mediators of the inﬂammatory response and/or apoptosis. Both metabolic inﬂammation and apoptosis play a central role in the pathogenesis of metabolic disease such as obesity and the progression of nonalcoholic steatohepatisis (NASH) to more severe liver disease. Obesity and nonalcoholic fatty liver disease (NAFLD) are the leading global health challenges associated with the development of numerous comorbidities including insulin resistance, type-2 diabetes and early mortality. Despite the high prevalence, current treatment strategies including lifestyle, dietary, pharmaceutical and surgical interventions, are often limited in their efﬁcacy to manage or treat obesity, and there are currently no clinical therapies for NAFLD/NASH. As mediators of inﬂammation and cell death, caspases are attractive therapeutic targets for the treatment of these metabolic diseases. As such, pan-caspase inhibitors that act by blocking apoptosis have reached phase I/II clinical trials in severe liver disease. However, there is still a lack of knowledge of the speciﬁc and differential functions of individual caspases. In addition, cross-talk between alternate cell death pathways is a growing concern for long-term caspase inhibition. Evidence is emerging of the important cell-death-independent, non- apoptotic functions of caspases in metabolic homoeostasis that may be of therapeutic value. Here, we review the current evidence for roles of caspases in metabolic disease and discuss their potential targeting as a therapeutic strategy. Facts Open questions (1) Caspases can mediate the inﬂammatory response and (1) How do caspases differentially regulate metabolism? apoptotic cell death to maintain organismal homo- (2) Which speciﬁc caspases have non-apoptotic roles in eostasis metabolic disease? (2) Caspase-dependent apoptosis is involved in the (3) What are the critical caspase substrates mediating pathogenesis of obesity and progression of severe metabolic functions? NASH (4) Can caspases or their speciﬁc substrates be therapeu- (3) Blocking apoptosis in NASH with pan-caspase inhibi- tically targeted in obesity? tors shows therapeutic potential in clinical studies (4) Caspase inhibition has not been investigated in the context of obesity Introduction Caspases are a family of evolutionary conserved cysteine- dependent aspartate-speciﬁc proteases that play crucial roles Edited by G. Melino. in maintaining organismal homoeostasis throughout life . Mammalian caspases are broadly classiﬁed as being * Claire H Wilson inﬂammatory/pyroptotic (human caspase-1, -4, -5 and -12, email@example.com murine caspase-1, -11 and -12), or as initiators (human and * Sharad Kumar murine caspase-2, -8, -9 and human caspase-10) and firstname.lastname@example.org executioners (human and murine caspase-3, -6 and -7) of apoptotic cell death . Caspase-14 is more difﬁcult to Centre for Cancer Biology, University of South Australia & SA Pathology, Adelaide, SA 5001, Australia classify but is not involved in cell death . In mice, 1234567890();,: 1234567890();,: Caspases in metabolic disease and their therapeutic potential 1011 Normal Liver Normal Adipose Adipocyte Metabolic Crosstalk Energy homeostasis Macrophage Metabolic Blood Vessel dysregulation Excessive fat NAFLD accumulation Diet, Obese Adipose Lipodystrophy Lipid Adipocyte Accumulation Lipid FFA droplet macrophage Lipotoxicity NASH Apoptosis Apoptotic Adipocyte, TNF-α Collagen IL-6 Crown-like structure deposition TNF-α IL-6 Activated HSC Disease progression macrophage NASH severity Scar formation, fibrosis, cirrhosis, HCC, end-stage liver failure Fig. 1 Metabolic cross-talk and apoptosis in the progression of obesity inﬂammation, metabolic dysfunction and hepatocyte cell death. Pro- and NAFLD/NASH. In obesity, excessive fat accumulation results in gression of NAFLD to NASH to more severe NASH involves marked overexpansion of adipose tissue, resulting in adipocyte cell death increases in hepatocyte apoptosis, resulting in hepatic stellate activa- which promotes inﬂammatory macrophage inﬁltration and adipose/ tion, collagen deposition, scarring and ﬁbrosis. Abbreviations: FFA metabolic dysfunction. Increases in circulating FFA from obese adi- free fatty acids, HCC hepatocellular carcinoma, HSC hepatic stellate pose tissue and/or from diet contribute to the excessive accumulation cell, IL interleukin, NAFLD nonalcoholic fatty liver disease, NASH of lipids in the liver and development of NAFLD. Release of nonalcoholic steatohepatitis, TNF tumour necrosis factor inﬂammatory cytokines from adipose tissue further contributes to liver caspase-11 is the murine orthologue of human caspase-4 initiator caspases following their dimerisation through cas- and -5. Apoptosis occurs via two main pathways: the pase activation platforms leads to subsequent effector cas- extrinsic death-receptor pathway (e.g. Fas/CD95, TNFR) or pase activation, cleavage of a large number of cellular the intrinsic pathway via mitochondrial outer membrane substrates and apoptosis . In response to damage- permeabilisation (MOMP). In both cases, the activation of associated molecular patterns (DAMPs) or pathogen- 1012 C. H. Wilson, S. Kumar Extrinsic Intrinsic Intracellular accumulation Circulating toxic lipid species/FFA FFA, FAS, TRAIL, TNF-α, etc Death-receptor Mitochondrial ER stress oligmerization dysfunction Caspase-8 Bid ROS tBid Caspase-2 Bid Caspase cascade MOMP Caspase-3/7 tBid Apoptosis Caspase-9 Caspase cascade Caspase-3/7 Apoptosis Fig. 2 Apoptotic pathways in metabolic disease. Caspase-dependent via many pathways involving caspase-dependent and independent extrinsic and intrinsic apoptotic pathways are induced in response to pathways. Upstream of the MOMP, caspase-2 is activated in response increases in circulating FFAs, cytokines and intracellular accumulation to stress-induced signals including ROS. Precise mechanisms of of toxic lipid species/FFA via increases in mitochondrial dysfunction apoptotic pathways in metabolic disease are still unknown. Abbre- and ER stress. Extrinsic apoptosis mainly involves activation of the viations: ER endoplasmic reticulum, FFA free fatty acids, MOMP initiator caspase-8, followed by activation of executioner caspases and/ mitochondria outer membrane permeabilisation, TNF tumour necrosis or cleavage of Bid, followed by induction of MOMP and subsequent factor, TRAIL TNF-related apoptosis-inducing ligand, ROS reactive executioner caspase activation. Intrinsic apoptotic pathways can occur oxygen species associated molecular patterns (PAMPs), the activation of compounds have been patented [6, 7]. Therefore, novel inﬂammatory caspases, particularly caspase-1, results in targeting strategies involving nanoparticle siRNA delivery maturation of inﬂammatory cytokines and pyroptosis, an or future utilisation of CRISPR–Cas9 gene-editing approa- alternative form of cell death . Caspases also have roles ches may be required to therapeutically ablate individual independent of inﬂammation and cell death with importance caspases. Firstly, however, better knowledge of the non- of their non-apoptotic functions increasingly becoming apoptotic roles and the precise mechanism of individual apparent . caspase functions need to be established. Furthermore, Apoptosis plays a central role in the pathogenesis of elucidation of the mechanism of caspase functions with obesity and progression of nonalcoholic steatohepatitis identiﬁcation of their speciﬁc proteolytic substrates may (NASH) to more severe liver disease (e.g. cirrhosis, hepa- prove to be of beneﬁt for future therapeutics. tocellular carcinoma and end-stage liver failure) [3, 4]. The therapeutic potential of blocking apoptosis through use of pan-caspase inhibitors in liver injury and disease has been Apoptosis in the pathogenesis of obesity demonstrated in animal models and initial clinical studies and progression of NASH (some ongoing)  but is yet to be investigated in the context of obesity. However, the precise function of speciﬁc Obesity, characterised by excessive accumulation of fat, caspases is still unclear, and evidence of cross-talk between involves impaired lipid storage with dysfunction and over- alternative cell death pathways may complicate therapeutic expansion of white adipose tissue (WAT), leading to toxic blocking of apoptosis to treat metabolic diseases. Due to accumulation of lipids in non-adipose tissue (e.g. liver and high overlap in substrate selectivity among caspase family skeletal muscle) (Fig. 1)[8, 9]. In obesity, WAT over- members, there is currently a lack of available speciﬁc expansion primarily occurs via increases in adipocyte size small-molecule inhibitors, although a number of (hypertrophy) and number (hyperplasia) . Hypertrophy Caspases in metabolic disease and their therapeutic potential 1013 occurs to a certain limit before it triggers cell death that do not necessarily infer changes in levels of apoptosis precedes hyperplasia to maintain or increase lipid-storage which is a post-translational mechanism. As the primary capacity [4, 10, 11]. Adipocyte size positively correlates initiator of the extrinsic apoptotic pathway, caspase-8 has a with increased caspase activation, adipocyte apoptosis, central role in obesity and NASH. Extrinsic apoptosis insulin resistance and inﬂammation in obese mice and typically involves ligand-dependent activation of cell death humans [4, 10, 12]. Adipocyte cell death promotes inﬁl- receptors (e.g. Fas, TNF-α and TRAIL) via binding of their tration of adipose tissue macrophages that aggregate in cognate ligands, triggering the formation of an intracellular crown-like structures to remove apoptotic bodies and lipid- death-inducing signalling complex (DISC) that recruits and droplet remnants. This coincides with the release of proin- activates caspase-8 . Caspase-8 can then directly activate ﬂammatory cytokines and mediators (TNF-α, IL-6 and caspase-3 or cleave Bid to trigger MOMP, indirectly acti- iNOS) that promote a state of low-grade chronic inﬂam- vating caspases and apoptosis similar to the intrinsic path- mation (Fig. 1)[10, 13]. Morphologically, adipocyte cell way (Fig. 2). Notably, caspase-8 can also be activated death resembles the ultrastructural features of necrosis and by intrinsic signals . Increases in circulating FasL cor- pyroptosis [13, 14], although involvement of apoptosis in relate with hepatocyte apoptosis and disease severity in this process is also well documented . Targeted apoptotic NASH patients . The toxic accumulation of FFA, par- deletion of adipocytes via induction of caspase-8 activation ticularly long-chain saturated fatty acids and other lipid using the FAT-ATTAC mouse model has demonstrated the species (e.g. free cholesterol, ceramide), can lead to role of apoptosis in the recruitment of inﬂammatory mac- increased reactive oxygen species (ROS) formation, mito- rophages and formation of crown-like structures [15, 16]. chondria dysfunction, TNF-α production and activation of Nonalcoholic fatty liver disease (NAFLD), characterised several stress pathways, including ER stress and JNK acti- by hepatic accumulation of lipids in the absence of alcohol, vation [17, 23, 24]. Release of TNF-α from adipose tissue results from imbalances in the uptake of circulating free into the circulation, can further promote local cell death by fatty acids (FFA) and/or increases in hepatic de novo lipo- the extrinsic TNFR-mediated pathway contributing sig- genesis accompanied by decreased fatty-acid output . niﬁcantly to hepatocellular death in the liver (Fig. 1). Although obesity is one of the major risk factors for the Increases in circulating FFA can also trigger extrinsic development of NAFLD, it also occurs in lean people and apoptosis by stimulating TRAIL death-receptor 2-mediated disorders where primary lipid storage is impaired or absent signalling [25, 26] and by sensitising cells to cytokine (e.g. lipodystrophies) (Fig. 1). NASH, a more severe toxicity (Fig. 2). Intrinsic apoptosis typically involves form of NAFLD, is distinguished from simple steatosis by oligomerisation of BAK or BAX at the mitochondria, the presence of massive hepatocellular death (primarily resulting in MOMP and release of cytochorome c that binds apoptosis), hepatocellular ballooning, inﬂammation and to apoptotic protease-activating factor 1 (APAF1) to form ﬁbrosis . Although multiple forms of cell death (pyr- the apoptosome. Apoptosome activates caspase-9 which optosis, necroptosis and autophagy-dependent cell death) then cleaves and activates caspase-3 to trigger apoptosis have been identiﬁed, hepatocyte apoptosis plays a key role (Fig. 2). Caspase-2 has been implicated to be important in driving disease progression with the formation of apop- for hepatocellular apoptosis in NASH, as discussed further totic bodies further promoting inﬂammatory inﬁltration and below [27, 28]. Although not required for intrinsic apop- activation of collagen-producing hepatic stellate cells that tosis, caspase-2 is activated in response to intrinsic signals promote ﬁbrogenesis (Fig. 1)[19, 20]. and is known to cleave Bid and function both upstream and downstream of MOMP to initiate or propagate the apoptotic signal (Fig. 2). As mentioned above, some caspases are Mechanisms of apoptosis in metabolic also involved in alternative cell death pathways, thus pro- disease viding cross-talk between apoptosis and other means of cellular demise. Furthermore, the roles of several caspases, In obesity and NASH, caspase-dependent apoptotic cell including caspase-1, -2, -3 and -8 in metabolic homo- death can be initiated by both the extrinsic and intrinsic eostasis and disease have been reported. signalling pathways (Fig. 2), although the precise mechan- ism is still unknown [4, 12, 21]. In obese human adipose tissue, increased levels of active caspase-3, -7 and -9 pro- Cross-talk between caspases and alternate tein and caspase-3/7 activity along with decreased phos- cell death pathways in metabolic disease phorylation of anti-apoptotic Bcl2 protein have been detected . Induction of CASP9 and CASP3/7 transcripts For therapeutic targeting of caspases addressed later in this was also detected in the obese adipose tissue , although paper, it is important to discuss the issue of alternate cell it is important to note that changes in CASP gene expression death pathways and cross-talk between them. Due to cross- 1014 C. H. Wilson, S. Kumar talk between different cell death pathways and morpholo- Mitochondrial DNA in the cytosol triggers the innate gical similarities, it is very difﬁcult to differentiate between immune cGAS/STING pathway, resulting in type-I different modalities of cell death in vivo . Death via interferon production by dying cells. The apoptotic cas- both pyroptosis and necroptosis results in lytic cell death, pase cascade generally suppresses this mtDNA-induced and apoptosis can result in secondary necrosis if the cGAS/STING signalling to ensure that cell death does not resulting apoptotic bodies are not removed by scavenger result in an immune response. Thus, an inhibition of cells . It has become increasingly evident that failure of caspases can result in increased secretion of IFN-β . one mode of cell death can result in alternative 'back-up' This can in turn promote DAMP signalling and may fur- modes of death being triggered, both dependent and inde- ther exacerbate local inﬂammation and progression of pendent of caspases, that are important to acknowledge metabolic disease. when considering blocking of apoptosis via caspase inhi- While caspase-8 has a key role in extrinsic apoptosis, it is bition as a therapeutic option (discussed later in this paper). also critical in suppression of necroptosis. Like pyroptosis, With regard to metabolic disease, these alternate cell death necroptosis is a form of programmed lytic cell death and pathways have important roles in propagating local morphologically the two forms of cell death can be identical inﬂammation and trigger further apoptosis or pyroptosis of . Necroptosis involves activation of intracellular surrounding cells following the release of DAMPs and receptor-interacting protein kinase 3 (RIPK3) followed by inﬂammatory cytokines. In addition to apoptotic and regu- phosphorylation of mixed-lineage kinase domain-like lated cell death, accidental or necrotic death can be (MLKL) which then binds and oligmerises at the plasma increased following blockage of programmed cell death membrane, forming necroptotic pores that result in cellular . swelling, plasma membrane rupture (lysis) and release of Pyroptosis primarily occurs via cleavage of gasdermin D cytosolic DAMPs in an analogous manner to gasdermins in by caspases-1, -4, -5 and 11 . Cleavage results in release pyroptosis . While RIPK3 can be activated by intra- and translocation of a N-terminal fragment of gasdermin D cellular signals, extrinsic TNFR signalling via RIPK1 can that oligomerises at the plasma membrane forming pyr- trigger RIPK3 activation. Typically, RIPK1 recruits FADD, optotic pores that result in cell swelling and membrane resulting in caspase-8 activation, leading to apoptosis and rupture . Recently, it has been shown that other gas- blockade of the inﬂammatory necroptotic pathway via dermin family members (six in humans) also form pores at caspase-8-mediated cleavage of CYLD, and in this manner, the plasma membrane and induce pyroptosis and this can caspase-8 has a dominant pro-survival effect during involve caspase cleavage . Effector caspases-3 and -6 embryogenesis and hematopoiesis . Thus, necroptosis were reported to cleave gasdermin B and gasdermin D only occurs in the absence or inhibition of caspase-8 within their pore-forming domain, suggesting that during activity. Regulation and cross-talk of alternative cell death apoptosis, caspases may inactivate gasdermins to prevent pathways is tightly regulated and the mechanisms are still pyroptosis . However, caspase-3 has also been shown to widely unknown. While a number of pro- and anti-apoptotic cleave and activate gasdermin E, resulting in a switch from molecules are known, an important protein in the context of apoptotic to secondary necrotic/pyroptosis death in response metabolic disease is the anti-apoptotic caspase-8 homologue to chemotherapeutic drugs, TNF-α and viral infection . cellular FLICE-inhibitor (cFLIP) protein . cFLIP has a While highly relevant in the context of metabolic disease as high afﬁnity for binding to procaspase-8 and can inhibit its discussed in this paper, it needs to be fully established if binding and activation to FADD to supress apoptosis, while gasdermin E is present in hepatocytes and/or adipocytes as depending on the levels of cFLIP, it can also help promote its expression seems to be limited to only certain cell types activation of caspase-8 and inhibit necroptosis via formation . Caspase-8 has also been proposed to function of a caspase-8–c-FLIP complex . While more in-depth upstream and downstream of the NLRP3 inﬂammasome, discussion of multiple forms of cell death is reviewed and it has been suggested that a ﬁne-tuned balance may elsewhere , complexities and therapeutic implications of exist between pyroptosis and apoptosis . Furthermore, caspase inhibition are further discussed below. activation of the NLRP3 inﬂammasome has also been reported downstream of necroptosis induction . Thus, communication between these various cell death pathways Caspase-1 in metabolic inﬂammation and needs to be further deﬁned. metabolic disease In the context of caspase inhibition, when MOMP reaches a certain level, it has been shown that caspase As an important component of the NLRP3 inﬂammasome, inhibition can have limited effect in stopping apoptosis activation of caspase-1 has been shown to be associated and can indeed result in increased inﬂammatory signalling with metabolic inﬂammation and disease, however, its following the release of mitochondrial DNA . precise role remains unclear due to inconsistent ﬁndings Caspases in metabolic disease and their therapeutic potential 1015 Table 1 Caspase deﬁciency in mouse models of dietary-induced obesity, NAFLD and NASH Caspase Mouse model Dietary model Duration Phenotype/outcomes a −/− 129mt/129mt Caspase-1 Casp1 and Casp11 knockout mice; (Casp1 Casp11 HFD (42% kJ fat) 12 weeks Increased susceptibility to obesity but protected from NAFLD/NASH  C57BL/6) HFD (45% kJ fat) or LFD 52 weeks Increased susceptibility to obesity with sex-speciﬁc differences  (10% kJ fat) HFD (45% kJ fat) 16 weeks Protected from DIO and insulin resistance  HFD (45% kJ fat) 16 weeks Protected from obesity, NAFLD and insulin resistance  HFD (60% kJ fat) 8 weeks Increased susceptibility to obesity, greater adiposity and inﬂammation and similar insulin sensitivity to WT control  HFD (45% kJ fat) 16 weeks Developed obesity and NAFLD similar to WT controls  HFD (60% kJ fat) 12 weeks Increased susceptibility to obesity but similar insulin sensitivity to WT control  MCD 24 days Increased NASH, increased NAFLD activity score, steatosis and inﬂammation and inﬁltration and liver injury (increased ALT)  −/− tm1Yuan/J Caspase-2 Casp2 null mice; (Casp2 ;B6.129SY-Casp2 ) MCD; HFD (20% kJ fat); 8 weeks Protected from NAFLD on HFD; protected from development of NASH but HFD + MCD not steatosis on MCD or HFD + MCD  −/− Casp2 null mice (Casp2 C57BL/6J) Western diet (45% kJ fat) 16 weeks Protected from DIO, NAFLD and insulin resistance  HFD (60% kJ fat) 12 weeks Protected from DIO, NAFLD and insulin resistance  −/− Caspase-3 Casp3 null mice (Casp3 C57BL/6, exon3 deletion) MCD 6 weeks Protected from development of NASH but not steatosis or liver injury  Δhepa Caspase-8 Hepatocyte-speciﬁc Casp8 null mice (Casp8 , C57BL/6) MCD 10 weeks Protected from development of NASH and steatosis  Liver parenchymal (LPC;hepatocytes and cholangiocytes) MCD 8 weeks Increased liver injury (AST, ALT and GLDH glutamate dehydrogenase), LPC-KO speciﬁc Casp8 null mice—(Casp8 ,C57BL/6) compensatory proliferation of parenchymal liver cells, inﬂammation and ﬁbrosis  LPC- LPC-speciﬁc Casp8 null mice and Rip3 null mice (Casp8 MCD 8 weeks Reduced liver injury (decreased AST, ALT and GLD), compensatory KO −/− LPC-KO /RIP3 ; C57BL/6) proliferation, inﬂammation and ﬁbrosis compared to MCD-fed Casp8 and WT controls; increased hepatic steatosis compared to all groups  −/− −/− Casp8 and Rip3 null mice (Casp8/RIP3 C57BL/6) CD-HFD 16 weeks Casp8 deletion rescued the phenotype of RIPK3 mice, resulting in development of obesity, WAT inﬂammation and insulin resistance similar to CD-HFD WT mice  LPC-KO −/− LPC-speciﬁc Casp8 null mice—(Casp8 ,C57BL/6) CD-HFD 16 weeks Hepatocyte deletion of Casp8 did not rescue RIPK3 from glucose intolerance, insulin resistance or WAT inﬂammation  −/− Caspase-11 Casp11 null mice (Casp11 C57BL/6 background) HFD (45% kJ fat) 16 weeks Develop obesity similar to WT control  −/−(b6) −/−(129) Caspase-12 Casp12 null mice (Casp12 C57BL/6 and Casp12 HFD (45% kJ fat) 16 weeks Increased obesity, NAFLD and insulin resistance in both strains  SV 129) ALT alanine aminotransferase, AST aminotranasferase, CD-HFD choline-deﬁcient high-fat diet, DIO diet-induced obesity, GLDH glutamate dehydrogenase, HFD high-fat diet, LFD low-fat diet, MCD methionine–choline-deﬁcient diet, NAFLD nonalcoholic fatty liver disease, NASH nonalcoholic steatohepatitis Note that these mice also carry a deﬁciency in caspase-11 (see text for details) 1016 C. H. Wilson, S. Kumar from knockout animal studies (Table 1). Release of contributedtoprotectionfromDIO . Following this, DAMPs and lipotoxicity can result in activation of the it was reported that the knockout mice had reduced NLRP3 inﬂammasome and caspase-1 in macrophages and intestinal absorption of dietary lipids, enhance hepatic adipose tissue , and increased expression and activa- triglyceride excretion and increased clearance of circu- tion of caspase-1 has been observed in mouse models of lating triglycerides . Enhanced triglyceride clearance, diet-induced obesity (DIO) and genetically obese db/db but not altered intestinal lipid absorption or hepatic −/− and ob/ob mice [37–39]. Increased activation of caspase-1 clearance, was also observed in Casp1 mice by Kotas has also been demonstrated during hyperglycaemia in et al.; however, this study, along with several other stu- mouse and human adipose tissue . Activation of dies found Casp1 deﬁciency to be paradoxically more caspase-1 triggers an inﬂammatory response by its clea- susceptible to the development of HFD-induced obesity vages of proinﬂammatory interleukins (IL)1-β and IL-18 (Table 1)[47–50]. While it is possible that gut microbiota to their mature forms and initiates pyroptotic cell death and loss of Casp11 (as discussed above) can contribute to via cleavage of gasdermin-D [2, 40] as described above. these inconsistencies, further studies are clearly needed to In addition, caspase-1 has been linked with metabolism establish whether targeting caspase-1 is of therapeutic via its cleavage of sterol-regulatory binding proteins value in obesity. (SREBPs), peroxisome proliferator-activated gamma Metabolic inﬂammation involving NLRP3 inﬂammasome- (PPARγ) and glycolytic enzymes (discussed further mediated activation of caspase-1 and hepatocyte pyroptosis below) [41–43]. has been reported to play a crucial role in the progression of −/− −/− Importantly, the Casp1 mice used in a number of NAFLD to NASH , and Casp1 mice fed with a studies (Table 1) are also deﬁcient for Casp11, the murine methionine–choline diet (MCD) for 4 weeks showed signs of homologue of human CASP4/5, due to the strain of 129 exacerbated NASH . NLRP3 inﬂammasome activation mice used to ﬁrstly generate the initial knockouts, before following ER stress, triggers caspase-1-mediated pyroptosis backcrossing to C57BL/6 mice, containing a mutation in the and positively correlates with liver injury in NASH patients −/− Casp11 locus that attenuates its expression . Thus, these . In addition, Casp1 mice fed with a MCD diet for −/− 129mt/129mt mice are referred to as Casp1 Casp in Table 1 4 weeks showed signs of exacerbated NASH . Para- −/− −/− although they are discussed as being Casp1 in the below doxically, protection of Casp1 mice from HFD-induced text. Since the discovery of this dual knockout, caution is NASH has also been reported, although in that study, Casp1 −/− needed in interpreting results from Casp1 knockout animal mice still developed obesity . studies as there may be an exacerbated or reduced response Chronic ER stress is a common feature of metabolic from combined deﬁciency of Casp1 on Casp11-deﬁcient disease that can contribute to the development of insulin background. To date, only a single study has looked at resistance  and ER stress-induced apoptosis can be the potential separate role of caspase-11 in metabolism by induced by multiple pathways . Although caspase-2 utilising Casp11-speciﬁc knockout mice generated on a was initially reported to be a major effector of ER stress- C57/BL6 background expressing WT Casp1 . In that induced apoptosis  controversy now surrounds this . −/− study, Casp11 mice showed no difference in their sus- ER stress has also been linked with induction of pyroptosis ceptibility to HFD-induced obesity compared to WT with NLRP3 inﬂammasome and caspase-1 activation posi- mice and displayed similar changes in weight and total fat tively correlating with ER markers and liver injury in −/− 129mt/129mt mass to the Casp1 Casp mice although the NASH patients . double knockouts accumulated signiﬁcantly more epididy- Other inﬂammatory caspases have also been investi- mal adipose tissue . gated in the context of metabolic disease with knockout In mice, caspase-1 (combined with caspase-11,as animal studies, suggesting that murine caspase-12 may discussed above) deﬁciency has been shown to result in have a protective role in the progression of obesity . reduced total fat mass, smaller adipocyte size, better Caspase-12 has also been shown to inhibit caspase-1 and insulin sensitivity and enhanced adipogenesis [37, 39]. block the inﬂammatory response, although its catalytic The knockout mice were found to be protected from DIO activity is not required for this [58, 59]. Human caspase- reportedly due, in part, to an increase in whole-body 12 protein is catalytically inactive and thus considered to fatty-acid oxidation . As smaller adipocytes are be a pseudogene . Furthermore, the expression of known to be more insulin sensitive and more ‘metaboli- functional caspase-12 protein is absent in most human cally’ active, this study indicates that the absence of populations due to a premature stop codon in human caspase-1 leads to ‘healthier’ fat-mass/expansion of fat. CASP12 , except in a small population of sub-Saharan However this is yet to be fully validated. Subsequently, in African descendants carrying a SNP in CASP12 and who a separate study, the same group reported that increases consequently have weakened inﬂammatory and innate in energy expenditure and enhanced faecal output also immune responses . Caspases in metabolic disease and their therapeutic potential 1017 Caspase-2 function in metabolic disease tracing in mouse show that brown or ‘beige’-like adipocytes are generated mainly de novo from pre-adipocyte cells and Caspase-2 is the most evolutionarily conserved member of not via transdifferentiation of mature adipocytes . the caspase family [1, 62]. Recent evidence indicates a role Machado et al. also observed increased proliferation of −/− for caspase-2 in metabolic homoeostasis. In mice, caspase-2 adipose-derived stem cells from Casp2 mice . These deﬁciency results in reduced maximal body weight, studies indicate a potential role for caspase-2 as a target in decreased total fat mass [63, 64] and smaller white adipo- obesity, however, as with the other caspases, it is yet to be −/− cyte size [65–67]. Casp2 mice also have reduced fasting established if blocking caspase-2 can reverse pre-existing −/− blood glucose and are protected from the development of disease. Interestingly, Casp2 mice have also been shown −/− age-induced glucose intolerance. This is despite Casp2 to be protected from streptozocin diabetes-induced bone mice displaying a mild premature ageing phenotype, in part marrow adiposity  suggesting depot-speciﬁcdifferences. due to enhanced susceptibility to oxidative stress-induced As described above, activation of caspase-2 has been damage and an impaired antioxidant response system [63, reported to increase with severity of NASH in patients  −/− −/− 68]. Recent data suggest that Casp2 mice have an and Casp2 mice are protected from the development of increased preference for whole-body carbohydrate utilisa- MCD-induced NASH (Table 1). While this implicates tion , but the mechanism of this phenotype has not been caspase-2 as an important mediator of apoptosis in NASH, no established. Importantly, these metabolic phenotypes in signiﬁcant differences have been noticed in the levels of −/− −/− Casp2 mice appear to be independent of apoptosis, as no apoptosis in livers of normal Casp2 mice or following detectable differences in cell death have been found in ethanol-induced liver injury , paraquat toxicity or adipose tissue or liver under normal dietary conditions [63, DEN-induced HCC . In addition, although MCD-fed mice 65, 66]. However, the apoptotic function of caspase-2 are protected from NASH, they still develop fatty liver . appears to be important in the progression of severe NASH Unlike HFD-feeding, the MCD is a nutrient-deprivation model [27, 28]. of NASH resembling features of starvation . Consistent Caspase-2 has been implicated in saturated fatty-acid- with this, no differences were observed in fasting-induced liver induced apoptosis (lipoapoptosis)  and in addition to steatosis  or alcohol-induced liver steatosis . This caspase-8, is activated in response to ceramide-induced suggests that caspase-2 does not alter FFA uptake and/or de apoptosis . While one study reported protection from novo lipogenesis in vivo, consistent with the lack of observed −/− western diet-induced liver injury in Casp2 mice , this is differences in lipogenic/FFA pathways [65, 66]. In contrast to −/− likely to be a secondary effect of overall protection from these models, protection of Casp2 mice from the devel- obesity. In addition, no detectable difference in hepatocellular opment of DIO NAFLD is likely a secondary effect to overall −/− apoptosis has been found in other studies employing Casp2 protection from obesity. Thus, while these studies provide animals [63, 65, 66, 68, 71, 72]. However, increased activation support for caspase-2 as a potential therapeutic target in of caspase-2 is observed in patients with more severe NASH metabolic diseases, further preclinical animal studies are nee- −/− compared to those with simple steatosis and Casp2 dedtovalidatesuchanapproach. mice are protected from MCD-induced NASH . Caspase-2 is an important regulator of genomic stability −/− Two independent studies have found that Casp2 mice and maintenance of normal ploidy [75, 76]. Following are protected from the development of DIO, NAFLD and cytokinesis failure, caspase-2 has been shown to cleave insulin resistance (Table 1)[65, 67]. Although both studies MDM2, thus stabilising p53 and resulting in cell-cycle observed a decrease in obese adipocyte apoptosis, this is arrest . Other ﬁndings suggest that caspase-2 prevents likely a consequence of overall reduced susceptibility to the accumulation of mitotically aberrant cells, such as obesity and does not provide evidence for direct involvement aneuploidy cells, via its apoptosis function and caspase-2 of caspase-2 in this context [65, 67]. While Machado et al. deﬁciency in mice results in accumulation of aneuploidy suggested that protection from DIO partly involves increases cells in the bone marrow of aged knockout mice [76, 78]. −/− in fatty-acid oxidation in Casp2 mice , this was not As p53 and aneuploidy affect cellular metabolism, the link consistent with the indirect calorimetry studies . Never- between caspase-2-augmented genomic stability and meta- theless, ﬁndings from both studies suggest that caspase-2 bolism cannot be ruled out. alters mature adipocyte metabolism of fats and/or that adi- pogenesis is enhanced which would consequently result in healthier expansion of fat mass [65, 67]. This includes the Non-apoptotic roles of caspase-8 and presence of smaller adipocytes, maintenance of WAT necroptosis in metabolic disease PPARy, FABP4 and adiponectin levels and evidence of WAT −/− −/− browning (increased UCP1 expression) following HFD or Similar to Casp2 mice, MCD-fed Casp3 mice show western-diet feeding [65, 67]. Studies involving lineage reduced development of NASH but not steatosis (Table 1) 1018 C. H. Wilson, S. Kumar . In contrast, hepatocyte-speciﬁc deletion of caspase-8 susceptibility to liver injury that is accompanied by Δhep (Casp8 ) reduces both MCD-induced NASH and stea- enhanced caspase activation, apoptosis and inﬂammation tosis  and reverts the enhanced progression of MCD- . Furthermore, cFLIP has been identiﬁed as a critical induced NASH in c-Met knockout mice after 4 weeks of suppressor of NASH and metabolic syndrome in animal Δhep dietary feeding . In addition, Casp8 mice show models with CFLAR mimicking peptides reversing NASH reduced development of alcohol-induced steatosis . and metabolic disorder in mice and monkeys . How- Δhep Microarray analysis of liver from MCD-fed Casp8 ever, this occurs via direct blocking of ASK1/JNK1 acti- mice provided evidence that caspase-8 may also have a vation  and may not involve its interaction with non-apoptotic role in de novo lipogenesis and hepatic lipid caspase-8. storage and export . However, in conﬂict with these ﬁndings, Gautheron et al. reported an increase in MCD- induced NASH (with no difference in steatosis) in liver Caspase substrates inﬂuencing de novo parenchymal and hepatocyte-speciﬁc Casp8 knockout mice lipogenesis and adipogenesis LPC−KO (LPC, hepatocytes and cholangiocytes; Casp8 ). This was reportedly due to increases in RIP3-dependent Increased hepatic de novo lipogenesis is a common feature hepatocyte necroptosis which was suggested to promote of NAFLD that can arise following activation of transcrip- NASH-induced liver ﬁbrosis . In addition to apoptosis, tion factors such as SREBP-1 and PPARγ . SREBP caspase-8 is required for suppression of necroptosis. transcription factors are master regulators of lipid homo- Necroptosis (programmed necrosis) is a highly speciﬁc eostasis that are activated in response to cholesterol deple- alternative cell death programme, dependent on RIP1 and tion, insulin stimulation and ER stress to transcriptionally RIP3 kinases, that is triggered downstream of the TNF upregulated genes involved in fatty-acid triglyceride, receptor . However, the role of necroptosis in human phospholipid and cholesterol synthesis . Caspase- liver disease is controversial as it requires a block or mediated cleavage of SREBP occurs during apoptosis  absence of caspase-8 and there is no evidence to suggest and has been observed following in vitro incubation with that caspase-8 would be blocked in patients . Addi- recombinant caspase-3, -7, -4 and -12 [91, 92]. Caspase-1 tionally, intrinsic apoptosis can still be triggered in the has also been shown to cleave SREBP independent of cell absence of caspase-8, and as such, enhanced activation of death to enhance de novo lipogenesis and membrane bio- caspase-9-mediated apoptosis was recently reported in genesis to possibly promote cell survival following hepatitis Δhepa hepatocytes of Casp8 mice following alcohol-induced C viral-infection  or in response to the bacterial pore- liver injury that was ameliorated using a pan-caspase forming toxin aerolysin . Whether NLRP3 inﬂamma- inhibitor . Recently, in vivo pan-caspase inhibition some activation of caspase-1 also results in SREBP clea- using ZVAD-fmk was shown to initially reduce lipopoly- vage in the setting of obesity and/or NAFLD/NASH is not saccharide/D-galactosamine (GalN)-induced hepatocellular known; however, if cleavage results in activation of apoptosis before triggering necrotic death after prolonged SREBP, and enhanced lipogenesis, then this is not a caspase inhibition . Interestingly, pharmacological desirable outcome in the treatment of these metabolic dis- inhibition of RIPK1 by necrostatin or by genetic deletion of eases. Some evidence suggests that caspase-3 may cleave RIPK3 in the presence of ZVAD-fmk did not prevent SREBP2 in a non-apoptotic manner in response to NGF and necrosis but rather, exacerbated LPS/GalN/ZVD-induced pro-NGF stimulation of p75 neurotrophin receptor signal- liver injury. This study suggested that inhibition of cas- ling to regulate low-density lipoprotein receptors (LDLRs) pases in liver injury can trigger a non-necroptotic caspase- and lipid uptake . Although human CASP2 gene was independent form of cell death . reported to be under transcriptional control of the SREBPs Further adding to the controversy, a subsequent study by and involved in positive-feedback loop, recombinant Gautheron et al. showed upregulation of RIP3 in obese caspase-2 does not cleave SREBPs . WAT and by using choline-deﬁcient HFD (CD-HFD) based PPARγ is also a key regulator of lipid metabolism and is on knockout animal studies, proposed that RIP3 plays a role an essential mediator of adipogenesis and adipose tissue in suppressing, not inducing, inﬂammation by blocking function . Caspase-mediated cleavage of PPARγ, caspase-8-dependent adipocyte apoptosis . However, it independent of apoptosis, has been observed in 3T3-L1 is still unclear how RIP3 acts to block caspase-8-induced adipocytes with cleavage, resulting in deactivation of apoptosis in this context. PPARγ and disruption of adipocyte metabolism by reducing As described above, cFLIP is an important regulator of the expression of lipogenic genes [97, 98]. He et al. caspase-8 and helps maintain a balance between apoptotic observed caspase-1, but not caspase-3, -8 or -9-mediated and necroptotic modes of cell death . In mice, cleavage of PPARγ, following TNF-α and cyclohexamide hepatocyte-speciﬁc deletion of cFLIP increases treatment of 3T3-L1 adipocytes . In contrast, Guilherme Caspases in metabolic disease and their therapeutic potential 1019 Table 2 Preclinical and clinical studies of caspase inhibitors in metabolic disease Inhibitor Company Speciﬁcity Stage in development Outcome Pralnacasan Vertex Pharmaceuticals Caspase-1 Preclinical/proof-of-concept obese Ob/Ob mouse Improved insulin sensitivity and attenuated increase in body weight VX-740 model (2 weeks) [37, 38] Phase IIb in rheumatoid arthritis and osteoarthritis Terminated in phase IIb in RA due to liver toxicity in animals Ac-YVAD-cmk Non-pharma Caspase-1 Preclinical in DIO and NASH mouse model Attenuated development of NASH, ﬁbrosis, insulin resistance and inﬂammation  Nivocasan Gilead Sciences Caspase-1, -8 and -9 Phase II in NASH (4 weeks) Completed; reduced ALT levels in NASH patients  GS-9450 Phase IIa in chronic HCV (5 weeks) Completed, reduced ALT levels  Phase II in chronic HCV (6 months) Terminated due to toxicity Emricasan Conatus Pharmaceuticals Broad- spectrum pan- Preclinical liver injury (bile-duct ligation (10 days); Reduced liver injury, inﬂammation and ﬁbrosis  IDN-6556; caspase Preclinical liver injury (α-FAS induced) Hepatoprotective, reduced ALT  PF-03491390 Preclinical DIO and NASH mouse model Reduced development of liver injury, inﬂammation and ﬁbrosis; no (20 weeks) change in steatosis  Phase 1 (7 days) Completed; reduced ALT levels  Phase II in chronic HCV, HBV, NASH, PBC and Completed; reduced ALT levels  PSC (14 days) Completed; reduced markers of liver injury  Phase II in chronic HCV (12 weeks) Completed; reduced ALT activity (NCT02077374) Phase II in NAFLD Active; not recruiting (NCT02686762) Phase II in NASH ﬁbrosis Active; recruiting (NCT03205345) Phase II in decompensated NASH cirrhosis Active; recruiting (NCT02960204) Phase II in NASH cirrhosis and severe portal hypertension VX-166 Vertex Pharmaceuticals Broad- spectrum pan- Preclinical mouse models of NASH and NAFLD Suppressed development of ﬁbrosis but did not improve liver injury caspase  Reduced inﬂammation and liver injury but not steatosis  ALT alanine aminotransferase, DIO diet-induced obesity, HBV hepatitis B virus, HCV hepatitis C virus, NAFLD nonalcoholic fatty liver disease, NASH nonalcoholic steatohepatitis, PBC primary biliary cirrhosis, PSC primary sclerosing cholangitis. NCT ClinicalTrials.gov registry number. 1020 C. H. Wilson, S. Kumar et al. observed caspase-3, -6 and -8, but not caspase-1, -2, metabolic stress . While promising, the long-term effects -5, -7 or -9 that cleave PPARγ in response to TNF-α sti- of pan-caspase inhibition need to be examined, especially mulation . TRAIL was also shown to regulate adipocyte with recent ﬁndings, also discussed above, from animal metabolism by activating caspase-8 and -3, which subse- studies indicating enhancement of alternative cell death quently led to non-apoptotic cleavage of PPARγ [98, 99]. pathways. [82, 85] While evidence suggests that caspase-1, TRAIL can inhibit adipogenic differentiation of human caspase-2 and caspase-8 can inﬂuence metabolism, inde- SGBS preadipocytes and stromal–vascular cells isolated pendent of cell death, whether and how they may lead to from human WAT in a dose-dependent manner by acti- metabolic disease onset and/or progression is not known. vating caspase-8 and -3, resulting in downregulation of C/ In the context of obesity, the utility of targeting adipo- EBP-α, CEBP-σ and PPARγ . Caspase-1 cleavage of cyte apoptosis is yet to be investigated; however, tissue- PPARγ in tumour-associated macrophages, promotes speciﬁc targeting of non-apoptotic roles via selective tumour differentiation, progression and metastasis . caspase-2 or caspase-1 inhibition may be of more value. As indicated from animal studies, if ablation of caspase-2 and/ or caspase-1 can modulate the size of adipocytes and adi- Therapeutic targeting of caspases in pogenesis in obese adipose tissue, then their therapeutic metabolic disease inhibition may result in the promotion of healthier fat mass that may help improve the reduction of fat accumulation in Several preclinical and clinical studies provide promising combination with dietary and lifestyle interventions. In evidence that pan-caspase inhibitors are of therapeutic value support of this concept, short-term (2-week) treatment of in blocking apoptosis in liver disease (Table 2). genetically obese ob/ob mice with the caspase-1 inhibitor Emricasan (IDN-6556), an irreversible pan-caspase inhi- Pralnacasan has been shown to improve insulin sensitivity bitor, has shown efﬁcacy in blocking hepatocellular apop- and attenuate increases in body weight (Table 2). tosis and attenuating liver injury, inﬂammation and ﬁbrosis However, a phase-IIb trial of Pralnacasan in rheumatoid in animal models of liver injury and HFD-induced NASH arthritis patients was terminated due to liver toxicity in (Table 2)[102–104]. Evaluation of Emricasan in phase I/II animals, although no adverse side effects were observed in clinical trials has demonstrated that it is well-tolerated in trial participants . In a separate preclinical study, caspase-1 humans following short-term treatment and improves the inhibition, using AC-YVAD-cmk, in HFD-fed −/− levels of liver marker enzymes, although these quickly obese–diabetic LDLR Leiden mice, reduced adipose tis- returned to pre-treatment levels after drug discontinuation sue inﬂammation, prevented development of NASH and [105–107]. Phase-II trials investigating its use in NAFLD attenuated progression of insulin resistance but did not alter (NCT02077374) show reduction in liver injury (Table 2). body-weight or dyslipidemia (Table 2). Phase-II trials in NASH-associated ﬁbrosis (but not cir- Despite being implicated in tumour suppression in mice, −/− rhosis) (NCT02686762), decompensated NASH cirrhosis inhibition of caspase-2 appears to be safe as Casp2 mice (NCT03205345) and in patients with NASH cirrhosis and do not spontaneously develop tumours and have no overtly severe portal hypertension (NCT02960204) they are cur- adverse phenotype . Supporting the safety in targeting rently active (Table 2). Another irreversible pan-caspase caspase-2, QPI-1007, a therapeutic naked siRNA targeting inhibitor, VX-166, has also been shown to reduce hepato- caspase-2, is currently being used in human phase-III clin- cellular apoptosis, inﬂammation and ﬁbrosis in MCD- and ical trials for the treatment of non-arteritic anterior ischae- HFD-models of NASH (Table 2)[108, 109]. VX-166 did mic optic neuropathy (Quark Pharmaceuticals; http://qua not reduce hepatic steatosis in the MCD-diet model  rkpharma.com). In mouse studies, infusing an anti- but reduced hepatic triglyceride levels in the HFD-model, caspase-2 morpholino into mouse brain reduced caspase-2 although no improvement in liver-injury markers was protein levels and its cleavage of tau to reverse memory observed [108, 109]. GS-9450, an irreversible selective deﬁcits without adverse effects . To the best of our inhibitor of caspases-1, -8 and -9, evaluated in a phase-II knowledge, there are currently no caspase-2 selective inhi- clinical trial in patients with NASH, is well tolerated in bitors available although a patent has been ﬁled for nasal patients and reduces serum ALT levels (Table 2). administration of a peptide inhibitor, with the amino acid However, a larger 6-month phase-II clinical trial in hepatitis sequence AFDAFC, targeting caspase-2 for treatment of C patients was later terminated due to drug-induced liver neurodegenerative disorders such as ALS, toxicity concerns (Table 2). As a stand-alone therapy, pan- Creutzfeldt–Jacob disease, AD, MCI, PD and HD . caspase inhibitors may have limited efﬁcacy and would best Due to the high overlap in substrate selectivity among be administered with other metabolic target therapies, such caspases, the development of small-molecule inhibitors that as thiazolidinediones (TZDs) or synthetic PPARγ agonist, speciﬁcally target caspases has remained challenging. that are geared to reduce hepatic fat accumulation and Therefore, novel approaches utilising advanced drug Caspases in metabolic disease and their therapeutic potential 1021 delivery systems such as nanoparticles or liposomes in Acknowledgements The caspase work in our laboratory was sup- ported by the National Health and Medical Research Council combination with siRNA-mediated knockdown or (NHMRC) of Australia project grants 1021456 and 1043057, a CRISPR–Cas9 gene editing should be a focus in future NHMRC Early Career Research Fellowship to CHW (1073771) and a studies. 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