IL-1β priming triggers an adaptive stress response that enhances pancreatic β-cell resilience to subsequent cytotoxic inflammatory insult

IL-1β priming triggers an adaptive stress response that enhances pancreatic β-cell resilience to subsequent cytotoxic inflammatory insult | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article IL-1β priming triggers an adaptive stress response that enhances pancreatic β-cell resilience to subsequent cytotoxic inflammatory insult Marcelo Perone, Carolina Setula, Andrea Scelza-Figueredo, Ingrid Pensado-Evans, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6378229/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Oct, 2025 Read the published version in Cell Death & Disease → Version 1 posted You are reading this latest preprint version Abstract Pancreatic β-cells fine-tune glucose homeostasis through insulin secretion. The endoplasmic reticulum (ER) is critical for insulin production, relying on the unfolded protein response (UPR) to adapt to the body's fluctuating demands. Islets from both type 1 (T1D) and type 2 diabetes (T2D) exhibit inflammation, β-cell dysfunction, and loss. ER stress is present in the inflamed islets of autoimmune diabetes-prone mice and individuals with T1D and T2D. Inflammatory cytokines induce ER stress and disrupt UPR regulation, driving β-cell apoptosis and contributing to diabetes development. Inflammatory cytokines, e.g., IL-1β, impair β-cell function and survival, contributing to diabetes pathogenesis by inducing stress, altering gene expression, driving dedifferentiation, and reducing insulin production. Paradoxically, β-cells exhibit a high density of IL-1R1, and IL-1R1/KO mice display impaired glucose tolerance and reduced insulin secretion. Postprandial IL-1β secreted by macrophages helps maintain blood glucose homeostasis. These observations suggest that circulating low IL-1β concentrations may have physiologically relevant roles; however, their effects on β-cell function and survival remain unclear due to conflicting reports. Preconditioning β-cells with physiological circulating levels of IL-1β (IL-1β low ) induced a resilient state, protecting them from pro-inflammatory cytokine (CYT)-induced cell death while preserving glucose-stimulated insulin secretion through hormesis. IL-1β low -treated INS-1E cells reduced CYT-induced NO secretion by suppressing NF-κB signaling and decreasing iNOS expression, correlating with reduced β-cell death. IL-1β low conditioning reduced ER stress and upregulated p-eIF2a in response to CYT, thereby enhancing the expression of ER chaperones and biomarkers linked to improved β-cell identity/functionality. Transcriptomic analysis revealed that IL-1β low preconditioning mitigated the CYT-induced loss of genes involved in β-cell function/identity, and suppressed the expression of genes linked to NF-κB signaling, cytokine-induced inflammation, and apoptosis. IL-1β low treatment counteracted the upregulation of stress-related genes triggered by pro-inflammatory stimuli. Enhancing IL-1βlow-induced stress-response hormesis may provide a novel strategy to sustain β-cell function and survival during harmful diabetic inflammation. Biological sciences/Cell biology Health sciences/Diseases/Endocrine system and metabolic diseases insulin apoptosis islets diabetes UPR hormesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Pancreatic β-cells play a crucial role in maintaining glucose homeostasis by secreting insulin. Type 1 (T1D) and type 2 diabetes (T2D), the most common clinical presentations of diabetes, are both characterized by β-cell dysfunction and loss. The endoplasmic reticulum (ER) in β-cells is essential for protein folding and insulin synthesis, with the unfolded protein response (UPR) helping to manage fluctuating insulin production demands [ 1 ]. ER stress markers are present in the inflamed islets of autoimmune diabetes-prone nonobese diabetic mice [ 2 ] and individuals with T1D [ 3 ], and T2D [ 4 ]. Hormesis is a phenomenon in which a cytotoxic agent, in small doses, benefits organisms. Cells exposed to low toxin levels can resist subsequent high-dose exposures [ 5 ]. It is suggested that the hormetic response to lifestyle detrimental factors such as poor diet, sedentarism, and stress may influence protection levels and impact T2D progression [ 6 ]. Inflammatory cytokines, such as IL-1β, TNF-α and IFN-γ negatively affect β-cell function and survival, contributing to the pathogenesis of both T1D and T2D [ 7 – 10 ]. Inflammation exacerbates ER stress and activates the UPR, which, when prolonged or dysregulated, leads to β-cell apoptosis [ 11 – 14 ]. Therefore, restoring ER homeostasis in β-cells has been proposed as a potential strategy to alleviate T1D [ 15 ]. Pro-inflammatory cytokines, particularly IL-1β, drive β-cell dedifferentiation by inducing cellular stress, altering gene expression, and reducing insulin production. This involves the downregulation of key transcription factors essential for β-cell identity (Pdx-1, Mafa, FoxO1, Nkx6.1). IL-1β and other inflammatory cytokines induce the expression of inducible nitric oxide synthase (iNOS) in β-cells, leading to nitric oxide (NO) accumulation. Elevated NO disrupts insulin secretion, protein synthesis, oxidative metabolism, and causes DNA damage, impairing β-cell function and health [ 16 , 17 ]. IFN-γ amplifies the harmful effects of IL-1β on β-cells [ 18 ]. In contrast, acute low concentrations of IL-1β stimulate insulin release in rat islets [ 19 ], underscoring its complex and context-dependent effects on β-cell function and insulin regulation. Meanwhile, the long-term impact of very low IL-1β concentrations on β-cells remains uncertain. In this study, we explored the role of IL-1β-mediated hormesis in defending β-cells against dysfunction and death induced by pro-inflammatory cytokines. Our findings show that IL-1β, at basal physiological concentrations, triggers a hormetic response in β-cells, enhancing their resilience to future cytotoxic cytokine challenges. Inducing hormetic responses in vivo offers a promising strategy to prevent β-cell decline in diabetes and warrants further investigation. Results Preconditioning INS-1E cells with low concentrations of IL-1β mitigates NO secretion in response to a cytotoxic pro-inflammatory cytokine challenge Increases in iNOS-derived NO act as a trigger for pro-inflammatory cytokine-mediated ER stress and death in a β-cell-specific manner [20]. INS-1E cells exposed to IL-1β (200 pg/ml) for 16h secreted significant amounts of NO (228 +/- 33.9 pmol/μg protein) into the culture medium. Notably, priming these cells with IL-1β (7.5 and 15 pg/ml for 72h) reduced NO secretion in response to IL-1β (200 pg/ml/16h) (Figure 1A). In a similar experiment, IL-1β preconditioning reduced NO secretion (Suppl. Figure 1A) in INS-1E cells compromised by a 16h exposure to a cytokine mixture containing IL-1β (200 pg/ml) and TNF-a (8 ng/ml). TNF-a and IL-1β, together with IFN-g trigger similar signaling pathways [21]. Thus, INS-1E cells were challenged with IL-1β 100 pg/ml + IFN-g 5 ng/ml (CYT). IL-1β preconditioning (10 pg/ml/72h) reduced CYT-induced NO secretion (Figure 1B), with effects starting at 48h and persisting through 72h (Figure 1C). Hereafter, IL-1β (10 pg/ml, 72h) will be referred to as IL-1β low . To explore the mechanisms behind reduced NO secretion in IL-1β low -preconditioned INS-1E cells, we examined iNOS expression and found decreased mRNA (p<0.01; Figure 1D) and protein levels (p<0.01; Figure 1E) following CYT-challenge. The iNOS-specific inhibitor SMT abolished NO secretion, confirming CYT acts via iNOS in INS-1E cells (Figure 1F). Unlike IL-1b low , IFN-g preconditioning did not reduce CYT-induced NO secretion (Suppl. Figure 1B). Priming INS-1E cells with IL-1 b low impairs the NF-kB pathway activation triggered by the pro-inflammatory cytokine mixture NF-κB activation links CYT-induced NO production, β-cell dysfunction, and apoptosis. IL-1b low reduced CYT-induced IκBa phosphorylation, abolishing the second peak of p-IκBa levels triggered by the pro-inflammatory cytokine mixture (p<0.01; Fig. 2A, B&C), and reducing NF-κB p65 nuclear translocation in INS-1E cells (p<0.05, IL-1b low + CYT vs. CYT; Fig. 2D,E). Additionally, preincubation of INS-1E cells with IL-1b low attenuated CYT-induced NF-κB transcriptional activity, as determined by a reporter assay using a plasmid with NF-κB response elements upstream of the luciferase gene (p<0.05, CYT vs. IL-1b low + CYT; Figure 2F). IL-1β promotes its own synthesis, partly via NF-κB [22;23]. Its autocrine or paracrine production by β-cells may contribute to their damage [24]. IL-1b low attenuated CYT-induced IL-1β mRNA levels (p<0.05, Fig. 2G) while upregulating the transcript expression of IL-1Ra (p<0.05, Fig. 2H), an endogenous IL-1β antagonist. Similar results were obtained in isolated mouse islets under IL-1b low regimen and iCYT (IL-1β 100 pg/ml + IFN-g 5 ng/ml + TNF-a 8 ng/ml) challenge (Suppl. Fig. 2A,B). The increase in IL-1Ra and the decrease in IL-1β mRNA levels induced by IL-1b low likely play a role in mitigating CYT-induced adverse effects on β-cells. IL1-R1 and IL1-R2 mRNA expression levels showed an increasing trend in INS-1E cells pretreated with IL-1b low before CYT challenge (Suppl. Fig. 2C,D). IL1-R2, a decoy receptor for IL-1β, may reduce signal transduction by increasing expression in response to its cognate ligand [25]. IL-1 b low preconditioning enhances resilience to pro-inflammatory cytokine-induced death in INS-1E cells IL-1b low reduced apoptosis/death in INS-1E cells after CYT/16h, with protection lasting up to 48h (p<0.05 and p<0.01 vs. CYT/16h and CYT/48h, respectively; Fig. 3A,B). These findings were further validated by assessing apoptosis through annexin-V/PI staining (p<0.05 vs. CYT at 48h; Fig. 3C,D). To investigate IL-1b low 's pro-survival mechanisms, we analyzed Bcl-2 family members involved in apoptosis. IL-1b low treatment hampered the CYT-induced increases in DP5 and PUMA mRNA (63.2% and 47.7%, respectively, vs. IL-1b low +CYT, Fig. 3 E,F) and counteracted the CYT-induced increase in Bax/Bcl-2 mRNA ratio, suggesting reduced apoptosis susceptibility (p<0.01 vs. CYT , Fig. 3G). Additionally, IL-1b low reduced CYT-mediated upregulation of CHOP (86.6% vs. CYT; Fig. 3H,I) and cleaved caspase-3 (52.9 % vs. CYT; Figure 3H,J), both key mediators in the final steps of apoptosis. These results support the notion that IL-1b low triggers a hormetic response, as evidenced by minimal apoptosis compared to INS-1E cells exposed to CYT alone. IL-1 b low triggers a stress-response hormesis We investigated if IL-1b low ‘s protective effect on β-cells requires baseline ER stress for hormesis by adding TUDCA, an ER stress alleviator [15], during preconditioning. Under this condition, CYT-induced NO secretion by INS-1E cells was comparable to that observed in cells treated only with TUDCA, without IL-1b low preconditioning (Figure 4A). As expected, TUDCA reduced levels of the ER stress sensor ATF4, along with CHOP and cleaved caspase-3, which are effectors of CYT-induced ER stress-mediated cell death. Interestingly, TUDCA during the IL-1b low preconditioning eliminates the stress-response hormesis, as evidenced by the unchanged expression levels of ATF4, CHOP, and cleaved caspase-3 under CYT stimulation (Fig. 4C-E). These results highlight that a minimal level of ER stress is required to elicit an effective, adaptive pro-survival stress-response hormesis in INS-1E cells against CYT insult. IL-1 b low preconditioning boosts CYT-induced eIF2 a phosphorylation promoting cellular stress adaptation In β-cells, pro-inflammatory cytokines activate the PERK branch of the UPR, leading to phosphorylation of eIF2a at Ser51 (p-eIF2a) [14,26]. While initially protective, prolonged activation can cause β-cell dysfunction and apoptosis [27,28]. p-eIF2a suppresses global protein synthesis to conserve energy while facilitating gene reprogramming and the induction of key ER chaperones like BiP to restore protein homeostasis. IL-1b low preconditioning further amplified the CYT-induced elevation of p-eIF2a levels in INS-1E cells (p<0.05 vs. CYT; Figure 5A,B) [14]. Both IL-1b low preconditioning alone and followed by CYT stimulation led to a significant increase in BiP expression, as assessed by WB (p<0.05 IL-1b low vs. control; p<0.01 IL-1b low + CYT vs. CYT; Figure 5C,D). BiP mRNA expression also increased following IL-1b low , requiring a 24h washout period to return to baseline upon CYT stimulation p<0.01 IL-1b low + CYT vs. CYT; Figure 5E). In addition, IL-1b low enhanced the expression of PDI, a chaperone essential for β-cell function (p<0.01 vs. untreated), and restored the expression of chaperones downregulated by CYT, including GRP94 (p<0.01), ORP150 (p<0.05), and PDI (p<0.01) (Figure 5F-H). This IL-1b low -induced upregulation of key chaperones suggests an improved capacity for protein folding, processing, and secretion, potentially reinforcing β-cell resilience. Regarding UPR sensor activation, CYT differentially affected the transcriptional activity of IRE1a-XBP1 and ATF6 pathways in INS-1E cells. While ATF6 transcriptional activity was reduced by CYT (p<0.001), IRE1a-mediated XBP1 splicing remained unchanged, consistent with previous findings [14] (Figure 5I,K). IL-1b low treatment alone increased ATF6 transcriptional activity (p<0.01 vs. control, Figure 5I), but IL-1b low preconditioning did not alter the transcriptional activity of ATF6 or IRE1a-mediated XBP1 splicing in response to CYT. XBP1s mRNA levels were downregulated by CYT; however, this reduction remained unaffected by IL-1b low conditioning (Figure 5J). Overall, the INS-1E response to IL-1b low suggests that, upon CYT stimulation, the induced proapoptotic ER stress is primarily regulated through the PERK/p-eIF2a pathway, leading to decreased expression of ATF4 (Figure 4C), CHOP, and cleaved caspase-3 (Figure 3H-J). Notably, IL-1b low pre-treatment significantly attenuated CYT-induced expression of these proapoptotic drivers while simultaneously increasing the mRNA levels of antiapoptotic genes (Figure 3E-G). IL-1b low -induced intracellular signaling highlights the PERK/p-eIF2a pathway as a key mediator of an adaptive response that helps preserve β-cell integrity. IL-1 b low attenuates CYT-induced downregulation of gene transcripts associated with β-cell identity/function, as well as the expression of Pdx-1 and insulin proteins The harmful islet microenvironment during diabetes progression disrupts β-cell identity and maturity [29]. CYT stimulation reduced the expression of β-cell identity mRNA transcripts ( Pdx-1 , MafA , Ucn3 , Ins1/2 , p<0.05 vs. untreated) in INS-1E cells (Fig. 6A-E). IL-1b low mitigated these reductions, particularly for Pdx-1 , MafA and Ins1/2 , and enhanced their recovery, including Ucn3 mRNA, after a 24h washout. In line with these findings, IL-1b low prevented the CYT-induced reduction (23% vs. untreated) in immune-reactive insulin in INS-1E cells (p<0.05 IL-1b low +CYT vs. CYT; Fig. 6F,G). Nuclear localization of Pdx-1 immunoreactivity was enhanced by IL-1b low , both in the absence of challenge (p<0.05 vs. untreated) and under CYT exposure (p<0.05 vs. CYT; Figure 6H,I). The pro-inflammatory cytokine mixture iCYT (IL-1β 100 pg/ml + IFN-g 5 ng/ml + TNF-a 8 ng/ml) reduced mRNA expression of β-cell identity and functionality markers in isolated murine islets. IL-1b low treatment facilitated their recovery in most cases, particularly after the washout period (Fig. 7A–F). IL-1b low alone increased the expression of Pdx-1 , GLUT2 and BiP mRNA compared to untreated islets (Fig. 7B,C,F) and showed a trend toward mitigating the CYT-induced increase in the β-cell dedifferentiation marker Aldh1a3 mRNA [30] (Fig. 7G). Collectively, the results indicate that IL-1b low helps preserve β-cell identity in pancreatic islets under harmful CYT-induced stimuli. IL-1 b low enhances glucose-stimulated insulin secretion impaired by pro-inflammatory cytokines To determine whether the beneficial effects of IL-1b low on β-cells observed thus far translate into improved insulin secretion, we assessed glucose-stimulated insulin secretion (GSIS). CYT exposure impaired GSIS (0.86 ± 0.29-fold vs. 3.2 ± 1.7-fold untreated, p<0.05; Fig. 8A), an effect partially counteracted by IL-1b low treatment in INS-1E cells (p<0.05; Fig. 8A). IL-1b low alone had no effect on insulin secretion. Remarkably, IL-1b low restored GSIS in iCYT-challenged isolated islets, further supporting its role in improving islet health and enhancing insulin secretion (p<0.05; Fig. 8B). This finding is particularly significant as it underscores IL-1b low ‘s protective role in preserving β-cell function in a cytokine-induced harmful environment, closely resembling the inflammatory microenvironment of islets in diabetes. RNA-seq reveals protective transcriptome modulation in IL-1 b low -conditioned INS-1E cells challenged with cytotoxic cytokines To identify the genetic mechanisms underlying phenotypic changes following IL-1b low and CYT-treated cells conditioned with IL-1b low , we performed transcriptome analysis (Fig. 8C). We generated and sequenced bulk RNA-seq libraries from INS-1E cells subjected to these treatments, along with untreated and CYT-challenged cells as controls. Principal component analysis reveals distinct clustering among experimental conditions, highlighting differences in transcriptional profiles. IL-1b low -treated cells exhibited a gene expression pattern similar to that of untreated cells. However, IL-1b low preserved the expression of β-cell identity and functionality genes ( Rfx6 , Nkx6-1 , Ins1 , Pdx-1 , Mafa , Pcsk1 ) in INS-1E cells that were subsequently exposed to a CYT challenge. The CYT challenge induced significant transcriptional changes. However, IL-1b low -treated cells exhibited a distinct response to the proinflammatory challenge, displaying a transcriptional profile that set them apart from CYT-treated cells. IL-1b low treatment downregulated apoptosis-related genes ( Fas , Ddit3 , Bid , Bbc3 ), attenuated the expression of genes associated with NF-κB signaling ( Nfkb2 , Relb , Nfkb1 , Nfkbil1 ) and cellular stress ( Sod2 , Trib3 , Hmox1 , Atf4 , Ern1 , Atf3 , Atf6 , Nfe2l2 , Hsp90b1 , Edem1 ), and reduced the expression of cytokine-related inflammatory genes ( Nos2 ). The heatmap of selected genes further supports our qPCR findings (Figs. 1, 3 and 6). Gene Set Enrichment Analysis (GSEA) evaluated the enrichment of selected Hallmark Gene Sets in IL-1b low -treated cells challenged with CYT, compared to those receiving the CYT challenge (Fig. 8E). IL-1b low -treated cells showed positive enrichment in the pancreas beta-cell gene set, suggesting an upregulation of genes that support β-cell phenotype. Core enrichment genes, such as Pcsk1 and Isl1 , were identified as key contributors. A positive enrichment score for the G2M Checkpoint pathway suggests that IL-1b low -stimulated genes promote cell cycle progression, potentially enhancing proliferation or cell cycle regulation. The UPR pathway exhibited negative enrichment in IL-1b low -treated cells, indicating reduced ER stress with Atf3 , Chac1 , and Ern1 contributing to this effect. Other negatively enriched Hallmark gene sets included the inflammatory response, TNF-a signaling via NF-kB, and apoptosis pathways. Discussion In this study, we show that IL-1β at basal physiological concentrations (IL-1β low ), triggers a stress-response hormesis in vitro , strengthening pancreatic β-cell resilience and enhancing insulin secretion under inflammatory and cytotoxic conditions. Priming β-cells with IL-1β low activates survival mechanisms by modulating gene expression and promoting an adaptive response, reducing β-cell death/apoptosis triggered by pro-inflammatory cytokines in models mimicking the diabetic islet microenvironment. β-cells exhibit a high density of IL-1R1 [ 31 ], prompting the question: why do these cells express abundant receptors that, when activated by their ligand, can initiate cell death? β-cells express components of the IL-1 signaling system, including IL-1α/β, IL-1R1, IL-1Ra, and IL-1R2, with the latter serving as a decoy receptor [ 25 ]. IL-1R1 knockout mice display impaired glucose tolerance and reduced insulin secretion [ 32 ]. Additionally, postprandial IL-1β secreted by macrophages contributes to blood glucose homeostasis [ 33 ]. Based on these observations, we investigated IL-1β priming's effects on β-cells. Prolonged inflammation disrupts the specialized phenotype of β-cells, leading to transdifferentiation and/or dedifferentiation [ 34 ]. This is clinically significant, as dedifferentiated β-cells are observed in patients with T1D and T2D, likely driven by chronic inflammation [ 35 ]. IL-1β (and/or TNF-α), combined with IFN-γ, disrupts cell function through NF-κB-regulated gene networks, ultimately leading to β-cell death. NF-κB-driven IL-1β transcriptional reprogramming reciprocally regulates chemokine and insulin secretion [ 36 ]. Cytokine-driven activation of the IKK complex triggers IκB phosphorylation, ubiquitination, and degradation, enabling NF-κB nuclear migration to induce inflammatory gene expression and mediators like iNOS [ 37 , 38 ]. NO is a major driver of β-cell dysfunction and apoptosis [ 17 , 18 ], impairs oxidative metabolism and insulin secretion, induces ER stress and activates signaling pathways culminating in β-cell apoptosis [ 11 , 14 ]. However, depending on intracellular levels, NO can also suppress apoptosis via caspase-3-dependent DNA damage repair [ 20 , 39 – 41 ]. We found that INS-1E cells preconditioned with IL-1β low exhibited reduced NO secretion in response to CYT challenge (Fig. 1 ), mediated by suppression of the NF-κB signaling pathway, leading to decreased iNOS expression (Figs. 1 & 2 ). Since CYT-induced NF-κB activation drives pro-apoptotic signaling in β-cells [ 14 , 36 ], our findings suggest a protective role for IL-1β low against CYT-induced cell death. We further assessed β-cell viability following IL-1β low preconditioning and found that it significantly enhanced cell survival after both short-term and long-term CYT exposure (Fig. 3 ). A previous study reported a cytoprotective effect of IL-1β on β-cells. Research involving rat β-cells indicated that IL-1β may protect against necrosis caused by STZ or alloxan, although it did not protect against cytokine-induced apoptosis. This protection, however, came at the cost of β-cell phenotype integrity, mediated through an NO-dependent mechanism [ 42 ]. This apparent discrepancy with our results may be explained by the differences in experimental conditions. In this study, β-cells were exposed to very low concentrations (∼10 pg/ml) of IL-1β for 72h. In contrast, the referenced study used higher IL-1β concentrations over a shorter exposure period. While some studies dismiss a direct link between CHOP and cytokine-induced β-cell death [ 43 ], others highlight its key role in CYT-induced apoptosis, as its knockdown significantly reduces this effect in INS-1E cells [ 37 ]. Additionally, studies suggest that CHOP also has a pro-inflammatory function [ 44 ]. Consistently, CYT challenge led to increased CHOP expression levels. However, in IL-1β low -conditioned INS-1E cells, both CHOP and cleaved caspase-3 were downregulated. These findings align with previous research identifying CHOP as a key regulator of β-cell apoptosis. CHOP contributes to CYT-induced NF-κB-dependent pathways ( e.g. , NO production, induction of pro-apoptotic mediators) and regulates mitochondrial-mediated apoptosis ( e.g. , caspase-3) [ 37 ]. In addition to confirming CHOP's relevance as a mediator in CYT-induced activation of the intrinsic apoptotic pathway, we provide new insights into how IL-1β low attenuation of CHOP impacts β-cell survival under CYT/inflammatory challenge. The UPR preserves cellular homeostasis under stress; however, excessive or prolonged ER stress compromises β-cell function and survival. β-cells rely on the ER and UPR machinery to process excess nutrients and ensure proper insulin folding and secretion [ 45 ]. Unresolved UPR contributes to T1D and T2D [ 3 , 46 ]. The UPR cascade is initiated upon BiP dissociation by the autophosphorylation of PERK and IRE1, along with the proteolysis of ATF6 [ 47 ]. While XBP1 and ATF6 manage ER stress, their reduced expression may limit adverse effects, support metabolic adaptation, and mitigate inflammation. However, persistent ER stress can still lead to ATF4-mediated CHOP activation [ 48 ]. Cytokine-induced ER stress shifts β-cell energy priorities, promoting survival mechanisms at the expense of normal cellular functions, including protein folding, synthesis, and insulin secretion. However, exacerbated ER stress activates PERK/eIF2α/ATF4/CHOP pathway leading to β-cell dysfunction and apoptosis [ 1 ]. The PERK/p-eIF2α pathway plays a crucial role in cell survival under stress by reducing global protein synthesis while selectively translating specific mRNAs, such as ATF4, which can drive either pro-survival or pro-apoptotic responses depending on the cellular context [ 49 ]. Notably, IL-1β low enhanced CYT-induced eIF2α phosphorylation in INS-1E cells, accompanied by reduced ATF4 and CHOP protein levels (Fig. 5 ). At the same time, IL-1β low favored the expression of ER chaperones crucial for insulin folding, processing, and handling, such as BIP [ 50 ] and GRP94 [ 51 ], respectively. These findings suggest that IL-1β low induces a distinct adaptive response and that its precise molecular mechanism requires further investigation. However, it clearly contributes to preserving β-cell integrity under pro-inflammatory cytokine-induced ER stress, with the pro-survival effects of the PERK/p-eIF2α pathway mediated through mechanisms involving ATF4/CHOP downregulation, although additional protective mechanisms cannot be ruled out. Future experiments should determine whether IL-1β low -induced PERK/p-eIF2α signaling promotes survival by reducing oxidative stress or enhancing autophagy while avoiding ATF4/CHOP upregulation [ 49 , 52 ]. IL-1β low mitigated the CYT-induced decline in β-cell identity and maturity markers ( e.g. , Ucn3, MafA, Pdx-1 , and GLUT2 ) in both INS-1E cells and mouse islets (Figs. 6 & 7 ) while preserving Pdx-1 and insulin expression (Fig. 6 ). This contrasts with previous reports suggesting that low concentrations of IL-1β drive β-cell dedifferentiation and dysfunction [ 53 ], possibly due to variations in its concentration and exposure duration. IL-1β low improved β-cell insulin secretion despite the acute impairment of glucose-stimulated insulin release by pro-inflammatory cytokines [ 9 , 11 , 14 , 42 , 54 ], with a stronger effect in isolated murine islets. This suggests IL-1β low may also support other islet-resident cells, warranting further investigation into its broader islet benefits. RNA-seq revealed a protective transcriptomic profile in IL-1β low -preconditioned INS-1E cells under CYT stimulation. DEGs showed preserved β-cell identity and reduced expression of inflammation, NF-κB signaling, ER stress, and apoptosis-related genes. IL-1β low and IL-1β low + CYT cells exhibited increased Rfx6 gene transcript expression, encoding a protein essential for islet cell development and insulin production [ 55 ]. Mutations in Rfx6 are associated with maturity-onset diabetes of the young [ 56 ], and its expression is dysregulated in human β- and α-cells in both T1D and T2D [ 57 ], while β-cell-specific Rfx6 knockout mice exhibit impaired insulin secretion [ 58 ]. Isl1 regulates genes essential for β-cell differentiation and maturation, such as Pdx-1 and Slc2a2 ( GLUT2 ) vital for β-cell function and glucose sensing, respectively [ 59 ]. IL-1β low -treatment preserved Isl1 expression in INS-1E cells. The ER chaperone Edem1 supports insulin processing and β-cell function by mitigating ER stress. [ 60 ]. Additionally, GSEA indicates that IL-1β low may enhance β-cell resilience by upregulating the cell cycle under inflammatory conditions. The enrichment of the G2M Checkpoint pathway may reflect an adaptive mechanism that counterbalances stress-induced β-cell loss by enhancing proliferative capacity or reinforcing cell cycle control. The observed negative enrichment in the UPR, inflammatory response, and apoptosis pathways indicates that IL-1β low may reduce ER stress and inflammatory signaling, contributing to improved β-cell survival. The downregulation of genes involved in inflammation and cell death aligns with a protective role for IL-1β low in modulating stress responses, ultimately fostering β-cell adaptation in a pro-inflammatory environment. qPCR validation of DEGs and GSEA-enriched genes in IL-1β low -treated cells is needed given their role in β-cell identity, function, and survival. Collectively, we describe a novel aspect of IL-1β low 's effects on β-cells, highlighting its ability to induce gene expression changes, modulate ER stress and UPR. These changes enhance cellular resilience against inflammatory cytotoxic challenges triggered by cytokines. However, several questions remain to be addressed: 1) Does β-cell resilience result from a single adaptive molecular pathway in response to IL-1β low ? 2) Could β-cell resilience be induced by other stress-inducing agents? 3) Since IL-1β low induces an increase in p-eIF2α levels, and mammalian stress granules (SGs) are known to assemble in response to stress-induced p-eIF2α [ 61 ], could the β-cell response to IL-1β low be associated with the protective effect mediated by SGs formation? and 4) Could IL-1β low 's effects be replicated in vivo ? Given that individuals genetically predisposed to T1D, T2D, obesity, or metabolic syndrome do not always progress to overt diabetes, it is plausible that, under certain conditions, β-cells activate protective defense mechanisms [ 6 ]. Our findings suggest that mild or transient stress induced by IL-1β low can trigger such protective responses. Future research should focus on identifying novel hormesis inducers (hormetins) in β-cells and uncovering their mechanisms to develop therapies that enhance β-cell function and survival in diabetes. Materials and methods Reagents Culture media, supplements and antibiotics were purchased from Gibco (Thermo Fisher Scien- tific, Carlsbad, CA, USA). Fetal bovine serum was obtained from Natocor (Córdoba, Argentina). Recombinant cytokines were purchased from R&D Systems (Minneapolis, MN, USA). Tauroursodeoxycholic acid (TUDCA), 5-methylisothiourea sulfate (SMT) and other analytical-grade reagents were purchased from Sigma-Aldrich. Animals C57BL/6NCrl mice were bred in a controlled environment (20–22°C, 12 h light–dark cycle) at the IIMT (Austral University-CONICET) animal facility and given ad libitum access to food and water. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, Eighth edition (2011). The study was approved by the Animal Research and Care Committee (CICUAL #2023-03) at Austral University. INS-1E cell line The rat β-cell line INS-1E (Prof. Wollheim, University Medical Centre, Geneva, Switzerland) was used between passages 63 and 90, and cultured at 37°C in a humidified atmosphere containing 5% (vol./vol.) CO2 in complete RPMI 1640 culture medium [11 mM glucose, 10% (vol./vol.) heat-inactivated fetal bovine serum (FBS), penicillin (50 IU/ml), streptomycin (50 µg/ml), L-glutamine (2 mmol/l), 2-mercaptoethanol (50 µmol/l), HEPES (10 mmol/l) and sodium pyruvate (1 mmol/l)]. The presence of mycoplasma was periodically checked by PCR. INS-1E were seeded at a density of 11 × 10 3 cells/cm 2 in multiwell plates (Nunc, Thermo Scientific, Denmark) in complete medium. Mice islets isolation and culture Islets (C57BL/6NCrl) were isolated by collagenase digestion and handpicked after density gradient centrifugation [ 62 ]. For standardization purposes, islets with a diameter of 100–125 µm were defined as one islet equivalent (IEQ). Islets were cultured on ultra-low attachment plates (Corning Costar, Kennebunk, ME, USA), at 37°C in humidified atmosphere containing 5% (vol./vol.) CO2 in RPMI 1640 medium containing 5.5 mM glucose, 10% FBS, penicillin (50 IU/ml), streptomycin (50 µg/ml), L-glutamine (2 mmol/l) and HEPES (10 mmol/l) for 24 h prior to performing experiments. Hormesis induction by IL-1β treatment INS-1E cells were conditioned with IL-1β 10 pg/ml for 72h (IL-1β low ), with fresh cytokine added every 24h without replacing the culture media. Then, the culture media was renewed, and cells were challenged with a proinflammatory cytokine mixture (CYT: IL-1β 100 pg/ml + IFN-γ 5 ng/ml). When INS-1E cells were allowed to recover, CYT-containing media was removed after 16h, followed by PBS washing and a 24h incubation in CYT-free RPMI with 10% FBS before harvesting (24h washout). Mouse islets were treated with IL-1β, similar to INS-1E cells. After 72h, the culture medium was refreshed, and the islets were challenged with a proinflammatory cytokine mixture (iCYT: IL-1β 100 pg/ml, IFN-γ 5 ng/ml, TNF-α 8 ng/ml) for 16h. For recovery, the iCYT-containing medium was removed, and the islets cultured for 24h, as with INS-1E cells. Alternatively, for GSIS experiments, islets were treated with IL-1β 10 pg/ml every 72h, with media replaced each time IL-1β was added. After three IL-1β treatments, islets were challenged with iCYT for 16h before starting the GSIS protocol. SDS-PAGE and Western blot INS-1E cells were harvested on ice-cold PBS, washed and lysed in lysis buffer [50 mM Tris–HCl pH 7.4, 250 mM NaCl, 25 mM NaF, 2 mM EDTA, 0.1% Triton-X, protease inhibitors mix (Complete ULTRA, Roche)]. Protein concentration was determined using the BCA assay Kit (Pierce, Thermo Fisher Scientific, Carlsbad, CA, USA) and samples were stored at -20°C. Proteins were separated by 8–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), blotted onto nitrocellulose or PVDF membranes (GE-Healthcare, Amersham, UK) and incubated with primary antibodies: IκBα (#4814, 1:1000), p-IκBα (#9246, 1:1000), β-actin (#3700, 1:1000), ATF4 (#11815, 1:1000), CHOP (#2895, 1:1000), Cleaved caspase-3 (#9664, 1:1000), eIF2α (#2103, 1:1000), p-eIF2α (#9721, 1:1000), PDI (#3501, Cell Signaling Technology, Danvers, MA, USA, 1:1000); iNOS (#610332, BD Biosciences, San Jose, CA, USA, 1:1000), ORP150 (#ab124884, 1:1000), GRP94 (#ab13509, 1:1000), BIP (#ab21685, Abcam, Cambridge, MA, USA, 1:1000). Blots were incubated with HRP-conjugated secondary antibodies: Goat anti-Mouse IgG (H + L) (#62-6520; Thermo Fisher Scientific, Carlsbad, CA, USA, 1:5000) and Goat Anti-Rabbit IgG (H + L) (#BA1054, Boster Biological Technology, Pleasanton, CA, USA, 1:5000), followed by visualization using ECL (Supersignal; Thermo Fisher Scientific, Carlsbad, CA, USA). Immunofluorescent microscopy INS-1E were cultured for 72 h on fibronectin-coated coverslips, treated as described in the figures, fixed by cold methanol and incubated with primary antibodies: monoclonal mouse anti-insulin (clone HB125); NFκB p65 (RelA, #sc-109, Santa Cruz Biotechnology, 1:60) or Pdx-1 (#5679, Cell Signaling Technology, Danvers, MA, USA, 1:100). Secondary antibodies were used at a 1:200 dilution: anti-mouse Alexa Fluor 488 or anti-rabbit Alexa Fluor 647 conjugated dye (Thermo Fisher Scientific, Carlsbad, CA, USA). Coverslips were mounted on slides with Mowiol and images were acquired on a NIKON Eclipse Ni microscope (Nikon, Tokyo, Japan). Image quantification was performed with Fiji software. Nitric oxide quantification Nitrite levels were measured as an indicator of nitric oxide (NO) production using the Griess reagent (1% sulfanilamide and 0.1% naphthyl ethylenediamine dihydrochloride in 2.5% phosphoric acid) at 570 nm [ 14 ]. Quantitative real-time PCR Total RNA was extracted from INS-1E cells using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) following the manufacturer's instructions. Nucleic acid quantification and quality control were assessed with a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Carlsbad, CA, USA). For cDNA synthesis, 1 µg of RNA was reverse-transcribed using RevertAid Reverse Transcriptase in the presence of RiboLock RNase Inhibitor (Thermo Fisher Scientific, Carlsbad, CA, USA) and oligo(dT) primers. All primers were designed using Primer3 and BLAST (NIH) (Suppl. Table 1). Real-time PCR was performed on an AriaMx Real-Time PCR Detection System (Agilent Technologies, Santa Clara, CA, USA), using Master Mix qPCR 2.0 Sybr Rox (Embiotec, BA, Argentina). Each reaction was carried out in triplicate, using HPRT as the normalization control. Relative gene expression was determined by the 2 − ΔΔCT method. Transient transfections and luciferase reporter assays NF-κB transcriptional activity was evaluated by transfecting INS-1E cells with a plasmid containing multimerized NF-κB-binding sites linked to a minimal promoter upstream of the luciferase gene (κB-Luc promoter) [ 63 ]. ATF6 pathway activation was assessed using a reporter plasmid in which the firefly luciferase gene is driven by five copies of the ATF6 consensus binding site (5xATF6-LUC). To quantitatively measure XBP1 splicing, we employed a splicing-specific reporter plasmid where the firefly luciferase coding sequence is fused to the second ORF of unspliced XBP1 (XBP1u-LUC); luciferase expression occurs only upon IRE1-mediated splicing that removes the 26-nt intron. All transfections included a CMV-Renilla LUC expression vector for normalization. Plasmids were transfected into INS-1E cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific) in Opti-MEM medium following IL-1β low conditioning. Thirty hours post-transfection, cells were challenged with pro-inflammatory cytokines. After treatment, cells were lysed and firefly and Renilla luciferase activities were sequentially measured using the Dual-Glo Luciferase Reporter Assay System (Promega) on a Centro LB963 luminometer (Berthold, Germany). Assessment of cell viability and apoptosis For cell viability assays, INS-1E cells were seeded in 96-well plates. After treatment, the medium was replaced with fresh medium containing 0.5 mg/mL MTT (Thermo Fisher Scientific, Carlsbad, CA, USA). After 3 h at 37°C, the medium was removed and replaced with 100 µL of acidified isopropanol (40 mM HCl), followed by incubation at room temperature for 15 min. Absorbance was measured at 570 nm [ 14 ]. For apoptosis assessment, INS-1E cells were seeded onto fibronectin-coated coverslips and treated as described in the figures. After treatment, cells were washed and stained with Hoechst 33342 (20 µg/ml) and propidium iodide (PI; 20 µg/mL) for 30 min at 37°C. Coverslips were mounted on slides with Mowiol, and images were immediately acquired using a NIKON Eclipse Ni microscope (Nikon, Tokyo, Japan). The percentage of apoptotic cells was analyzed by two investigators blinded to the experiment using Fiji software. Additionally, apoptosis was evaluated by phosphatidylserine exposure analysis using PE-Annexin V and 7-AAD staining (BD Biosciences) according to the manufacturer’s instructions, followed by flow cytometry analysis (BD Accuri C6 Plus). Insulin quantification and Glucose-Stimulated Insulin Secretion (GSIS) Insulin secretion from INS-1E and islets was quantified using a sandwich ELISA [ 14 ]. For GSIS, cells/islets were incubated in Krebs–Ringer phosphate buffer (KRB: 135 mmol/l NaCl, 0.5 mmol/L NaH2PO4, 3.6 mmol/l KCl, 0.5 mmol/L MgCl2, 1.5 mmol/L CaCl2, 5 mM NaHCO3, pH 7.4) supplemented with 10 mmol/L HEPES and 0.1% BSA. Cells/islets were first incubated in glucose-free medium for 2 h, followed by a 1-h incubation in fresh KRB-HEPES-BSA containing 2 mmol/l glucose. The supernatant was discarded, and cells/islets were incubated again in fresh KRB-HEPES-BSA with 2 mmol/L glucose. The supernatant was collected, and the cells/islets were subsequently incubated in KRB-HEPES-BSA with 20 mmol/l glucose for an additional 1 h before collecting the solution. Secreted insulin was normalized to total protein content in cell/islet lysates and stimulation index was calculated as the ratio of insulin released under high glucose versus low glucose condition. Protein concentration was determined using the BCA assay Kit (Pierce). RNAseq and bioinformatic analysis Total RNA was extracted from INS-1E cells, and RNA sequencing (RNA-seq) libraries were prepared using the TruSeq RNA Library Prep Kit (Illumina). Sequencing was performed on the Illumina platform. Analyses were conducted in RStudio (R version 4.3.3) using Bioconductor packages. Raw sequencing reads underwent quality control using FastQC (version v0.11.9) to assess read quality [ 64 ]. Preprocessing, including adapter trimming and filtering of low-quality reads was performed using the rfastp package (version 1.12.0) [ 65 ]. The reference index was generated with BSgenome.Rnorvegicus.UCSC.rn7 (version 1.4.3) [ 66 ] and subsequently applied for read alignment to the rat reference genome (mRatBN7.2) using the Rsubread package (version 2.16.1) [ 67 ]. Read quantification was carried out with the featureCounts function from the Rsubread package, using the Rattus norvegicus gene annotation file (mRatBN7.2 GTF). The resulting count matrix was exported for further statistical analysis. The edgeR package (version 4.0.16) was used to normalize sequencing counts and perform differential expression analysis between selected conditions [ 68 ]. To filter out lowly expressed genes, only genes with counts per million (CPM) > 1 in at least two samples were retained. Library sizes were recalculated, and normalization was performed using TMM (trimmed mean of M-values) normalization. Dispersion estimation was conducted, followed by the generation of a biological coefficient of variation (BCV) plot to assess variability across samples. For variance stabilization, the normalized expression data derived from the RNA-seq count matrix was voom-transformed using the limma package (version 3.58.1) [ 69 ]. The transformed matrix was subsequently used for principal component analysis (PCA). The normalized count matrix was log2-transformed (log2-CPM) for heatmap generation. The heatmap was generated from a pre-filtered count matrix based on a list of differentially expressed genes (DEGs) that included all pairwise comparisons performed. DEGs were identified using exactTest, with different thresholds depending on the comparison: in IL-1β low -treated cells versus untreated cells, differentially expressed genes were selected using a false discovery rate (FDR) 0.6. This more permissive threshold was used because the untreated and IL-1β low -treated samples were highly similar, and the small differences between them required a less stringent log2FC cutoff to allow for the selection of statistically significant genes with low variation in expression. For all other comparisons, a threshold of FDR 1 was applied. GSEA (Gene Set Enrichment Analysis) was conducted using the msigdbr package (version 10.0.1) to obtain gene sets from the Hallmark Gene Set specific to the rat species ( Rattus norvegicus ). The ranked gene list, based on the log fold change (logFC) was used to perform the enrichment analysis with the fgsea package (version 1.28.0). The analysis aimed to identify pathways with significant positive or negative enrichment. Results were filtered to retain only those meeting a statistical significance threshold of p < 0.05. The gggsea package was used to visualize the GSEA results. Statistical analysis Results are presented as mean ± SD. Comparison between groups was carried out using paired or unpaired Student ́s t -test or ANOVA followed by Bonferroni ́s multiple comparison test, as appropriate. A p < 0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using GraphPad Prism version 10.2.3 Software. Declarations Acknowledgements: The 5xATF6-LUC and XBP1u-LUC plasmids were a kind gift from Prof. Dr. Sarah Gerlo (Univ. Ghent, Belgium). Conflict of Interest Statement : The authors have no relevant financial or non-financial interests to disclose. Author Contribution Statement C.S. and M.J.P. conceived and designed the study. C.S., M.S.O., A.S-F., I.P-E., and I.R-V. developed the methodology. C.S., L.A., and M.J.P. conducted data analysis and performed statistical analyses. C.S., R.G.M., L.A., and M.J.P. interpreted the results. L.A., E.S., and M.J.P. secured funding. L.A. and M.J.P. were responsible for project administration, funding management, and supervision. C.S., L.A., and M.J.P. contributed to writing, reviewing, and editing the manuscript. MJP is the guarantor of this work, has full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final version of the paper. Prior Presentation. Parts of this study were presented in abstract form at the Annual Meeting of The Endocrine Society, Boston (MA), USA, 1-4 June 2024. Declaration of Generative AI and AI-assisted technologies in the writing process. The author(s) used ChatGPT to check English grammar and improve the language; they reviewed and edited the content as needed and took full responsibility for the publication's content. Funding Statement Supported by ANPCyT-FONCyT (PICT-2018-1577 to MJP / -2021-GRF-TII-241 to LA), FPREDM 2024 (to ES); Universidad Austral (#2024 to MJP and #2023 to LA); and Sociedad Argentina de Diabetes (#2022/#2024 to MJP and #2022 to LA). We thank the support of Facultad de Ciencias Biomédicas (Universidad Austral), Fundación Marjorie para la Investigación en Diabetes (www.fumdiab.org.ar) and The Sugar Science & DKNET-2022 (USA). Data Availability Statement : The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. 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Additional Declarations There is no duality of interest Supplementary Files originalwesternblots.pdf original western blots Supplementaryfile.docx Supplementary file Cite Share Download PDF Status: Published Journal Publication published 21 Oct, 2025 Read the published version in Cell Death & Disease → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6378229","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":441064704,"identity":"176a8dfa-92b8-4369-899a-b621707cbd83","order_by":0,"name":"Marcelo Perone","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9764-9196","institution":"Instituto de Investigaciones en Medicina Traslacional (IIMT), CONICET-Universidad Austral","correspondingAuthor":true,"prefix":"","firstName":"Marcelo","middleName":"","lastName":"Perone","suffix":""},{"id":441064705,"identity":"82e944b9-cf23-41a1-948f-5c6502bc6ee8","order_by":1,"name":"Carolina Setula","email":"","orcid":"","institution":"Instituto de Investigaciones en Medicina Traslacional (IIMT), CONICET-Universidad Austral","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Setula","suffix":""},{"id":441064706,"identity":"aa8dc06a-b58e-4f6c-8ae9-9c1485f133ed","order_by":2,"name":"Andrea Scelza-Figueredo","email":"","orcid":"","institution":"Instituto de Investigaciones en Medicina Traslacional (IIMT), CONICET-Universidad Austral","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Scelza-Figueredo","suffix":""},{"id":441064707,"identity":"87c34113-508b-4397-943b-d0ab2b928a19","order_by":3,"name":"Ingrid Pensado-Evans","email":"","orcid":"","institution":"Instituto de Investigaciones en Medicina Traslacional (IIMT), CONICET-Universidad Austral","correspondingAuthor":false,"prefix":"","firstName":"Ingrid","middleName":"","lastName":"Pensado-Evans","suffix":""},{"id":441064708,"identity":"5413a756-421c-4171-8c7d-0c1f7c10c2e6","order_by":4,"name":"Miranda Orellano","email":"","orcid":"","institution":"Instituto de Investigaciones en Medicina Traslacional (IIMT), CONICET-Universidad Austral","correspondingAuthor":false,"prefix":"","firstName":"Miranda","middleName":"","lastName":"Orellano","suffix":""},{"id":441064709,"identity":"4ab24c66-f54e-469f-8f57-6ab2e8e2cacb","order_by":5,"name":"Ignacio Rodriguez-Valero","email":"","orcid":"","institution":"Instituto de Investigaciones en Medicina Traslacional (IIMT), CONICET-Universidad Austral","correspondingAuthor":false,"prefix":"","firstName":"Ignacio","middleName":"","lastName":"Rodriguez-Valero","suffix":""},{"id":441064710,"identity":"68b3fe63-ec80-47a8-bb3f-4b23427bfc84","order_by":6,"name":"Eduardo Spinedi","email":"","orcid":"","institution":"La Plata Medical School","correspondingAuthor":false,"prefix":"","firstName":"Eduardo","middleName":"","lastName":"Spinedi","suffix":""},{"id":441064711,"identity":"e3c5bff2-2971-4749-be99-2aba87d664e7","order_by":7,"name":"Raghavendra Mirmira","email":"","orcid":"","institution":"The University of Chicago","correspondingAuthor":false,"prefix":"","firstName":"Raghavendra","middleName":"","lastName":"Mirmira","suffix":""},{"id":441064712,"identity":"9a1bc8e7-f4d7-4408-be0d-3c1e9f094017","order_by":8,"name":"Luz Andreone","email":"","orcid":"https://orcid.org/0000-0003-0080-1273","institution":"Instituto de Investigaciones en Medicina Traslacional (IIMT), CONICET-Universidad Austral","correspondingAuthor":false,"prefix":"","firstName":"Luz","middleName":"","lastName":"Andreone","suffix":""}],"badges":[],"createdAt":"2025-04-04 18:20:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6378229/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6378229/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-025-08059-0","type":"published","date":"2025-10-21T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81534049,"identity":"9aa8a9db-0482-4626-b5d5-71008ddb9d66","added_by":"auto","created_at":"2025-04-28 09:56:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1492081,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePreconditioning with low concentrations of IL-1b reduces pro-inflammatory cytokine-induced iNOS expression and activity in INS-1E cells. A-B.\u003c/strong\u003e INS-1E cells were treated for 72 h with IL-1β (3.75, 7.5, 15 pg/ml or 10 pg/ml as indicated) and subsequently challenged or not with IL-1β 200 pg/ml or IL-1β 100 pg/ml + IFN-g 5 ng/ml (CYT) for 16 h, n=5. \u003cstrong\u003eC.\u003c/strong\u003e INS-1E cells were conditioned with IL-1β 10 pg/ml (24, 48 or 72 h as indicated) and subsequently challenged or not with CYT for 16 h, cuadruplicate determinations from one representative experiment. \u003cstrong\u003eD-E.\u003c/strong\u003e INS-1E were conditioned for 72 h with IL-1β 10 pg/ml (IL-1β\u003csup\u003elow\u003c/sup\u003e) and subsequently challenged or not with CYT for 6 h, \u003cem\u003eiNOS\u003c/em\u003e mRNA and protein expression were analyzed by RT-qPCR and Western blot, respectively. \u003cstrong\u003eD. \u003c/strong\u003eRelative \u003cem\u003eiNOS\u003c/em\u003e mRNA levels, normalized to HPRT, n=3.\u003cstrong\u003e E. \u003c/strong\u003eRepresentative blots and quantitative analysis of iNOS\u003cstrong\u003e \u003c/strong\u003eprotein expression, β-actin was used as loading control, n=3. \u003cstrong\u003eF. \u003c/strong\u003eCYT-induced NO secretion in the presence of 5-methylisothiourea sulfate (SMT, iNOS selective inhibitor), two independent experiments.\u003cstrong\u003e A-C, F.\u003c/strong\u003e NO levels in the conditioned media were assessed by Griess reaction and normalized to total cell protein content. Data are shown as mean ± SD. Relevant experiments were performed independently at least 3 times. (**) p \u0026lt; 0.01, (***) p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6378229/v1/1fecb456a3ac6c83c18ec88d.png"},{"id":81535728,"identity":"e1c86833-d2f8-43b1-a5a6-2b27b76b6b40","added_by":"auto","created_at":"2025-04-28 10:12:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5326906,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePriming with IL-1β\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003elow\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e impairs cytokine-triggered NF-κB pathway activation in INS-1E cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-C.\u003c/strong\u003e INS-1E cells were conditioned for 72 h with IL-1β 10 pg/ml (IL-1β\u003csup\u003elow\u003c/sup\u003e) and subsequently challenged or not with IL-1β 100 pg/ml + IFN-g 5 ng/ml (CYT). After indicated time, levels of phospho-IkBa and total IkBa were analyzed by Western blot. Representative blots \u003cstrong\u003e(A)\u003c/strong\u003e and quantitative analysis of phospho-IkBa \u003cstrong\u003e(B) \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003eIkBa \u003cstrong\u003e(C) \u003c/strong\u003eprotein expression, β-actin was used as loading control, n=3. \u003cstrong\u003eD-E.\u003c/strong\u003e INS-1 were treated as described in (A) with 30 min CYT stimulation. NF-kB (RelA) cellular localization was analyzed by fluorescence microscopy.\u003cstrong\u003e D. \u003c/strong\u003eRepresentative microscopy images of INS-1E cells immunostained for NF-kB (red) in different experimental conditions as indicated; nuclei were stained with Hoechst (blue); scale bars 10 μm. \u003cstrong\u003eE.\u003c/strong\u003e Quantification of nuclear:cytoplasmic ratio of NF-kB staining from analysis of 5 separate high-power field images for each experimental condition, n=3. \u003cstrong\u003eF.\u003c/strong\u003e INS-1 were preconditioned as described in (A) and transiently transfected with kB-LUC\u003cstrong\u003e \u003c/strong\u003eand CMV-RL reporter plasmids. At 24 h post-transfection, cells were challenged or not with CYT for 16 h, collected and firefly luciferase (LUC) activity was measured and normalized against renilla luciferase (RL) activity for transfection efficiency n=3. \u003cstrong\u003eG-H.\u003c/strong\u003e INS-1 were preconditioned as described in (A) and challenged or not with CYT for 16 h. \u003cstrong\u003eG.\u003c/strong\u003e \u003cem\u003eIL-1\u003c/em\u003eβ mRNA and \u003cstrong\u003eH.\u003c/strong\u003e \u003cem\u003eIL-1Ra\u003c/em\u003e mRNA expression were analyzed by RT-qPCR. Relative mRNA levels normalized to \u003cem\u003eHPRT\u003c/em\u003e, n=3. Data are shown as mean ± SD. Experiments were performed independently at least 3 times. (*) p \u0026lt; 0.05, (**) p \u0026lt; 0.01, (***) p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6378229/v1/96c15cf705c0d2566f107450.png"},{"id":81535729,"identity":"eb1a1e0d-3c06-490e-b712-351750ceea7e","added_by":"auto","created_at":"2025-04-28 10:12:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6357899,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-1β\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003elow\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e enhances resilience to cytokine-induced death in INS-1E cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B.\u003c/strong\u003e INS-1E cells were conditioned for 72 h with IL-1β 10 pg/ml (IL-1β\u003csup\u003elow\u003c/sup\u003e) and subsequently challenged or not with IL-1β 100 pg/ml + IFN-g 5 ng/ml (CYT). After indicated time, cell death was analyzed by fluorescence microscopy. \u003cstrong\u003eB.\u003c/strong\u003e Representative fluorescence microscopy images of cells stained with Hoecshst (blue) and propidium iodide (red) in different experimental conditions as indicated; scale bars 10 μm. Representative cells considered as apoptotic are shown at a higher magnification. \u003cstrong\u003eA.\u003c/strong\u003e Percentage of apoptotic cells after 16h and 48h of CYT challenging obtained from analysis of 5 separate high-power field images for each experimental condition. \u003cstrong\u003eC-D.\u003c/strong\u003e INS-1 were preconditioned as described in (A) and challenged or not with CYT for 48h. \u003cstrong\u003eC.\u003c/strong\u003e Apoptosis was quantified by flow cytometry after Annexin-V/7AAD dual staining. \u003cstrong\u003eD. \u003c/strong\u003eRepresentative dot plots of cells under each experimental condition.\u003cstrong\u003e \u003c/strong\u003ePercentage of early (Q3) and late (Q2) apoptotic cells, n=3. \u003cstrong\u003eE-G.\u003c/strong\u003e INS-1 were preconditioned as described in (A) and challenged or not with CYT for 16h. \u003cstrong\u003eE.\u003c/strong\u003e \u003cem\u003eDP5\u003c/em\u003e mRNA expression, \u003cstrong\u003eF.\u003c/strong\u003e \u003cem\u003ePUMA\u003c/em\u003e mRNA expression and \u003cstrong\u003eG.\u003c/strong\u003e \u003cem\u003eBax\u003c/em\u003e mRNA to \u003cem\u003eBcl-2\u003c/em\u003e mRNA ratio were analyzed by RT-qPCR. Relative mRNA levels normalized to \u003cem\u003eHPRT\u003c/em\u003e, n=3. \u003cstrong\u003eH-J.\u003c/strong\u003e Levels of CHOP and cleaved caspase-3 were analyzed by Western blot. Representative blots (\u003cstrong\u003eH\u003c/strong\u003e) and quantitative analysis of \u003cstrong\u003eI.\u003c/strong\u003e CHOP and\u003cstrong\u003e J.\u003c/strong\u003e cleaved caspase-3 protein expression, β-actin was used as loading control, n=3. Data are shown as mean ± SD. Experiments were performed independently at least 3 times. (*) p \u0026lt; 0.05, (**) p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6378229/v1/8b59c510fa5b9734636d0d38.png"},{"id":81534654,"identity":"9831faff-686a-4b75-8afc-b44c4f8600f1","added_by":"auto","created_at":"2025-04-28 10:04:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1193711,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMild ER stress is needed for an effective IL-1β\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003elow\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e - induced hormesis in INS-1E cells. A-E.\u003c/strong\u003e INS-1E cells were conditioned for 72h with IL-1β 10 pg/ml (IL-1β\u003csup\u003elow\u003c/sup\u003e) in the absence or presence of tauroursodeoxycholic acid (TUDCA, 5 mM) and subsequently challenged or not with IL-1β 100 pg/ml + IFN-g 5 ng/ml (CYT) for 16h. \u003cstrong\u003eB.\u003c/strong\u003e NO levels in the conditioned media were assessed by Griess reaction and normalized to total cell protein content, n=3. \u003cstrong\u003eC-E.\u003c/strong\u003e Levels of ATF4, CHOP and cleaved caspase-3 were analyzed by Western blot. Representative blots (\u003cstrong\u003eB\u003c/strong\u003e) and quantitative analysis of \u003cstrong\u003eC.\u003c/strong\u003e ATF4, \u003cstrong\u003eD.\u003c/strong\u003e CHOP and\u003cstrong\u003e E.\u003c/strong\u003e cleaved caspase-3 protein expression, b-actin was used as loading control, n=3-5. Data are shown as mean ± SD. Experiments were performed independently at least 3 times. (*) p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6378229/v1/13912a0ff85b1798cb664187.png"},{"id":81534061,"identity":"3233afa4-b929-424e-be94-6954846dbced","added_by":"auto","created_at":"2025-04-28 09:56:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1968932,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-1β\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003elow\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e boosts cytokine-induced eIF2a phosphorylation promoting cellular stress adaptation.\u003c/strong\u003e INS-1E cells were conditioned for 72 h with IL-1β 10 pg/ml (IL-1β\u003csup\u003elow\u003c/sup\u003e) and subsequently challenged or not with IL-1β 100 pg/ml + IFN-g 5 ng/ml (CYT) for 16h. \u003cstrong\u003eA-D\u003c/strong\u003e \u0026amp; \u003cstrong\u003eF-H\u003c/strong\u003e. Levels of phospho-eIF2a and total eIF2a , BIP, GRP94, ORP150 and PDI were analyzed by Western blot. Representative blots (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eC\u003c/strong\u003e) and quantitative analysis of \u003cstrong\u003eB\u003c/strong\u003e. phospho- to total eIF2a ratio, \u003cstrong\u003eD.\u003c/strong\u003e BIP, \u003cstrong\u003eF.\u003c/strong\u003e GRP94, \u003cstrong\u003eG.\u003c/strong\u003e ORP150 and H. PDI expression, b-actin was used as loading control, n=3-5. E. \u003cem\u003eBIP\u003c/em\u003e mRNA expression was analyzed by RT-qPCR. Relative mRNA levels normalized to \u003cem\u003eHPRT\u003c/em\u003e, n=4. A 24 h washout condition (without CYT) was also evaluated. \u003cstrong\u003eJ.\u003c/strong\u003e \u003cem\u003eXBP1s\u003c/em\u003e mRNA to \u003cem\u003eXBP1t\u003c/em\u003e mRNA ratio were analyzed by RT-qPCR. Relative mRNA levels normalized to \u003cem\u003eHPRT\u003c/em\u003e, n=5. \u003cstrong\u003eI\u003c/strong\u003e \u0026amp; \u003cstrong\u003eK.\u003c/strong\u003e INS-1 were preconditioned as described in (A) and transiently transfected with 5xATF6-LUC or XBP1u-LUC, and CMV-RL reporter plasmids. At 24 h post-transfection, cells were challenged or not with CYT for 16 h, collected and firefly luciferase (LUC) activity was measured and normalized against renilla luciferase (RL) activity for transfection efficiency n=3. Tunicamycin (2 mg/mL,Tn) or Thapsigargin (50 nM, Tg) for 16h were used as positive controls. Data are shown as mean ± SD. Experiments were performed independently at least 3 times. (*) p \u0026lt; 0.05, (**) p \u0026lt; 0.01, (***) p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6378229/v1/08210b3ec92d0a82f03ab980.png"},{"id":81534656,"identity":"2845e369-0d05-4cdd-96b5-9550b4115573","added_by":"auto","created_at":"2025-04-28 10:04:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9391509,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of IL-1b\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003elow\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e on β-cell identity and function markers.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-E.\u003c/strong\u003e INS-1E cells were conditioned for 72 h with IL-1β 10 pg/ml (IL-1β\u003csup\u003elow\u003c/sup\u003e) and subsequently challenged or not with IL-1β 100 pg/ml + IFN-g 5 ng/ml (CYT) for 16h. A subsequent 24 h washout condition (without CYT) was also evaluated. \u003cstrong\u003eA.\u003c/strong\u003e \u003cem\u003ePdx-1\u003c/em\u003e mRNA, \u003cstrong\u003eB.\u003c/strong\u003e \u003cem\u003eMafA\u003c/em\u003e mRNA, \u003cstrong\u003eC.\u003c/strong\u003e \u003cem\u003eUcn3\u003c/em\u003e mRNA, \u003cstrong\u003eD.\u003c/strong\u003e \u003cem\u003eIns1\u003c/em\u003e mRNA and \u003cstrong\u003eE.\u003c/strong\u003e \u003cem\u003eIns2\u003c/em\u003e mRNA expression was analyzed by RT-qPCR. Relative mRNA levels normalized to \u003cem\u003eHPRT\u003c/em\u003e, n=3-4. F-I. INS-1E cells were treated as described in (A).\u003c/p\u003e\n\u003cp\u003eImmunoreactive insulin expression (\u003cstrong\u003eF-G\u003c/strong\u003e) and the nuclear localization of immunoreactive Pdx-1 (\u003cstrong\u003eH-I\u003c/strong\u003e) were analyzed by fluorescence microscopy. \u003cstrong\u003eF, H.\u003c/strong\u003e Representative microscopy images of INS-1E cells immune-stained for insulin (\u003cstrong\u003eF\u003c/strong\u003e, green) or for Pdx1 (\u003cstrong\u003eH\u003c/strong\u003e, red) in different experimental conditions as indicated; nuclei were stained with Hoechst (blue); scale bars 10 μm. Quantification of \u003cstrong\u003eG.\u003c/strong\u003e Insulin staining or \u003cstrong\u003eI.\u003c/strong\u003e nuclear Pdx-1 staining from analysis of 5 separate high-power field images for each experimental condition, n=3. Data are shown as mean ± SD. Experiments were performed independently at least 3 times. (*) p \u0026lt; 0.05, (**) p \u0026lt; 0.01, (***) p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6378229/v1/196eee689b9d35323eeefe51.png"},{"id":81534067,"identity":"ff4b03aa-aff4-4196-b319-74e995ba4c3d","added_by":"auto","created_at":"2025-04-28 09:56:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1718780,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-1b\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003elow\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e's effects on gene transcripts associated with β-cell identity and function in murine islets.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMurine islets (50 IEQ/well) were conditioned for 72 h with IL-1β 10 pg/ml (IL-1β\u003csup\u003elow\u003c/sup\u003e) and subsequently challenged or not with IL-1β 100 pg/ml + IFN-g 5 ng/ml + TNF-a 8 ng/mL (iCYT) for 16h. A subsequent 24 h washout condition (without iCYT) was also evaluated. \u003cstrong\u003eA.\u003c/strong\u003e \u003cem\u003eUcn3\u003c/em\u003e mRNA, \u003cstrong\u003eB.\u003c/strong\u003e \u003cem\u003ePdx-1 \u003c/em\u003emRNA, \u003cstrong\u003eC.\u003c/strong\u003e \u003cem\u003eGlut2\u003c/em\u003e mRNA, \u003cstrong\u003eD.\u003c/strong\u003e \u003cem\u003eIns1\u003c/em\u003e mRNA, \u003cstrong\u003eE.\u003c/strong\u003e \u003cem\u003eIns2\u003c/em\u003e mRNA, \u003cstrong\u003eF.\u003c/strong\u003e \u003cem\u003eBIP\u003c/em\u003e mRNA and and \u003cstrong\u003eG.\u003c/strong\u003e \u003cem\u003eAldh1a3\u003c/em\u003e mRNA expression were analyzed by RT-qPCR. Relative mRNA levels normalized to \u003cem\u003eHPRT\u003c/em\u003e, n=3-8. Data are shown as mean ± SD. Experiments were performed independently at least 3 times. (*) p \u0026lt; 0.05, (**) p \u0026lt; 0.01, (***) p \u0026lt; 0.001, (ns) non-significative.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6378229/v1/89cce25ad7b8e52cb329a27e.png"},{"id":81534063,"identity":"99b2174f-6c6f-4f59-abbd-3b3758a67f6a","added_by":"auto","created_at":"2025-04-28 09:56:25","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2175481,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-1β\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003elow\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e helps preserve GSIS and modulates the transcriptomic profile.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA, C-D.\u003c/strong\u003e\u0026nbsp;INS-1E cells were conditioned for 72h with IL-1β 10 pg/ml (IL-1β\u003csup\u003elow\u003c/sup\u003e)\u0026nbsp;and subsequently challenged or not with IL-1β\u0026nbsp;100 pg/ml + IFN-g\u0026nbsp;5 ng/ml (CYT) for 16h.\u0026nbsp;\u003cstrong\u003eB.\u0026nbsp;\u003c/strong\u003e\u0026nbsp;Isolated mouse islets (5 IEQ/well) were stimulated three times with\u0026nbsp;IL-1β 10 pg/ml every 72h\u0026nbsp;and then challenged or not with\u0026nbsp;IL-1β\u0026nbsp;100 pg/ml + IFN-g\u0026nbsp;5 ng/ml + TNF-a\u0026nbsp;8 ng/ml (iCYT) for 16h. \u0026nbsp;\u003cstrong\u003eA-B.\u0026nbsp;\u003c/strong\u003eGlucose-Stimulated Insulin Secretion (GSIS) was assessed by ELISA in the conditioned media of (\u003cstrong\u003eA\u003c/strong\u003e) INS-1E cells and (\u003cstrong\u003eB\u003c/strong\u003e) isolated mouse islets cultured in the presence of low (2mM) or high (20 mM) glucose. The insulin secretion index (20mM/2mM) is expressed as mean\u0026nbsp;±\u0026nbsp;SD, n=6.\u0026nbsp;(*) p \u0026lt; 0.05. \u003cstrong\u003eC-D.\u003c/strong\u003e\u0026nbsp;Bulk RNA-seq was performed on n=3 samples per experimental condition.\u0026nbsp;\u003cstrong\u003eC.\u0026nbsp;\u003c/strong\u003ePrincipal component analysis (PCA) of transcriptomic profiles, showing distinct clustering among experimental conditions. Each point represents an individual sample, with the percentage of variance explained by each principal component indicated on the axis labels.\u0026nbsp;\u003cstrong\u003eD.\u003c/strong\u003e\u0026nbsp;Heatmap generated using log2CPM normalized data, followed by Z-score transformation. Log2CPM data were filtered based on a list of differentially expressed genes (DEGs) derived from all pairwise comparisons. Red indicates genes with higher expression relative to the mean (positive Z-scores), while blue represents genes with lower expression relative to the mean (negative Z-scores). Hierarchical clustering of genes and samples was performed based on similarities in gene expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Gene set enrichment analysis GSEA of IL-1β\u003csup\u003elow\u003c/sup\u003e-treated INS-1E cells exposed to CYT compared to CYT-challenged cells, using Hallmark gene sets: pancreas beta cells, G2M checkpoint, inflammatory response, TNF-a\u0026nbsp;signaling via NF-kB, unfolded protein response and apoptosis. Normalized enrichment score (NES) and p value are displayed for each pathway.\u003c/p\u003e","description":"","filename":"figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6378229/v1/bb1213bafcb1ecaffc6b1865.png"},{"id":81534050,"identity":"f2673b50-c050-469d-a370-07a693047838","added_by":"auto","created_at":"2025-04-28 09:56:24","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1652256,"visible":true,"origin":"","legend":"original western blots","description":"","filename":"originalwesternblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6378229/v1/03cf43741b790fd96d223cf9.pdf"},{"id":81536213,"identity":"e3836b7d-5ef9-4ab3-b69f-90ede0c6fded","added_by":"auto","created_at":"2025-04-28 10:20:25","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":404007,"visible":true,"origin":"","legend":"Supplementary file","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-6378229/v1/1b0193ae1bb9c0d2c88e6f67.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"IL-1β priming triggers an adaptive stress response that enhances pancreatic β-cell resilience to subsequent cytotoxic inflammatory insult","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic β-cells play a crucial role in maintaining glucose homeostasis by secreting insulin. Type 1 (T1D) and type 2 diabetes (T2D), the most common clinical presentations of diabetes, are both characterized by β-cell dysfunction and loss.\u003c/p\u003e \u003cp\u003eThe endoplasmic reticulum (ER) in β-cells is essential for protein folding and insulin synthesis, with the unfolded protein response (UPR) helping to manage fluctuating insulin production demands [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. ER stress markers are present in the inflamed islets of autoimmune diabetes-prone nonobese diabetic mice [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and individuals with T1D [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and T2D [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHormesis is a phenomenon in which a cytotoxic agent, in small doses, benefits organisms. Cells exposed to low toxin levels can resist subsequent high-dose exposures [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It is suggested that the hormetic response to lifestyle detrimental factors such as poor diet, sedentarism, and stress may influence protection levels and impact T2D progression [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInflammatory cytokines, such as IL-1β, TNF-α and IFN-γ negatively affect β-cell function and survival, contributing to the pathogenesis of both T1D and T2D [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Inflammation exacerbates ER stress and activates the UPR, which, when prolonged or dysregulated, leads to β-cell apoptosis [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, restoring ER homeostasis in β-cells has been proposed as a potential strategy to alleviate T1D [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Pro-inflammatory cytokines, particularly IL-1β, drive β-cell dedifferentiation by inducing cellular stress, altering gene expression, and reducing insulin production. This involves the downregulation of key transcription factors essential for β-cell identity (Pdx-1, Mafa, FoxO1, Nkx6.1). IL-1β and other inflammatory cytokines induce the expression of inducible nitric oxide synthase (iNOS) in β-cells, leading to nitric oxide (NO) accumulation. Elevated NO disrupts insulin secretion, protein synthesis, oxidative metabolism, and causes DNA damage, impairing β-cell function and health [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. IFN-γ amplifies the harmful effects of IL-1β on β-cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In contrast, acute low concentrations of IL-1β stimulate insulin release in rat islets [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], underscoring its complex and context-dependent effects on β-cell function and insulin regulation. Meanwhile, the long-term impact of very low IL-1β concentrations on β-cells remains uncertain.\u003c/p\u003e \u003cp\u003eIn this study, we explored the role of IL-1β-mediated hormesis in defending β-cells against dysfunction and death induced by pro-inflammatory cytokines. Our findings show that IL-1β, at basal physiological concentrations, triggers a hormetic response in β-cells, enhancing their resilience to future cytotoxic cytokine challenges. Inducing hormetic responses \u003cem\u003ein vivo\u003c/em\u003e offers a promising strategy to prevent β-cell decline in diabetes and warrants further investigation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003ePreconditioning INS-1E cells with low concentrations of IL-1β mitigates NO secretion in response to a cytotoxic pro-inflammatory cytokine challenge\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIncreases in iNOS-derived NO act as a trigger for pro-inflammatory cytokine-mediated ER stress and death in a β-cell-specific manner\u0026nbsp;[20].\u0026nbsp;INS-1E cells exposed to IL-1β (200 pg/ml) for 16h secreted significant amounts of NO (228 +/- 33.9 pmol/μg protein) into the culture medium. Notably, priming these cells with IL-1β (7.5 and 15 pg/ml for 72h) reduced NO secretion in response to IL-1β (200 pg/ml/16h) (Figure 1A). In a similar experiment, IL-1β preconditioning reduced NO secretion (Suppl. Figure 1A) in INS-1E cells compromised by a 16h exposure to a cytokine mixture containing IL-1β (200 pg/ml) and TNF-a\u0026nbsp;(8 ng/ml).\u003c/p\u003e\n\u003cp\u003eTNF-a\u0026nbsp;and IL-1β, together with IFN-g\u0026nbsp;trigger similar signaling pathways [21]. Thus, INS-1E cells were challenged with IL-1β 100 pg/ml + IFN-g\u0026nbsp;5 ng/ml (CYT). IL-1β preconditioning (10 pg/ml/72h) reduced CYT-induced NO secretion (Figure 1B), with effects starting at 48h and persisting through 72h (Figure 1C). Hereafter, IL-1β (10 pg/ml, 72h) will be referred to as IL-1β\u003csup\u003elow\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo explore the mechanisms behind reduced NO secretion in IL-1β\u003csup\u003elow\u003c/sup\u003e-preconditioned INS-1E cells, we examined iNOS expression and found decreased mRNA (p\u0026lt;0.01; Figure 1D) and protein levels (p\u0026lt;0.01; Figure 1E) following CYT-challenge.\u0026nbsp;The iNOS-specific inhibitor SMT abolished NO secretion, confirming CYT acts via iNOS in INS-1E cells (Figure 1F). Unlike IL-1b\u003csup\u003elow\u003c/sup\u003e, IFN-g\u0026nbsp;preconditioning did not reduce CYT-induced NO secretion (Suppl. Figure 1B).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePriming INS-1E cells with IL-1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e\u003cem\u003e\u003csup\u003elow\u003c/sup\u003e\u003c/em\u003e\u003cem\u003eimpairs the NF-kB pathway activation triggered by the pro-inflammatory cytokine mixture\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNF-κB activation links CYT-induced NO production, β-cell dysfunction, and apoptosis.\u003c/p\u003e\n\u003cp\u003eIL-1b\u003csup\u003elow\u003c/sup\u003e reduced CYT-induced IκBa\u0026nbsp;phosphorylation, abolishing the second peak of p-IκBa\u0026nbsp;levels triggered by the pro-inflammatory cytokine mixture (p\u0026lt;0.01; Fig. 2A, B\u0026amp;C), and reducing NF-κB p65 nuclear translocation in INS-1E cells (p\u0026lt;0.05, IL-1b\u003csup\u003elow\u003c/sup\u003e + CYT vs. CYT; Fig. 2D,E). Additionally, preincubation of INS-1E cells with IL-1b\u003csup\u003elow\u003c/sup\u003e attenuated CYT-induced NF-κB transcriptional activity, as determined by a reporter assay using a plasmid with NF-κB response elements upstream of the luciferase gene (p\u0026lt;0.05, CYT vs. IL-1b\u003csup\u003elow\u003c/sup\u003e + CYT; Figure 2F).\u003c/p\u003e\n\u003cp\u003eIL-1β promotes its own synthesis, partly via NF-κB [22;23]. Its autocrine or paracrine production by β-cells may contribute to their damage [24]. IL-1b\u003csup\u003elow\u003c/sup\u003e attenuated CYT-induced IL-1β mRNA levels (p\u0026lt;0.05, Fig. 2G) while upregulating the transcript expression of IL-1Ra (p\u0026lt;0.05, Fig. 2H), an endogenous IL-1β antagonist. Similar results were obtained in isolated mouse islets under IL-1b\u003csup\u003elow\u003c/sup\u003e regimen and iCYT (IL-1β 100 pg/ml + IFN-g\u0026nbsp;5 ng/ml + TNF-a\u0026nbsp;8 ng/ml)\u0026nbsp;challenge (Suppl. Fig. 2A,B). The increase in \u003cem\u003eIL-1Ra\u003c/em\u003e and the decrease in \u003cem\u003eIL-1β\u003c/em\u003e mRNA levels induced by IL-1b\u003csup\u003elow\u003c/sup\u003e likely play a role in mitigating CYT-induced adverse effects on β-cells. \u003cem\u003eIL1-R1\u003c/em\u003e and \u003cem\u003eIL1-R2\u003c/em\u003e mRNA expression levels showed an increasing trend in INS-1E cells pretreated with IL-1b\u003csup\u003elow\u003c/sup\u003e before CYT challenge (Suppl. Fig. 2C,D). IL1-R2, a decoy receptor for IL-1β, may reduce signal transduction by increasing expression in response to its cognate ligand [25].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIL-1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e\u003cem\u003e\u003csup\u003elow\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;preconditioning enhances resilience to pro-inflammatory cytokine-induced death in INS-1E cells\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIL-1b\u003csup\u003elow\u003c/sup\u003e reduced apoptosis/death in INS-1E cells after CYT/16h, with protection lasting up to 48h (p\u0026lt;0.05 and p\u0026lt;0.01 vs. CYT/16h and CYT/48h, respectively; Fig. 3A,B). These findings were further validated by assessing apoptosis through annexin-V/PI staining (p\u0026lt;0.05 vs. CYT at 48h; Fig. 3C,D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo investigate IL-1b\u003csup\u003elow\u0026nbsp;\u003c/sup\u003e's pro-survival mechanisms, we analyzed Bcl-2 family members involved in apoptosis. IL-1b\u003csup\u003elow\u003c/sup\u003e treatment hampered the CYT-induced increases in \u003cem\u003eDP5\u003c/em\u003e and \u003cem\u003ePUMA\u003c/em\u003e mRNA (63.2% and 47.7%, respectively, vs. IL-1b\u003csup\u003elow\u003c/sup\u003e+CYT, Fig. 3 E,F) and counteracted the CYT-induced increase in \u003cem\u003eBax/Bcl-2\u003c/em\u003e mRNA ratio, suggesting reduced apoptosis susceptibility (p\u0026lt;0.01 vs. CYT\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eFig. 3G).\u0026nbsp;Additionally, IL-1b\u003csup\u003elow\u003c/sup\u003e reduced CYT-mediated upregulation of CHOP (86.6% vs. CYT; Fig. 3H,I) and cleaved caspase-3 (52.9 % vs. CYT; Figure 3H,J), both key mediators in the final steps of apoptosis. These results support the notion that IL-1b\u003csup\u003elow\u003c/sup\u003e triggers a hormetic response, as evidenced by minimal apoptosis compared to INS-1E cells exposed to CYT alone.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIL-1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e\u003cem\u003e\u003csup\u003elow\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;triggers a stress-response hormesis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated if IL-1b\u003csup\u003elow\u0026nbsp;\u003c/sup\u003e‘s protective effect on β-cells requires baseline ER stress for hormesis by adding TUDCA, an ER stress alleviator [15], during preconditioning. Under this condition, CYT-induced NO secretion by INS-1E cells was comparable to that observed in cells treated only with TUDCA, without IL-1b\u003csup\u003elow\u003c/sup\u003e preconditioning (Figure 4A). As expected, TUDCA reduced levels of the ER stress sensor ATF4, along with CHOP and cleaved caspase-3, which are effectors of CYT-induced ER stress-mediated cell death. Interestingly, TUDCA during the IL-1b\u003csup\u003elow\u003c/sup\u003e preconditioning eliminates the stress-response hormesis, as evidenced by the unchanged expression levels of ATF4, CHOP, and cleaved caspase-3 under CYT stimulation (Fig. 4C-E). These results highlight that a minimal level of ER stress is required to elicit an effective, adaptive pro-survival stress-response hormesis in INS-1E cells against CYT insult.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIL-1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e\u003cem\u003e\u003csup\u003elow\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;preconditioning boosts CYT-induced eIF2\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e\u0026nbsp;phosphorylation promoting cellular stress adaptation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn β-cells, pro-inflammatory cytokines activate the PERK branch of the UPR, leading to phosphorylation of eIF2a\u0026nbsp;at Ser51 (p-eIF2a) [14,26]. While initially protective, prolonged activation can cause β-cell dysfunction and apoptosis [27,28]. p-eIF2a\u0026nbsp;suppresses global protein synthesis to conserve energy while facilitating gene reprogramming and the induction of key ER chaperones like BiP to restore protein homeostasis. IL-1b\u003csup\u003elow\u003c/sup\u003e preconditioning further amplified the CYT-induced elevation of p-eIF2a\u0026nbsp;levels in INS-1E cells (p\u0026lt;0.05 vs. CYT; Figure 5A,B) [14]. Both IL-1b\u003csup\u003elow\u003c/sup\u003e preconditioning alone and followed by CYT stimulation led to a significant increase in BiP expression, as assessed by WB (p\u0026lt;0.05 IL-1b\u003csup\u003elow\u003c/sup\u003e vs. control; p\u0026lt;0.01\u0026nbsp;IL-1b\u003csup\u003elow\u003c/sup\u003e + CYT vs. CYT; Figure 5C,D). \u003cem\u003eBiP\u003c/em\u003e mRNA expression also increased following\u0026nbsp;IL-1b\u003csup\u003elow\u003c/sup\u003e, requiring a 24h washout period to return to baseline upon CYT stimulation p\u0026lt;0.01\u0026nbsp;IL-1b\u003csup\u003elow\u003c/sup\u003e + CYT vs. CYT; Figure 5E).\u003c/p\u003e\n\u003cp\u003eIn addition, IL-1b\u003csup\u003elow\u003c/sup\u003e enhanced the expression of PDI, a chaperone essential for β-cell function (p\u0026lt;0.01 vs. untreated), and restored the expression of chaperones downregulated by CYT, including GRP94 (p\u0026lt;0.01), ORP150 (p\u0026lt;0.05), and PDI (p\u0026lt;0.01) (Figure 5F-H). This\u0026nbsp;IL-1b\u003csup\u003elow\u003c/sup\u003e-induced upregulation of key chaperones suggests an improved capacity for protein folding, processing, and secretion, potentially reinforcing β-cell resilience.\u003c/p\u003e\n\u003cp\u003eRegarding UPR sensor activation, CYT differentially affected the transcriptional activity of IRE1a-XBP1 and ATF6 pathways in INS-1E cells. While ATF6 transcriptional activity was reduced by CYT (p\u0026lt;0.001), IRE1a-mediated XBP1 splicing remained unchanged, consistent with previous findings [14] (Figure 5I,K). IL-1b\u003csup\u003elow\u003c/sup\u003e treatment alone increased ATF6 transcriptional activity (p\u0026lt;0.01 vs. control, Figure 5I), but\u0026nbsp;IL-1b\u003csup\u003elow\u003c/sup\u003e preconditioning did not alter the transcriptional activity of ATF6 or IRE1a-mediated XBP1 splicing in response to CYT. XBP1s mRNA levels were downregulated by CYT; however, this reduction remained unaffected by\u0026nbsp;IL-1b\u003csup\u003elow\u003c/sup\u003e conditioning (Figure 5J).\u003c/p\u003e\n\u003cp\u003eOverall, the INS-1E response to IL-1b\u003csup\u003elow\u003c/sup\u003e suggests that, upon CYT stimulation, the induced proapoptotic ER stress is primarily regulated through the PERK/p-eIF2a\u0026nbsp;pathway, leading to decreased expression of ATF4 (Figure 4C), CHOP, and cleaved caspase-3 (Figure 3H-J). Notably, IL-1b\u003csup\u003elow\u003c/sup\u003e pre-treatment significantly attenuated CYT-induced expression of these proapoptotic drivers while simultaneously increasing the mRNA levels of antiapoptotic genes (Figure 3E-G).\u0026nbsp;IL-1b\u003csup\u003elow\u003c/sup\u003e-induced intracellular signaling highlights the PERK/p-eIF2a\u0026nbsp;pathway as a key mediator of an adaptive response that helps preserve β-cell integrity.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIL-1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e\u003cem\u003e\u003csup\u003elow\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;attenuates CYT-induced downregulation of gene transcripts associated with β-cell identity/function, as well as the expression of Pdx-1 and insulin proteins\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe harmful islet microenvironment during diabetes progression disrupts β-cell identity and maturity\u0026nbsp;[29]. CYT stimulation reduced the expression of β-cell identity mRNA transcripts (\u003cem\u003ePdx-1\u003c/em\u003e, \u003cem\u003eMafA\u003c/em\u003e, \u003cem\u003eUcn3\u003c/em\u003e, \u003cem\u003eIns1/2\u003c/em\u003e, p\u0026lt;0.05 vs. untreated) in INS-1E cells (Fig. 6A-E). IL-1b\u003csup\u003elow\u003c/sup\u003e mitigated these reductions, particularly for \u003cem\u003ePdx-1\u003c/em\u003e, \u003cem\u003eMafA\u003c/em\u003e and \u003cem\u003eIns1/2\u003c/em\u003e, and enhanced their recovery, including \u003cem\u003eUcn3\u003c/em\u003e mRNA, after a 24h washout.\u003c/p\u003e\n\u003cp\u003eIn line with these findings,\u0026nbsp;IL-1b\u003csup\u003elow\u003c/sup\u003e prevented the CYT-induced reduction (23% vs. untreated) in immune-reactive insulin in INS-1E cells (p\u0026lt;0.05\u0026nbsp;IL-1b\u003csup\u003elow\u003c/sup\u003e+CYT vs. CYT; Fig. 6F,G). Nuclear localization of Pdx-1 immunoreactivity was enhanced by\u0026nbsp;IL-1b\u003csup\u003elow\u003c/sup\u003e, both in the absence of challenge (p\u0026lt;0.05 vs. untreated) and under CYT exposure (p\u0026lt;0.05 vs. CYT; Figure 6H,I).\u003c/p\u003e\n\u003cp\u003eThe pro-inflammatory cytokine mixture iCYT (IL-1β 100 pg/ml + IFN-g\u0026nbsp;5 ng/ml + TNF-a\u0026nbsp;8 ng/ml) reduced mRNA expression of β-cell identity and functionality markers in isolated murine islets.\u0026nbsp;IL-1b\u003csup\u003elow\u003c/sup\u003e treatment facilitated their recovery in most cases, particularly after the washout period (Fig. 7A–F). IL-1b\u003csup\u003elow\u003c/sup\u003e alone increased the expression of \u003cem\u003ePdx-1\u003c/em\u003e, \u003cem\u003eGLUT2\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;BiP\u003c/em\u003e mRNA compared to untreated islets (Fig. 7B,C,F) and showed a trend toward mitigating the CYT-induced increase in the β-cell dedifferentiation marker \u003cem\u003eAldh1a3\u003c/em\u003emRNA\u0026nbsp;[30]\u0026nbsp;(Fig. 7G). Collectively, the results indicate that\u0026nbsp;IL-1b\u003csup\u003elow\u003c/sup\u003e helps preserve β-cell identity in pancreatic islets under harmful CYT-induced stimuli.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIL-1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e\u003cem\u003e\u003csup\u003elow\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;enhances glucose-stimulated insulin secretion impaired by pro-inflammatory cytokines\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether the beneficial effects of IL-1b\u003csup\u003elow\u003c/sup\u003e on β-cells observed thus far translate into improved insulin secretion, we assessed glucose-stimulated insulin secretion (GSIS).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCYT exposure impaired GSIS (0.86 ± 0.29-fold vs. 3.2 ± 1.7-fold untreated, p\u0026lt;0.05; Fig. 8A), an effect partially counteracted by IL-1b\u003csup\u003elow\u003c/sup\u003e treatment in INS-1E cells (p\u0026lt;0.05; Fig. 8A). IL-1b\u003csup\u003elow\u003c/sup\u003e alone had no effect on insulin secretion.\u003c/p\u003e\n\u003cp\u003eRemarkably, IL-1b\u003csup\u003elow\u003c/sup\u003e restored GSIS in iCYT-challenged isolated islets, further supporting its role in improving islet health and enhancing insulin secretion (p\u0026lt;0.05; Fig. 8B). This finding is particularly significant as it underscores IL-1b\u003csup\u003elow\u0026nbsp;\u003c/sup\u003e‘s protective role in preserving β-cell function in a cytokine-induced harmful environment, closely resembling the inflammatory microenvironment of islets in diabetes.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRNA-seq reveals protective transcriptome modulation in IL-1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e\u003cem\u003e\u003csup\u003elow\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e-conditioned INS-1E cells challenged with cytotoxic cytokines\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the genetic mechanisms underlying phenotypic changes following IL-1b\u003csup\u003elow\u003c/sup\u003e and CYT-treated cells conditioned with IL-1b\u003csup\u003elow\u003c/sup\u003e, we performed transcriptome analysis (Fig. 8C). We generated and sequenced bulk RNA-seq libraries from INS-1E cells subjected to these treatments, along with untreated and CYT-challenged cells as controls.\u003c/p\u003e\n\u003cp\u003ePrincipal component analysis reveals distinct clustering among experimental conditions, highlighting differences in transcriptional profiles. IL-1b\u003csup\u003elow\u003c/sup\u003e-treated cells exhibited a gene expression pattern similar to that of untreated cells. However, IL-1b\u003csup\u003elow\u003c/sup\u003e preserved the expression of β-cell identity and functionality genes (\u003cem\u003eRfx6\u003c/em\u003e, \u003cem\u003eNkx6-1\u003c/em\u003e, \u003cem\u003eIns1\u003c/em\u003e, \u003cem\u003ePdx-1\u003c/em\u003e, \u003cem\u003eMafa\u003c/em\u003e, \u003cem\u003ePcsk1\u003c/em\u003e) in INS-1E cells that were subsequently exposed to a CYT challenge.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe CYT challenge induced significant transcriptional changes. However, IL-1b\u003csup\u003elow\u003c/sup\u003e-treated cells exhibited a distinct response to the proinflammatory challenge, displaying a transcriptional profile that set them apart from CYT-treated cells. IL-1b\u003csup\u003elow\u003c/sup\u003e treatment downregulated apoptosis-related genes (\u003cem\u003eFas\u003c/em\u003e, \u003cem\u003eDdit3\u003c/em\u003e, \u003cem\u003eBid\u003c/em\u003e, \u003cem\u003eBbc3\u003c/em\u003e), attenuated the expression of genes associated with NF-κB signaling (\u003cem\u003eNfkb2\u003c/em\u003e, \u003cem\u003eRelb\u003c/em\u003e, \u003cem\u003eNfkb1\u003c/em\u003e, \u003cem\u003eNfkbil1\u003c/em\u003e) and cellular stress (\u003cem\u003eSod2\u003c/em\u003e, \u003cem\u003eTrib3\u003c/em\u003e, \u003cem\u003eHmox1\u003c/em\u003e, \u003cem\u003eAtf4\u003c/em\u003e, \u003cem\u003eErn1\u003c/em\u003e, \u003cem\u003eAtf3\u003c/em\u003e, \u003cem\u003eAtf6\u003c/em\u003e, \u003cem\u003eNfe2l2\u003c/em\u003e, \u003cem\u003eHsp90b1\u003c/em\u003e, \u003cem\u003eEdem1\u003c/em\u003e), and reduced the expression of cytokine-related inflammatory genes (\u003cem\u003eNos2\u003c/em\u003e). The heatmap of selected genes further supports our qPCR findings (Figs. 1, 3 and 6).\u003c/p\u003e\n\u003cp\u003eGene Set Enrichment Analysis (GSEA) evaluated the enrichment of selected Hallmark Gene Sets in IL-1b\u003csup\u003elow\u003c/sup\u003e-treated cells challenged with CYT, compared to those receiving the CYT challenge (Fig. 8E). IL-1b\u003csup\u003elow\u003c/sup\u003e-treated cells showed positive enrichment in the pancreas beta-cell gene set, suggesting an upregulation of genes that support β-cell phenotype. Core enrichment genes, such as \u003cem\u003ePcsk1\u003c/em\u003e and \u003cem\u003eIsl1\u003c/em\u003e, were identified as key contributors. A positive enrichment score for the G2M Checkpoint pathway suggests that IL-1b\u003csup\u003elow\u003c/sup\u003e-stimulated genes promote cell cycle progression, potentially enhancing proliferation or cell cycle regulation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe UPR pathway exhibited negative enrichment in IL-1b\u003csup\u003elow\u003c/sup\u003e-treated cells, indicating reduced ER stress with \u003cem\u003eAtf3\u003c/em\u003e, \u003cem\u003eChac1\u003c/em\u003e, and \u003cem\u003eErn1\u003c/em\u003e contributing to this effect. Other negatively enriched Hallmark gene sets included the inflammatory response, TNF-a\u0026nbsp;signaling via NF-kB, and apoptosis pathways.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we show that IL-1β at basal physiological concentrations (IL-1β\u003csup\u003elow\u003c/sup\u003e), triggers a stress-response hormesis \u003cem\u003ein vitro\u003c/em\u003e, strengthening pancreatic β-cell resilience and enhancing insulin secretion under inflammatory and cytotoxic conditions.\u003c/p\u003e \u003cp\u003ePriming β-cells with IL-1β\u003csup\u003elow\u003c/sup\u003e activates survival mechanisms by modulating gene expression and promoting an adaptive response, reducing β-cell death/apoptosis triggered by pro-inflammatory cytokines in models mimicking the diabetic islet microenvironment.\u003c/p\u003e \u003cp\u003eβ-cells exhibit a high density of IL-1R1 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], prompting the question: why do these cells express abundant receptors that, when activated by their ligand, can initiate cell death? β-cells express components of the IL-1 signaling system, including IL-1α/β, IL-1R1, IL-1Ra, and IL-1R2, with the latter serving as a decoy receptor [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. IL-1R1 knockout mice display impaired glucose tolerance and reduced insulin secretion [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Additionally, postprandial IL-1β secreted by macrophages contributes to blood glucose homeostasis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Based on these observations, we investigated IL-1β priming's effects on β-cells.\u003c/p\u003e \u003cp\u003eProlonged inflammation disrupts the specialized phenotype of β-cells, leading to transdifferentiation and/or dedifferentiation [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This is clinically significant, as dedifferentiated β-cells are observed in patients with T1D and T2D, likely driven by chronic inflammation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. IL-1β (and/or TNF-α), combined with IFN-γ, disrupts cell function through NF-κB-regulated gene networks, ultimately leading to β-cell death. NF-κB-driven IL-1β transcriptional reprogramming reciprocally regulates chemokine and insulin secretion [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Cytokine-driven activation of the IKK complex triggers IκB phosphorylation, ubiquitination, and degradation, enabling NF-κB nuclear migration to induce inflammatory gene expression and mediators like iNOS [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. NO is a major driver of β-cell dysfunction and apoptosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], impairs oxidative metabolism and insulin secretion, induces ER stress and activates signaling pathways culminating in β-cell apoptosis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, depending on intracellular levels, NO can also suppress apoptosis via caspase-3-dependent DNA damage repair [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe found that INS-1E cells preconditioned with IL-1β\u003csup\u003elow\u003c/sup\u003e exhibited reduced NO secretion in response to CYT challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), mediated by suppression of the NF-κB signaling pathway, leading to decreased iNOS expression (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Since CYT-induced NF-κB activation drives pro-apoptotic signaling in β-cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], our findings suggest a protective role for IL-1β\u003csup\u003elow\u003c/sup\u003e against CYT-induced cell death. We further assessed β-cell viability following IL-1β\u003csup\u003elow\u003c/sup\u003e preconditioning and found that it significantly enhanced cell survival after both short-term and long-term CYT exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A previous study reported a cytoprotective effect of IL-1β on β-cells. Research involving rat β-cells indicated that IL-1β may protect against necrosis caused by STZ or alloxan, although it did not protect against cytokine-induced apoptosis. This protection, however, came at the cost of β-cell phenotype integrity, mediated through an NO-dependent mechanism [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This apparent discrepancy with our results may be explained by the differences in experimental conditions. In this study, β-cells were exposed to very low concentrations (\u0026sim;10 pg/ml) of IL-1β for 72h. In contrast, the referenced study used higher IL-1β concentrations over a shorter exposure period.\u003c/p\u003e \u003cp\u003eWhile some studies dismiss a direct link between CHOP and cytokine-induced β-cell death [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], others highlight its key role in CYT-induced apoptosis, as its knockdown significantly reduces this effect in INS-1E cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Additionally, studies suggest that CHOP also has a pro-inflammatory function [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Consistently, CYT challenge led to increased CHOP expression levels. However, in IL-1β\u003csup\u003elow\u003c/sup\u003e-conditioned INS-1E cells, both CHOP and cleaved caspase-3 were downregulated. These findings align with previous research identifying CHOP as a key regulator of β-cell apoptosis. CHOP contributes to CYT-induced NF-κB-dependent pathways (\u003cem\u003ee.g.\u003c/em\u003e, NO production, induction of pro-apoptotic mediators) and regulates mitochondrial-mediated apoptosis (\u003cem\u003ee.g.\u003c/em\u003e, caspase-3) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In addition to confirming CHOP's relevance as a mediator in CYT-induced activation of the intrinsic apoptotic pathway, we provide new insights into how IL-1β\u003csup\u003elow\u003c/sup\u003e attenuation of CHOP impacts β-cell survival under CYT/inflammatory challenge.\u003c/p\u003e \u003cp\u003eThe UPR preserves cellular homeostasis under stress; however, excessive or prolonged ER stress compromises β-cell function and survival. β-cells rely on the ER and UPR machinery to process excess nutrients and ensure proper insulin folding and secretion [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Unresolved UPR contributes to T1D and T2D [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The UPR cascade is initiated upon BiP dissociation by the autophosphorylation of PERK and IRE1, along with the proteolysis of ATF6 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. While XBP1 and ATF6 manage ER stress, their reduced expression may limit adverse effects, support metabolic adaptation, and mitigate inflammation. However, persistent ER stress can still lead to ATF4-mediated CHOP activation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCytokine-induced ER stress shifts β-cell energy priorities, promoting survival mechanisms at the expense of normal cellular functions, including protein folding, synthesis, and insulin secretion. However, exacerbated ER stress activates PERK/eIF2α/ATF4/CHOP pathway leading to β-cell dysfunction and apoptosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The PERK/p-eIF2α pathway plays a crucial role in cell survival under stress by reducing global protein synthesis while selectively translating specific mRNAs, such as ATF4, which can drive either pro-survival or pro-apoptotic responses depending on the cellular context [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, IL-1β\u003csup\u003elow\u003c/sup\u003e enhanced CYT-induced eIF2α phosphorylation in INS-1E cells, accompanied by reduced ATF4 and CHOP protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). At the same time, IL-1β\u003csup\u003elow\u003c/sup\u003e favored the expression of ER chaperones crucial for insulin folding, processing, and handling, such as BIP [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and GRP94 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], respectively. These findings suggest that IL-1β\u003csup\u003elow\u003c/sup\u003e induces a distinct adaptive response and that its precise molecular mechanism requires further investigation. However, it clearly contributes to preserving β-cell integrity under pro-inflammatory cytokine-induced ER stress, with the pro-survival effects of the PERK/p-eIF2α pathway mediated through mechanisms involving ATF4/CHOP downregulation, although additional protective mechanisms cannot be ruled out. Future experiments should determine whether IL-1β\u003csup\u003elow\u003c/sup\u003e-induced PERK/p-eIF2α signaling promotes survival by reducing oxidative stress or enhancing autophagy while avoiding ATF4/CHOP upregulation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIL-1β\u003csup\u003elow\u003c/sup\u003e mitigated the CYT-induced decline in β-cell identity and maturity markers (\u003cem\u003ee.g.\u003c/em\u003e, \u003cem\u003eUcn3, MafA, Pdx-1\u003c/em\u003e, and \u003cem\u003eGLUT2\u003c/em\u003e) in both INS-1E cells and mouse islets (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) while preserving Pdx-1 and insulin expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This contrasts with previous reports suggesting that low concentrations of IL-1β drive β-cell dedifferentiation and dysfunction [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], possibly due to variations in its concentration and exposure duration.\u003c/p\u003e \u003cp\u003eIL-1β\u003csup\u003elow\u003c/sup\u003e improved β-cell insulin secretion despite the acute impairment of glucose-stimulated insulin release by pro-inflammatory cytokines [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], with a stronger effect in isolated murine islets. This suggests IL-1β\u003csup\u003elow\u003c/sup\u003e may also support other islet-resident cells, warranting further investigation into its broader islet benefits.\u003c/p\u003e \u003cp\u003eRNA-seq revealed a protective transcriptomic profile in IL-1β\u003csup\u003elow\u003c/sup\u003e-preconditioned INS-1E cells under CYT stimulation. DEGs showed preserved β-cell identity and reduced expression of inflammation, NF-κB signaling, ER stress, and apoptosis-related genes. IL-1β\u003csup\u003elow\u003c/sup\u003e and IL-1β\u003csup\u003elow\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;CYT cells exhibited increased \u003cem\u003eRfx6\u003c/em\u003e gene transcript expression, encoding a protein essential for islet cell development and insulin production [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Mutations in \u003cem\u003eRfx6\u003c/em\u003e are associated with maturity-onset diabetes of the young [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], and its expression is dysregulated in human β- and α-cells in both T1D and T2D [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], while β-cell-specific \u003cem\u003eRfx6\u003c/em\u003e knockout mice exhibit impaired insulin secretion [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Isl1 regulates genes essential for β-cell differentiation and maturation, such as \u003cem\u003ePdx-1\u003c/em\u003e and \u003cem\u003eSlc2a2\u003c/em\u003e (\u003cem\u003eGLUT2\u003c/em\u003e) vital for β-cell function and glucose sensing, respectively [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. IL-1β\u003csup\u003elow\u003c/sup\u003e-treatment preserved \u003cem\u003eIsl1\u003c/em\u003e expression in INS-1E cells. The ER chaperone Edem1 supports insulin processing and β-cell function by mitigating ER stress.\u003c/p\u003e \u003cp\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Additionally, GSEA indicates that IL-1β\u003csup\u003elow\u003c/sup\u003e may enhance β-cell resilience by upregulating the cell cycle under inflammatory conditions. The enrichment of the G2M Checkpoint pathway may reflect an adaptive mechanism that counterbalances stress-induced β-cell loss by enhancing proliferative capacity or reinforcing cell cycle control. The observed negative enrichment in the UPR, inflammatory response, and apoptosis pathways indicates that IL-1β\u003csup\u003elow\u003c/sup\u003e may reduce ER stress and inflammatory signaling, contributing to improved β-cell survival. The downregulation of genes involved in inflammation and cell death aligns with a protective role for IL-1β\u003csup\u003elow\u003c/sup\u003e in modulating stress responses, ultimately fostering β-cell adaptation in a pro-inflammatory environment. qPCR validation of DEGs and GSEA-enriched genes in IL-1β\u003csup\u003elow\u003c/sup\u003e-treated cells is needed given their role in β-cell identity, function, and survival.\u003c/p\u003e \u003cp\u003eCollectively, we describe a novel aspect of IL-1β\u003csup\u003elow\u003c/sup\u003e's effects on β-cells, highlighting its ability to induce gene expression changes, modulate ER stress and UPR. These changes enhance cellular resilience against inflammatory cytotoxic challenges triggered by cytokines. However, several questions remain to be addressed: 1) Does β-cell resilience result from a single adaptive molecular pathway in response to IL-1β\u003csup\u003elow\u003c/sup\u003e? 2) Could β-cell resilience be induced by other stress-inducing agents? 3) Since IL-1β\u003csup\u003elow\u003c/sup\u003e induces an increase in p-eIF2α levels, and mammalian stress granules (SGs) are known to assemble in response to stress-induced p-eIF2α [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], could the β-cell response to IL-1β\u003csup\u003elow\u003c/sup\u003e be associated with the protective effect mediated by SGs formation? and 4) Could IL-1β\u003csup\u003elow\u003c/sup\u003e's effects be replicated \u003cem\u003ein vivo\u003c/em\u003e?\u003c/p\u003e \u003cp\u003eGiven that individuals genetically predisposed to T1D, T2D, obesity, or metabolic syndrome do not always progress to overt diabetes, it is plausible that, under certain conditions, β-cells activate protective defense mechanisms [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Our findings suggest that mild or transient stress induced by IL-1β\u003csup\u003elow\u003c/sup\u003e can trigger such protective responses. Future research should focus on identifying novel hormesis inducers (hormetins) in β-cells and uncovering their mechanisms to develop therapies that enhance β-cell function and survival in diabetes.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eReagents\u003c/p\u003e \u003cp\u003eCulture media, supplements and antibiotics were purchased from Gibco (Thermo Fisher Scien- tific, Carlsbad, CA, USA). Fetal bovine serum was obtained from Natocor (C\u0026oacute;rdoba, Argentina). Recombinant cytokines were purchased from R\u0026amp;D Systems (Minneapolis, MN, USA). Tauroursodeoxycholic acid (TUDCA), 5-methylisothiourea sulfate (SMT) and other analytical-grade reagents were purchased from Sigma-Aldrich.\u003c/p\u003e \u003cp\u003eAnimals\u003c/p\u003e \u003cp\u003eC57BL/6NCrl mice were bred in a controlled environment (20\u0026ndash;22\u0026deg;C, 12 h light\u0026ndash;dark cycle) at the IIMT (Austral University-CONICET) animal facility and given \u003cem\u003ead libitum\u003c/em\u003e access to food and water. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, Eighth edition (2011). The study was approved by the Animal Research and Care Committee (CICUAL #2023-03) at Austral University.\u003c/p\u003e \u003cp\u003eINS-1E cell line\u003c/p\u003e \u003cp\u003eThe rat β-cell line INS-1E (Prof. Wollheim, University Medical Centre, Geneva, Switzerland) was used between passages 63 and 90, and cultured at 37\u0026deg;C in a humidified atmosphere containing 5% (vol./vol.) CO2 in complete RPMI 1640 culture medium [11 mM glucose, 10% (vol./vol.) heat-inactivated fetal bovine serum (FBS), penicillin (50 IU/ml), streptomycin (50 \u0026micro;g/ml), L-glutamine (2 mmol/l), 2-mercaptoethanol (50 \u0026micro;mol/l), HEPES (10 mmol/l) and sodium pyruvate (1 mmol/l)]. The presence of mycoplasma was periodically checked by PCR. INS-1E were seeded at a density of 11 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e in multiwell plates (Nunc, Thermo Scientific, Denmark) in complete medium.\u003c/p\u003e \u003cp\u003eMice islets isolation and culture\u003c/p\u003e \u003cp\u003eIslets (C57BL/6NCrl) were isolated by collagenase digestion and handpicked after density gradient centrifugation [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. For standardization purposes, islets with a diameter of 100\u0026ndash;125 \u0026micro;m were defined as one islet equivalent (IEQ). Islets were cultured on ultra-low attachment plates (Corning Costar, Kennebunk, ME, USA), at 37\u0026deg;C in humidified atmosphere containing 5% (vol./vol.) CO2 in RPMI 1640 medium containing 5.5 mM glucose, 10% FBS, penicillin (50 IU/ml), streptomycin (50 \u0026micro;g/ml), L-glutamine (2 mmol/l) and HEPES (10 mmol/l) for 24 h prior to performing experiments.\u003c/p\u003e \u003cp\u003eHormesis induction by IL-1β treatment\u003c/p\u003e \u003cp\u003eINS-1E cells were conditioned with IL-1β 10 pg/ml for 72h (IL-1β\u003csup\u003elow\u003c/sup\u003e), with fresh cytokine added every 24h without replacing the culture media. Then, the culture media was renewed, and cells were challenged with a proinflammatory cytokine mixture (CYT: IL-1β 100 pg/ml\u0026thinsp;+\u0026thinsp;IFN-γ 5 ng/ml). When INS-1E cells were allowed to recover, CYT-containing media was removed after 16h, followed by PBS washing and a 24h incubation in CYT-free RPMI with 10% FBS before harvesting (24h washout).\u003c/p\u003e \u003cp\u003eMouse islets were treated with IL-1β, similar to INS-1E cells. After 72h, the culture medium was refreshed, and the islets were challenged with a proinflammatory cytokine mixture (iCYT: IL-1β 100 pg/ml, IFN-γ 5 ng/ml, TNF-α 8 ng/ml) for 16h. For recovery, the iCYT-containing medium was removed, and the islets cultured for 24h, as with INS-1E cells. Alternatively, for GSIS experiments, islets were treated with IL-1β 10 pg/ml every 72h, with media replaced each time IL-1β was added. After three IL-1β treatments, islets were challenged with iCYT for 16h before starting the GSIS protocol.\u003c/p\u003e \u003cp\u003eSDS-PAGE and Western blot\u003c/p\u003e \u003cp\u003eINS-1E cells were harvested on ice-cold PBS, washed and lysed in lysis buffer [50 mM Tris\u0026ndash;HCl pH 7.4, 250 mM NaCl, 25 mM NaF, 2 mM EDTA, 0.1% Triton-X, protease inhibitors mix (Complete ULTRA, Roche)]. Protein concentration was determined using the BCA assay Kit (Pierce, Thermo Fisher Scientific, Carlsbad, CA, USA) and samples were stored at -20\u0026deg;C. Proteins were separated by 8\u0026ndash;12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), blotted onto nitrocellulose or PVDF membranes (GE-Healthcare, Amersham, UK) and incubated with primary antibodies: IκBα (#4814, 1:1000), p-IκBα (#9246, 1:1000), β-actin (#3700, 1:1000), ATF4 (#11815, 1:1000), CHOP (#2895, 1:1000), Cleaved caspase-3 (#9664, 1:1000), eIF2α (#2103, 1:1000), p-eIF2α (#9721, 1:1000), PDI (#3501, Cell Signaling Technology, Danvers, MA, USA, 1:1000); iNOS (#610332, BD Biosciences, San Jose, CA, USA, 1:1000), ORP150 (#ab124884, 1:1000), GRP94 (#ab13509, 1:1000), BIP (#ab21685, Abcam, Cambridge, MA, USA, 1:1000). Blots were incubated with HRP-conjugated secondary antibodies: Goat anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (#62-6520; Thermo Fisher Scientific, Carlsbad, CA, USA, 1:5000) and Goat Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (#BA1054, Boster Biological Technology, Pleasanton, CA, USA, 1:5000), followed by visualization using ECL (Supersignal; Thermo Fisher Scientific, Carlsbad, CA, USA).\u003c/p\u003e \u003cp\u003eImmunofluorescent microscopy\u003c/p\u003e \u003cp\u003eINS-1E were cultured for 72 h on fibronectin-coated coverslips, treated as described in the figures, fixed by cold methanol and incubated with primary antibodies: monoclonal mouse anti-insulin (clone HB125); NFκB p65 (RelA, #sc-109, Santa Cruz Biotechnology, 1:60) or Pdx-1 (#5679, Cell Signaling Technology, Danvers, MA, USA, 1:100). Secondary antibodies were used at a 1:200 dilution: anti-mouse Alexa Fluor 488 or anti-rabbit Alexa Fluor 647 conjugated dye (Thermo Fisher Scientific, Carlsbad, CA, USA). Coverslips were mounted on slides with Mowiol and images were acquired on a NIKON Eclipse Ni microscope (Nikon, Tokyo, Japan). Image quantification was performed with Fiji software.\u003c/p\u003e \u003cp\u003eNitric oxide quantification\u003c/p\u003e \u003cp\u003eNitrite levels were measured as an indicator of nitric oxide (NO) production using the Griess reagent (1% sulfanilamide and 0.1% naphthyl ethylenediamine dihydrochloride in 2.5% phosphoric acid) at 570 nm [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eQuantitative real-time PCR\u003c/p\u003e \u003cp\u003eTotal RNA was extracted from INS-1E cells using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) following the manufacturer's instructions. Nucleic acid quantification and quality control were assessed with a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Carlsbad, CA, USA). For cDNA synthesis, 1 \u0026micro;g of RNA was reverse-transcribed using RevertAid Reverse Transcriptase in the presence of RiboLock RNase Inhibitor (Thermo Fisher Scientific, Carlsbad, CA, USA) and oligo(dT) primers. All primers were designed using Primer3 and BLAST (NIH) (Suppl. Table\u0026nbsp;1). Real-time PCR was performed on an AriaMx Real-Time PCR Detection System (Agilent Technologies, Santa Clara, CA, USA), using Master Mix qPCR 2.0 Sybr Rox (Embiotec, BA, Argentina). Each reaction was carried out in triplicate, using HPRT as the normalization control. Relative gene expression was determined by the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCT method.\u003c/p\u003e \u003cp\u003eTransient transfections and luciferase reporter assays\u003c/p\u003e \u003cp\u003eNF-κB transcriptional activity was evaluated by transfecting INS-1E cells with a plasmid containing multimerized NF-κB-binding sites linked to a minimal promoter upstream of the luciferase gene (κB-Luc promoter) [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. ATF6 pathway activation was assessed using a reporter plasmid in which the firefly luciferase gene is driven by five copies of the ATF6 consensus binding site (5xATF6-LUC). To quantitatively measure XBP1 splicing, we employed a splicing-specific reporter plasmid where the firefly luciferase coding sequence is fused to the second ORF of unspliced XBP1 (XBP1u-LUC); luciferase expression occurs only upon IRE1-mediated splicing that removes the 26-nt intron. All transfections included a CMV-Renilla LUC expression vector for normalization.\u003c/p\u003e \u003cp\u003ePlasmids were transfected into INS-1E cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific) in Opti-MEM medium following IL-1β\u003csup\u003elow\u003c/sup\u003e conditioning. Thirty hours post-transfection, cells were challenged with pro-inflammatory cytokines. After treatment, cells were lysed and firefly and Renilla luciferase activities were sequentially measured using the Dual-Glo Luciferase Reporter Assay System (Promega) on a Centro LB963 luminometer (Berthold, Germany).\u003c/p\u003e \u003cp\u003eAssessment of cell viability and apoptosis\u003c/p\u003e \u003cp\u003eFor cell viability assays, INS-1E cells were seeded in 96-well plates. After treatment, the medium was replaced with fresh medium containing 0.5 mg/mL MTT (Thermo Fisher Scientific, Carlsbad, CA, USA). After 3 h at 37\u0026deg;C, the medium was removed and replaced with 100 \u0026micro;L of acidified isopropanol (40 mM HCl), followed by incubation at room temperature for 15 min. Absorbance was measured at 570 nm [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor apoptosis assessment, INS-1E cells were seeded onto fibronectin-coated coverslips and treated as described in the figures. After treatment, cells were washed and stained with Hoechst 33342 (20 \u0026micro;g/ml) and propidium iodide (PI; 20 \u0026micro;g/mL) for 30 min at 37\u0026deg;C. Coverslips were mounted on slides with Mowiol, and images were immediately acquired using a NIKON Eclipse Ni microscope (Nikon, Tokyo, Japan). The percentage of apoptotic cells was analyzed by two investigators blinded to the experiment using Fiji software. Additionally, apoptosis was evaluated by phosphatidylserine exposure analysis using PE-Annexin V and 7-AAD staining (BD Biosciences) according to the manufacturer\u0026rsquo;s instructions, followed by flow cytometry analysis (BD Accuri C6 Plus).\u003c/p\u003e \u003cp\u003eInsulin quantification and Glucose-Stimulated Insulin Secretion (GSIS)\u003c/p\u003e \u003cp\u003eInsulin secretion from INS-1E and islets was quantified using a sandwich ELISA [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For GSIS, cells/islets were incubated in Krebs\u0026ndash;Ringer phosphate buffer (KRB: 135 mmol/l NaCl, 0.5 mmol/L NaH2PO4, 3.6 mmol/l KCl, 0.5 mmol/L MgCl2, 1.5 mmol/L CaCl2, 5 mM NaHCO3, pH 7.4) supplemented with 10 mmol/L HEPES and 0.1% BSA. Cells/islets were first incubated in glucose-free medium for 2 h, followed by a 1-h incubation in fresh KRB-HEPES-BSA containing 2 mmol/l glucose. The supernatant was discarded, and cells/islets were incubated again in fresh KRB-HEPES-BSA with 2 mmol/L glucose. The supernatant was collected, and the cells/islets were subsequently incubated in KRB-HEPES-BSA with 20 mmol/l glucose for an additional 1 h before collecting the solution. Secreted insulin was normalized to total protein content in cell/islet lysates and stimulation index was calculated as the ratio of insulin released under high glucose versus low glucose condition. Protein concentration was determined using the BCA assay Kit (Pierce).\u003c/p\u003e \u003cp\u003eRNAseq and bioinformatic analysis\u003c/p\u003e \u003cp\u003eTotal RNA was extracted from INS-1E cells, and RNA sequencing (RNA-seq) libraries were prepared using the TruSeq RNA Library Prep Kit (Illumina). Sequencing was performed on the Illumina platform. Analyses were conducted in RStudio (R version 4.3.3) using Bioconductor packages. Raw sequencing reads underwent quality control using FastQC (version v0.11.9) to assess read quality [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Preprocessing, including adapter trimming and filtering of low-quality reads was performed using the rfastp package (version 1.12.0) [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The reference index was generated with BSgenome.Rnorvegicus.UCSC.rn7 (version 1.4.3) [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] and subsequently applied for read alignment to the rat reference genome (mRatBN7.2) using the Rsubread package (version 2.16.1) [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Read quantification was carried out with the featureCounts function from the Rsubread package, using the Rattus norvegicus gene annotation file (mRatBN7.2 GTF). The resulting count matrix was exported for further statistical analysis.\u003c/p\u003e \u003cp\u003eThe edgeR package (version 4.0.16) was used to normalize sequencing counts and perform differential expression analysis between selected conditions [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. To filter out lowly expressed genes, only genes with counts per million (CPM)\u0026thinsp;\u0026gt;\u0026thinsp;1 in at least two samples were retained. Library sizes were recalculated, and normalization was performed using TMM (trimmed mean of M-values) normalization. Dispersion estimation was conducted, followed by the generation of a biological coefficient of variation (BCV) plot to assess variability across samples. For variance stabilization, the normalized expression data derived from the RNA-seq count matrix was voom-transformed using the limma package (version 3.58.1) [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. The transformed matrix was subsequently used for principal component analysis (PCA).\u003c/p\u003e \u003cp\u003eThe normalized count matrix was log2-transformed (log2-CPM) for heatmap generation. The heatmap was generated from a pre-filtered count matrix based on a list of differentially expressed genes (DEGs) that included all pairwise comparisons performed. DEGs were identified using exactTest, with different thresholds depending on the comparison: in IL-1β\u003csup\u003elow\u003c/sup\u003e-treated cells versus untreated cells, differentially expressed genes were selected using a false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and |log2 fold change (log2FC)| \u0026gt; 0.6. This more permissive threshold was used because the untreated and IL-1β\u003csup\u003elow\u003c/sup\u003e-treated samples were highly similar, and the small differences between them required a less stringent log2FC cutoff to allow for the selection of statistically significant genes with low variation in expression. For all other comparisons, a threshold of FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and |log2FC| \u0026gt; 1 was applied.\u003c/p\u003e \u003cp\u003eGSEA (Gene Set Enrichment Analysis) was conducted using the msigdbr package (version 10.0.1) to obtain gene sets from the Hallmark Gene Set specific to the rat species (\u003cem\u003eRattus norvegicus\u003c/em\u003e). The ranked gene list, based on the log fold change (logFC) was used to perform the enrichment analysis with the fgsea package (version 1.28.0). The analysis aimed to identify pathways with significant positive or negative enrichment. Results were filtered to retain only those meeting a statistical significance threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The gggsea package was used to visualize the GSEA results.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eResults are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Comparison between groups was carried out using paired or unpaired Student ́s \u003cem\u003et\u003c/em\u003e-test or ANOVA followed by Bonferroni ́s multiple comparison test, as appropriate. A \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using GraphPad Prism version 10.2.3 Software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 5xATF6-LUC and XBP1u-LUC plasmids were a kind gift from Prof. Dr. Sarah Gerlo (Univ. Ghent, Belgium).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e: The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.S. and M.J.P. conceived and designed the study. C.S., M.S.O., A.S-F., I.P-E., and I.R-V. developed the methodology. C.S., L.A., and M.J.P. conducted data analysis and performed statistical analyses. C.S., R.G.M., L.A., and M.J.P. interpreted the results. L.A., E.S., and M.J.P. secured funding. L.A. and M.J.P. were responsible for project administration, funding management, and supervision. C.S., L.A., and M.J.P. contributed to writing, reviewing, and editing the manuscript. MJP is the guarantor of this work, has full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final version of the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrior Presentation.\u003c/strong\u003e Parts of this study were presented in abstract form at the Annual Meeting of The Endocrine Society, Boston (MA), USA, 1-4 June 2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Generative AI and AI-assisted technologies in the writing process.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) used ChatGPT to check English grammar and improve the language; they reviewed and edited the content as needed and took full responsibility for the publication\u0026apos;s content.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupported by ANPCyT-FONCyT (PICT-2018-1577 to MJP / -2021-GRF-TII-241 to LA), FPREDM 2024 (to ES); Universidad Austral (#2024 to MJP and #2023 to LA); and Sociedad Argentina de Diabetes (#2022/#2024 to MJP and #2022 to LA). We thank the support of Facultad de Ciencias Biom\u0026eacute;dicas (Universidad Austral), Fundación Marjorie para la Investigación en Diabetes (www.fumdiab.org.ar) and The Sugar Science \u0026amp; DKNET-2022 (USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e: The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMeyerovich K, Ortis F, Allagnat F, Cardozo AK. Endoplasmic reticulum stress and the unfolded protein response in pancreatic islet inflammation. \u003cem\u003eJ Mol Endocrinol.\u003c/em\u003e 2016;57(1):R1-17.\u003c/li\u003e\n\u003cli\u003eTersey SA, Nishiki Y, Templin AT, Cabrera SM, Stull ND, Colvin SC, et al. 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Nucleic Acids Res. 2015;43(7):e47.\u003cbr\u003e \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"insulin, apoptosis, islets, diabetes, UPR, hormesis","lastPublishedDoi":"10.21203/rs.3.rs-6378229/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6378229/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePancreatic β-cells fine-tune glucose homeostasis through insulin secretion. The endoplasmic reticulum (ER) is critical for insulin production, relying on the unfolded protein response (UPR) to adapt to the body's fluctuating demands. Islets from both type 1 (T1D) and type 2 diabetes (T2D) exhibit inflammation, β-cell dysfunction, and loss. ER stress is present in the inflamed islets of autoimmune diabetes-prone mice and individuals with T1D and T2D.\u003c/p\u003e\n\u003cp\u003eInflammatory cytokines induce ER stress and disrupt UPR regulation, driving β-cell apoptosis and contributing to diabetes development. Inflammatory cytokines, \u003cem\u003ee.g.,\u003c/em\u003e IL-1β, impair β-cell function and survival, contributing to diabetes pathogenesis by inducing stress, altering gene expression, driving dedifferentiation, and reducing insulin production. Paradoxically, β-cells exhibit a high density of IL-1R1, and IL-1R1/KO mice display impaired glucose tolerance and reduced insulin secretion. Postprandial IL-1β secreted by macrophages helps maintain blood glucose homeostasis. These observations suggest that circulating low IL-1β concentrations may have physiologically relevant roles; however, their effects on β-cell function and survival remain unclear due to conflicting reports.\u003c/p\u003e\n\u003cp\u003ePreconditioning β-cells with physiological circulating levels of IL-1β (IL-1β\u003csup\u003elow\u003c/sup\u003e) induced a resilient state, protecting them from pro-inflammatory cytokine (CYT)-induced cell death while preserving glucose-stimulated insulin secretion through hormesis. IL-1β\u003csup\u003elow\u003c/sup\u003e-treated INS-1E cells reduced CYT-induced NO secretion by suppressing NF-κB signaling and decreasing iNOS expression, correlating with reduced β-cell death. IL-1β\u003csup\u003elow\u003c/sup\u003e conditioning reduced ER stress and upregulated p-eIF2a in response to CYT, thereby enhancing the expression of ER chaperones and biomarkers linked to improved β-cell identity/functionality.\u003c/p\u003e\n\u003cp\u003eTranscriptomic analysis revealed that IL-1β\u003csup\u003elow\u003c/sup\u003e preconditioning mitigated the CYT-induced loss of genes involved in β-cell function/identity, and suppressed the expression of genes linked to NF-κB signaling, cytokine-induced inflammation, and apoptosis. IL-1β\u003csup\u003elow\u003c/sup\u003e treatment counteracted the upregulation of stress-related genes triggered by pro-inflammatory stimuli.\u003c/p\u003e\n\u003cp\u003eEnhancing IL-1βlow-induced stress-response hormesis may provide a novel strategy to sustain β-cell function and survival during harmful diabetic inflammation.\u003c/p\u003e","manuscriptTitle":"IL-1β priming triggers an adaptive stress response that enhances pancreatic β-cell resilience to subsequent cytotoxic inflammatory insult","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-28 09:56:20","doi":"10.21203/rs.3.rs-6378229/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6b57532a-ae04-4bfa-a70a-8d9b5c7707fd","owner":[],"postedDate":"April 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46960844,"name":"Biological sciences/Cell biology"},{"id":46960845,"name":"Health sciences/Diseases/Endocrine system and metabolic diseases"}],"tags":[],"updatedAt":"2025-10-22T07:08:00+00:00","versionOfRecord":{"articleIdentity":"rs-6378229","link":"https://doi.org/10.1038/s41419-025-08059-0","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-10-21 04:00:00","publishedOnDateReadable":"October 21st, 2025"},"versionCreatedAt":"2025-04-28 09:56:20","video":"","vorDoi":"10.1038/s41419-025-08059-0","vorDoiUrl":"https://doi.org/10.1038/s41419-025-08059-0","workflowStages":[]},"version":"v1","identity":"rs-6378229","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6378229","identity":"rs-6378229","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}